Progress in Neuro-Psychopharmacology & Biological Psychiatry 48 (2014) 161–169

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Progress in Neuro-Psychopharmacology & BiologicalPsychiatry

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Brief maternal separation affects brain α1-adrenoceptors and apoptoticsignaling in adult mice

Roberto Coccurello a, Adam Bielawski b, Agnieszka Zelek-Molik b, Jerzy Vetulani b, Marta Kowalska b,Francesca R. D'Amato a, Irena Nalepa b,⁎a Institute of Cell Biology and Neurobiology (IBCN), National Research Council (CNR)/S. Lucia Foundation, Via del Fosso di Fiorano 64, 00143 Rome, Italyb Department of Brain Biochemistry, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Str, 31-343 Kraków, Poland

Abbreviations: Amy, amygdaloid nucleus; CB, clean becentral medial; CL, central lateral; CM, central medial; CtxCCtxM2, primary and secondary motor cortices; CtxPIR, pisomatosensory cortex;HSP, heat shock protein; IMD, intermMDC, medial central; MDL, medial lateral; MDM,mediodornoradrenergic; Nucleus Acb, nucleus accumbens; PC,preference test; Thal, thalamic nucleus.⁎ Corresponding author. Tel.: +48 12 662 32 25; fax: +

E-mail address: nfnalepa@cyf-kr.edu.pl (I. Nalepa).

0278-5846/$ – see front matter © 2013 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.pnpbp.2013.10.004

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 July 2013Received in revised form 21 September 2013Accepted 3 October 2013Available online 12 October 2013

Keywords:AnhedoniaBcl-2 proteinsEarly life adversityHeat shock proteinsOlfactory learningα1-Adrenoceptors

Exposure to adversity during early life is a risk factor for the development of different mood and psychiatricdisorders, including depressive-like behaviors. Here, neonatal mice were temporarily but repeatedly (day 1 today 13) separated frommothers and placed in a testing environment containing a layer of odorless clean bedding(CB). We assessed in adult animals the impact of this early experience on binding sites and mRNA expression ofα1-adrenergic receptor subtypes, heat shock proteins (HSPs) and proapoptotic and antiapoptoticmembers of theBcl-2 family proteins in different brain regions involved in processing of olfactory information and rewardingstimuli. We found that repeated exposure to CB experience produced anhedonic-like behavior in terms ofreduced saccharin intake and α1-adrenoceptor downregulation in piriform and somatosensory cortices,hippocampus, amygdala and discrete thalamic nuclei. We also found a selective decrease of α1B-adrenoceptorbinding sites in the cingulate cortex and hippocampus and an increase of hippocampal α1A and α1B receptor,but not of α1D-adrenoceptor, mRNA levels. Moreover, while a significant decrease of antiapoptotic heat shockproteins Hsp72 and Hsp90 was identified in the prefrontal cortex, a parallel increase of antiapoptotic membersof Bcl-2 family proteins was found at the hippocampal level. Together, these data provide evidence that theearly exposure to CB experience produced enduring downregulation of α1-adrenoceptors in the prefrontal–limbic forebrain/limbic midbrain network, which plays a key role in the processing of olfactory informationand reaction to rewarding stimuli. Finally, these data show that CB experience can “prime” the hippocampalcircuitry and promote the expression of antiapoptotic factors that can confer potential neuroprotectionto subsequent adversity.

© 2013 Elsevier Inc. All rights reserved.

1. Introduction

Compelling evidence from a variety of studies suggests that early lifeevents strongly influence brain function and constitute amajor risk factorfor the development and persistence ofmental disorders, includingmajordepression (Colman and Ataullahjan, 2010; Heim and Nemeroff, 2001).Early life adverse events occurring during critical neonatal periodsmay therefore increase susceptibility to psychopathology later in life.

Although the problem of modeling depression in rodents iscontroversial, some core symptoms of human depression seem tohave analogs in animal behavior. Thus, the loss of enjoyment in humans

dding; CEL, central lateral; CEM,G, cingulate cortex; CtxM1 andriform cortex; CtxS2, secondaryediodorsal; LC, locus coeruleus;sal; NA, noradrenaline; NAergic,paracentral; SPT, saccharine

48 12 637 45 00.

ghts reserved.

seems to be an analog of anhedonia in rodents and is measuredas a decline of the rewarding properties of drug of abuse, palatablefoods or sweetened drinks in animals. A variety of stressful proceduresexploited during early life experiences can induce depression-likebehavioral changes in rodents (Schmidt et al., 2011; Ventura et al.,2013). The alteration of postnatal rearing environment has beenshown to exert selective changes in several neurotransmitter systems,as for the activity of mesolimbic dopamine system (Coccurello et al.,2009; Piazza et al., 1996). For this reason, the neurochemical changesobserved later in life in animals subjected to neonatal adverse eventsseem to be important to understand comparable changes appearingin the course of depression in humans. The monoamine hypothesis ofdepression assumes that the symptoms of this disease are triggered bydeficits in monoaminergic transmission with several studies suggestingthat noradrenaline (NA) may play an essential role (Haenisch andBönisch, 2011; Nalepa and Sulser, 2004; Vetulani and Nalepa, 2000).

The locus coeruleus (LC), the main noradrenergic (NAergic) nucleusin the brain, is the major source of the NAergic perikarya projecting tothe forebrain. NA acts through three families of adrenergic receptors,α1-, α2- and β-adrenoceptors, all belonging to the class of G-protein-

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coupled receptors. α2-Adrenergic receptors are coupled through Gi/oproteins to the adenylyl cyclase and cyclic adenosine monophosphate(cAMP) second messenger system in inhibitory manner, whileβ-adrenoceptors through stimulatory Gs protein increase cAMP pro-duction. α1-Adrenergic receptors are coupled to phospholipase C(PLC) and phosphatidylinositol second messenger via Gq/11 proteins.There are three subtypes of α1-adrenergic receptors, namely α1A,α1B and α1D that function as stimulatory receptors in response tothe physiological agonist NA (Docherty, 2010). The NAergic systemand central α1-adrenoceptors are involved in the regulation of a largevariety of physiologic and pathologic emotional processes, includingpositively and aversively motivated behaviors, fear and anxiety(Morilak et al., 2005). It is therefore conceivable that changes inducedby early life adverse events may be reflected by the alteration of thecentral NAergic systemand expression of various subclasses of adrenergicreceptors in adult life.

Heat shock proteins (HSPs) are molecular chaperones whose rolein cell homeostasis and cytoprotection is increasingly recognized(Kim et al., 2006; Samali and Orrenius, 1998). HSP-70 and otherassociated chaperone molecules (e.g., HSP-90) have been shownto confer neuroprotection and exert antiapoptotic effects by actingat several levels of the cell death cascade as to inhibit caspaseactivation or reduce the release of cytochrome c from mitochondria(Brown, 2007). Remarkably, α1-adrenoceptors may mediate stress-induced extracellular elevation of the inducible form of HSP-70,Hsp72 (Johnson et al., 2005). Depressive behavior is also associatedto changes in neural plasticity and antidepressant therapy mayprevent apoptosis-induced cell death (Lee et al., 2001). Caspases are aclass of cysteine proteases playing a key contribution in programmedcell death but also in non-apoptotic processes such as cell plasticity andexperience-dependent remodeling of synaptic circuits (Li et al., 2010;McLaughlin, 2004). The apoptosis induced by the caspase-3 activationaffects the balance between proapoptotic (Bax, Bid) and antiapoptotic(Bcl-2, Bcl-xL)members of the B-cell-lymphoma2 (Bcl-2) family proteinsand the adverse effects of stress can be counterbalanced by the releaseof HSPs (Kiang and Tsokos, 1998).

The clean bedding (CB) procedure was previously used to show thatthe temporary isolation from the mother/nest odor information couldbe a model of short maternal separation (D'Amato and Cabib, 1987).In this protocol of brief maternal separation, not the classical handlingbut the privation of mother/nest olfactive cues during the separationappears responsible for the observed long-term effects on emotionalityand sensitivity of opioid and dopaminergic systems (Cabib et al., 1993;D'Amato and Cabib, 1987; D'Amato et al., 1999).

The primary goals of this studywere: (1) to investigatewhether earlyCB experience can induce depressive- or anhedonic-like symptoms inadult mice in the form of altered preference for sweetened solutions;(2) to examine the long-term effects of CB experience on density ofα1-adrenoceptors and mRNA expression of their subtypes in severalkey brain structures and, finally, (3) to evaluate the influence of thisshort-term maternal separation on the expression of HSP-70(Hsp72 and Hsc73), HSP-90 (Hsp90A and Hsp90B) and its impact onantiapoptotic Bcl-2 and Bcl-xL family proteins as functional antagonistof proapoptotic Bax mRNAs.

2. Methods

2.1. Animals

Outbred albino NMRI mice served as subjects of this study (Harlan,Italy). Nulliparous females aged 6 weeks and weighing 22–25 g werehoused in social groups on a 12-h light–dark cycle 7 AM–7 PM withfood andwater available ad lib in a colony room at constant temperature21±1°C. Seven days after their arrival, the femaleswere housed in pairsin 33 × 15 × 13 cm Plexiglas cages. A male was introduced and leftfor 15 days. Pregnant females were then removed to individual cages,

the floor of which was covered with sawdust. No other nesting materialwas supplied. Twice a day, aroundparturition time, cageswere inspectedfor live pups. The day of birth was considered as day 0. On day 1 litterswere culled to eight pups (four males and four females). Litters withless than eight pups were discarded from the sample. Data presentedin this paper are based on a total of 18 lactating females with theiroffspring. During the first two postpartum weeks, home-cage beddingwas changed only on the 10th day of pups' life: mother and offspringwere temporarily removed from their cage, soiled bedding was replacedwith cleanmaterial, except for a very small amount that was scattered inthe clean environment. Animals were then reintroduced into their cages.The experimentswere carried out in accordancewith the Italian nationallaws and regulations concerning the use of animal for research, theNational Institute of Health Guide for the Care and Use of LaboratoryAnimals (NIH Publications No. 80-23) revised 1996 and EU Directive2010/63/EU for animal experiments. All efforts were made to minimizethe number of animals used and their suffering.

2.2. Experimental procedure

Micewereweaned on day 28 of life and housed in groups of four. Thebehavioral test and biochemical measurements were conducted in 90-day-old male mice. Postnatal stress procedure was carried out aspreviously described (D'Amato et al., 1999). Litters were randomlyassigned to one of the two experimental conditions on day 1: briefpostnatalmaternal separation (clean bedding: CB, N=9) and unhandled(control: C, N=9). Once a day, from days 1 to 13, each whole CB litterwas transferred to a new cage, the floor of which was covered withclean bedding and left for 15 min. During the entire 15 min of theprocedure, the cage was placed on a hot plate set at a temperature of35 °C, to prevent cooling of pups. During this procedure, the motherwas left in its home cage. This experimental manipulationwas randomlyperformed between 11 AM and 5 PM. Control litters were leftundisturbed until weaning (except for cage cleaning as described).

2.3. Saccharin preference test (SPT)

2.3.1. FamiliarizationSaccharin solution was prepared using distilled water and pure

saccharin (Hermes Susstoff AG, Zurich). The solutions were freshlyprepared immediately before use and offered at room temperature.Animals living in pairs were singly housed in a test cage for 1 h. Hereanimals were exposed to a double choice (either saccharin solution(0.5%) or drinking water) from graduated tubes (10 ml volume). Theintake was measured to the nearest 0.1ml. The test cage was the sameup to the end of the experiment. Sample sizes were as follows: CB=11 and C=8. No more than 2 animals were taken from the same litter.

2.3.2. Experimental phaseAfter familiarization, every other day (day 1 up to day 5), animals

were moved into their own test cages and the 1-h saccharin intakemeasured. Then, animals were placed back in their home cages. Onlyanimals drinking at least 0.1ml on day 1 were included in the study.

2.4. α1-Adrenoceptors autoradiography

Mice were decapitated, their entire brains taken out, immersedin n-heptane (Sigma-Aldrich) and frozen on dry ice. Sample sizeswere as follows: CB=3 and C=2; each sample derived from a differentlitter. Each frozen brain tissue was mounted on tissue holders and tenconsecutive series of 12-μm thin coronal sections were taken across at250-μm intervals on a Shandon cryostat (UK). The sections were storedat −70°C till the time of the assay. α1-Adrenoceptors were assayedusing [3H]prazosin in the absence and the presence of 10 nM WB4101mask as previously described (Nalepa et al., 2005) and detailed inSupplementary material (Method SM1).

Fig. 1. Effects of postnatal exposure to CB procedure on 0.5% saccharin drinking in adultmice. Mean (± S.E.M.) of 1 h saccharin intake via an intermittent, every-other-day, accessperiod.

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2.4.1. α1-Adrenoceptor ligands[3H]prazosin was obtained from Radioactive Centre Amersham

(77 mCi/mmol) and 2-([2,6-dimetoxyphenoxy-ethyl]aminomethyl)-1,4-benzodioxane (WB4101) from Sigma (St. Louis, MO, USA).

2.5. Analysis of mRNA expression of α1-adrenoceptors, heat shockand Bcl-2 family proteins

2.5.1. Tissue and RNA isolationBrain structureswere dissected immediately after decapitation of rats

and then frozen at −70 °C for further studies. Total cellular RNA fromthe prefrontal cortex, hippocampus and thalamus of 6 litters × group(CB and C) was extracted and purified with spin columns by means ofRNeasy kit (Qiagen). RNA concentration and integrity were determinedby spectrophotometry and RNA gel electrophoresis.

2.5.2. Analysis of mRNAs level (RT-qPCR)The Chromo4 Four-Color Real-Time PCR (MJ Research) Detection

System for quantitative real-time detection of PCR products was applied.Total RNA was reversely transcribed (RT) to cDNA using 0.2 μg ofuniversal primer oligo(dT)15 and according to the parameters indicatedin Supplementary material (Table S1A). The 400ng of total RNA and 2Uof ribonuclease inhibitor (Fermentas, Vilnius, Lithuania) were incubatedin 0.2-ml nuclease-free tubes (Eppendorf, Hamburg, Germany). TheRT reaction was performed in a final volume of 20 μl containing 1XAMV reverse transcriptase buffer (50 mM Tris, pH 8.3; 6 mMMgCl2; 40 mM KCl; 4 mM 1, 4-dithiothreitol; Finzymes Oy, Finland),1mM deoxynucleotide-3-phosphate mixture (dNTP; Fermentas, Vilnius,Lithuania). After denaturation step 10 U of avian myeloblastosis virusreverse transcriptase (Finzymes Oy, Finland) was added. The RT productswere stored at −20 °C until further use. For α1-adrenoceptors andthe heat shock proteins' assays, the 12.5 ng of cDNA was amplifiedby quantitative polymerase chain reaction (qPCR) in the presence of0.3 μM of both sense and antisense primers using the kit containingSYBR Green I fluorescent dye (Roche Diagnostics) and accordingly tothe parameters indicated in Supplementary material (Table S1B). Amelting curve was analyzed at the end of PCR cycle to confirm that asingle product had been amplified. For an estimation of the Bcl2 proteinfamily, the same amount of cDNAwas amplified in presence of TaqManprobes at parameters shown in Supplementary material (Table S1C)and using the supplier's kit (Applied Biosystem) containing the probesand primers. All qPCR reactions were performed in a final volumeof 20 μl and the ROX dye as internal standard was used. Sequences ofused primers selected from adequate sequences of Mus musculus mRNAtogether with the amplicons' size and the details of TagMan probes areshown in Supplementary material (Table S2 and Table S3, respectively).Threshold value (Ct) for each sample was set in the exponential phaseof PCR, and standard curve (rank of dilution from 1.5 to 50 ng of cDNA)method was used for analyzing the data. The cDNA used to standardcurve was pooled from aliquot RT products of all animals. In all casesthe hypoxanthine–guanine phosphoribosyltransferase (HPRT) was usedas reference gene whose expression was observed at the constant levelin all experimental groups of animals.

2.6. Statistical analysis

Data from SPT were analyzed by repeated measures ANOVA withtwo between factor (early experience (control and CB) and saccharinconcentration (0.5%)) and one within factor (time, 5 levels: from day1 up to day 5). The specific [3H]prazosin binding was considered asindication of total α1-adrenoceptor subtypes binding, while the [3H]prazosin binding in the presence of WB4101 (10 nM) — as the specificindication of α1B-adrenoceptor subtype. The densities at the severalareaswere compared between control and CB animals. Autoradiographicdata (expressed as fmol/mg of tissue) were analyzed (separately foreach structure and receptor subtype) by one-way analysis of variance

(ANOVA) with treatment (control or CB) as factors. Similarly, the mRNAdata were analyzed by one-way ANOVA separately for each receptorsubtype. All statistical analyses were carried out with STATISTICA8.0 software. The individual differences were subsequently testedfor significance with the Fisher's LSD. The p values less than 0.05were regarded as significant.

3. Results

3.1. Saccharine preference test

For the SPT, the ANOVA analysis revealed a significant effect of briefmaternal separation in animals tested for a 0.5% saccharin intake(F1,68 = 4.52; p b 0.05), with mice underwent early CB experiencedrinking less sweetened solution than controls (Fig. 1).

3.2. Effect of postnatal CB experience on brain α1-adrenergicreceptors density

Postnatal CB experience led to changes in the α1-adrenoceptors asmeasured in brains of 90-day-old mice. Significant differences betweencontrol and CB mice were seen in almost all brain regions analyzedwhere the [3H]prazosin binding sites in CB brains were reduced(Figs. 2A, 3A, B, E, F). The most pronounced decrease of the total α1-adrenoceptor densities (including all the three receptor subtypes) wereobserved in paracentral and mediodorsal thalami (Thal PC-MDM; 35%decrease) (F1,20 = 32.65; p b 0.001), in centromedial and centrolateralamygdala (Amy CEM-CEL; 28% decrease) (F1,20 = 6.19; p b 0.05), inpiriform cortex (CtxPIR; 28% decrease) (F1,21 = 21.18; p b 0.001) andhippocampus (22%decrease) (F1,18=14.30; pb0.01). The only exceptionwas the cingulate cortex (CtxCG) where the α1-adrenoceptors' densitychanged in an opposite manner, with a small (17% vs. control mice)but significant increase (F1,18 = 11.00; p b 0.01). In relation to theuntreated control group, the [3H]prazosin binding sites assessed inthe presence of WB4101 were also diminished with changes limitedto four brain regions (Figs. 2B, 3C, D, G, H). Thus, the decrease inthe α1B receptor densities was less marked and the changes did notexceed 18% in the nucleus accumbens (F1,18 = 6.57; p b 0.05), inthe centromedial and intermediodorsal thalamic nuclei (Thal CM-IMD)(F1,20=7.78; pb 0.05) and hippocampus (F1,18=16.00; pb 0.001), andreached 20% in the case of the CtxCG (F1,18=4.80; pb0.05) (Fig. 2B).

3.3. Effect of postnatal CB experience on brain α1-adrenergicreceptor mRNA expression

Expression of mRNA of α1-adrenergic receptor subtypes wasassessed in three selected brain regions. In the hippocampus of adultmice that underwent CB procedure, the α1A and the α1B receptor

Fig. 2. Effect of postnatal exposure to CB procedure on the α1-adrenergic receptors' density in selected brain regions of adult mice. (A) Authoradiography of [3H]prazosin binding sitesrepresents the totalα1-adrenoceptors. (B) [3H]prazosin binding in the presence of 10nMWB4101mask represents theα1B-adrenoceptor population. Abbreviations: nucleus Acb, nucleusaccumbens (core). Ctx, cortical regions: CG, cingulate cortex; M1 andM2, primary and secondary motor cortices; S2, secondary somatosensory cortex; PIR, piriform cortex; Thal, thalamicnuclei: CL, central lateral, analyzed together with MDL, medial lateral and MDC, medial central; CM, central medial and IMD, intermedio-dorsal; PC, paracentral and MDM, mediodorsal;Amy, amygdaloid nuclei: CEM, central medial; CEL, central lateral. Data represent the mean values± SEM of n=8–10 and n=12–14 sections (for control and CB group, respectively).***p b 0.001, **p b 0.01, *p b 0.05— significantly different vs. control mice.

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mRNA levels were significantly increased (respectively by 59% and103% as compared to untreated control group, F1,10 = 8.34; p b 0.05and F1,10 = 5.02; p b 0.05, respectively), while the α1D-adrenoceptorremained unchanged (Fig. 4). No changes were observed in the mRNAexpression of α1-adrenoceptor subtypes in the prefrontal cortex andthe thalamus (data not shown).

3.4. Expression of mRNA level of the heat shock proteins in selectedbrain regions

The mRNA expression of heat shock proteins (HSP) belonging toHSP70 and HSP90 families were assessed in selected brain structuresof adult mice underwent CB experience. These proteins include

Fig. 3. Example of autoradiographic images of [3H]prazosin binding (upper panel) and the [3H]prazosin binding in the presence of 10 nMWB4101 mask (lower panel) in brain of adultmice. (A, C, E, G) — controls and (B, D, F, H)— mice exposed to CB procedure. Brain section equivalent to Paxinos and Franklin (2001). Bregmas: 1.7 (A–D) and–1.7mm (E–H).

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the inducible forms of the HSP70 (Hsp72) and HSP90 (Hsp90A) andthe Hsc73 and Hsp90B, which are their constitutive representatives. Inthe prefrontal cortex of CBmice, themRNA expression of both inducibleforms (Hsp72 and Hsp90A) was reduced (by 15%) compared to thecontrols animals (F1,17 = 8.38; p b 0.05 and F1,9 = 4.88; p b 0.05,respectively) (Fig. 5) while the Hsc73 and Hsp90B were not affected.No changes in the tested HSPs were noticed in the hippocampus (datanot shown).

3.5. Expression of mRNA level of the Bcl-2 family proteinsin the hippocampus

The mRNA expression of three proteins, the antiapoptotic Bcl-2 andBcl-xL and the proapoptotic Bax, all belonging to the Bcl-2 family, wereassessed. The changes were observed only in the antiapoptotic proteinsin the hippocampus of CBmice (Fig. 6A–C). The level of Bcl-2mRNAwassignificantly elevated (by 36% vs. control; F1,10=7.85; pb0.05) (Fig. 6A).The anti-apoptotic index, calculated as the ratio of anti-apoptotic Bcl-2and Bcl-xL proteins to the pro-apoptotic Bax protein mRNA expression,was also elevated in the hippocampus of adult mice that underwentearly CB experience (F1, 10= 4,90; p b 0.05 and F1, 10= 5,10; p b 0.05)(Fig. 6D, E). No changes were found in the thalamus (not shown).

4. Discussion

The experiments described here provide the first evidence that arepeated and transitory postnatal experience, in the formof perturbationof early mother-pup bonding (via CB experience), is liable to produce

Fig. 4. Effect of postnatal exposure to CB procedure on mRNA level of the α1-adrenoceptor subreceptor. (C) α1D-Adrenergic receptor. Data are expressed as mean± S.E.M. (n= 6). *p b 0.05

marked changes in α1-adrenoceptors binding density and expressionof different α1-adrenoceptors subtypes in selected brain regions andreduce preference for a palatable fluid in adult life. This study alsoprovides a demonstration that this early social experience may producelong-term changes in the levels of expression of HSPs in the prefrontalcortex. Moreover, while proapoptotic Bax proteins did not change,this study reports that antiapoptotic Bcl-2 but not Bcl-xL proteinsare upregulated in the hippocampus of adult animals that underwentpostnatal CB experience.

4.1. Hedonic experience and NAergic signaling: role of α1-adrenoceptors inreward and olfactory information processing

The NAergic system is involved in the processing of drug-associatedrewards (Weinshenker and Schroeder, 2007) and the interaction ofNAergic transmission with mesolimbic dopamine system has beenshown to mediate the behavioral response to stress and the reinforcingeffects of psychostimulants (Ventura et al., 2003). The relevance ofNAergic system for hedonic experience is further suggested by thepotent NA release evoked by amphetamine administration (Rothmanet al., 2001) aswell as by the suppression of cocaine-induced conditionedplace preference in dopamine beta-hydroxylase (Dbh) null mice (Jasminet al., 2006). Of particular importance, the administration of the selectiveα1-adrenoceptor antagonist prazosin reduced reinstatement ofdrug-seeking behavior (Zhang and Kosten, 2005), thus demonstratingthe importance of this class of receptor for cocaine and heroinself-administration (Greenwell et al., 2009; Wee et al., 2008).The involvement of α1-adrenoceptors in the mechanism of action

types in the hippocampus of adult mice. (A) α1A-Adrenergic receptor. (B) α1B-Adrenergiccompared to control mice.

Fig. 5.Effect of postnatal exposure to CB procedure onmRNA level of Hsp72 andHsp90A—

the inducible forms of heat shock proteins HSP70 and HSP90, respectively, in theprefrontal cortex of adult mice. Their constitutive forms, Hsc73 and Hsp 90B, were notaltered (not shown). Data are expressed asmean±S.E.M. (n=6–10). *pb0.05 comparedto control mice.

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of drug of abuse was also showed by our earlier studies demonstratingthat acute injection of cocaine enhances the α1-adrenergic receptorresponsiveness to NA (Wieczerzak et al., 2008) while cocaine sen-sitization produced significant alterations in the α1-adrenoceptorsdensity (Nalepa et al., 2006).

The current study, using [3H]prazosin labeling, demonstrates that thisrepeated early life experience can induce long-term changes of NAergicsystem, which is reflected by the alteration of α1-adrenoceptor densityand expression in several brain regions. Our data provide also evidencethat pups which underwent repeated CB experience showed reducedpropensity to seek rewarding stimuli in adult life. In most of the regionsexplored, the CB experience produced a decrease of α1-adrenoceptorbinding sites that was more pronounced in CtxPIR and in discretethalamic nuclei (PC-MDM) but also evident in CEM-CEL amygdala,CtxS2 and hippocampus (Fig. 2A). Being one of the core symptomsof depression, the notion of anhedonia assumes a central role forthe diagnosis of this disorder. Exposure to stress can desensitize theα1-adrenoceptors (Stone et al., 1986) and despite some controversialresults there is evidence of a decrease in the density of α1-adrenoceptors in depressed subjects (Gross-Isseroff et al., 1990;Underwood et al., 2004). The increase in the number and the affinityof α1-adrenoceptors after chronic antidepressant treatment supportsthe idea that these receptors may be impaired in depression (Nalepaet al., 2002; Stone et al., 2003).

The reduction of α1-adrenoceptors binding at CtxPIR level isparticularly attractive in the light of the type of early experience usedin this study and for the importance of this structure for olfactorylearning and the formation of odor memory. The piriform/olfactorycortex receives direct projections from the olfactory bulb (Shipleyand Ennis, 1996) and represents a main component of the olfactorysystem network. The key role of odor information for pups' survival

Fig. 6. (A–C) Effect of postnatal exposure to CB procedure onmRNA levels of Bcl family proteinsanti-apoptotic Bcl-2 and Bcl-xL to pro-apoptotic Bax protein mRNA expression. Data are expre

and response to emotional experiences is underlined by the fact thatolfactory bulb sends direct inputs to cortical amygdaloid complex, fromwhich also originates the projections to hippocampus via the entorhinalcortex (McDonald, 1998; Miyamichi et al., 2011; Vanderwolf, 2001).Notably, CEM-CEL amygdala, where α1-adrenoceptors binding wasfound significantly reduced, receives olfactory information from corticalamygdala and appears functionally connected with the olfactory bulb(McDonald, 1998). In this view, amygdala andhippocampus are integralcomponents of the olfactory system (Swanson and Petrovich, 1998;Vanderwolf, 2001).

Especially during early postnatal period, environmental odors conveycrucial information for learning of mother–pup attachment and infantsurvival (Cheslock et al., 2000). There is evidence that olfactory cuesconveyed by the mother and the rapid learning of odor preference areboth encoded in the CtxPIR in spite of the abusive behavior of stressedmothers (Roth and Sullivan, 2005). Most interestingly, during thematurational period, olfactory-mediated attachment to caregiversrequires high levels of NAergic neurotransmission from LC to olfactorysystem (Harley et al., 2006). In the light of CB-induced decrease of α1-adrenoceptor binding sites, it is tempting to hypothesize that an earlyperturbation of olfactory information processing might have producedthe downregulation of postsynaptic excitatory α1 receptors, whoseactivationunderliesNAergic-mediated learning signal inneonatal rodents(Harley et al., 2006). Thus, brief but repeated deprivation of mothers'odor during CB experience might cause a mismatch between thehyperesponsiveness of LC neurons to external sensory stimuli that isobserved during early infancy (Nakamura et al., 1987) and the scarcityof the olfactory signals conveyed by the environment. It is also ofnote that the decrease of NA released in the olfactory bulb depends onthe maturation of the LC, which involves the development of functionalα2-adrenergic inhibitory autoreceptors that offset the LC drive(Nakamura and Sakaguchi, 1990). CB-induced temporary deprivation ofmothers' odor occurred during early postnatal development (days 1–13), when α2 inhibitory autoreceptors are not fully functional andNAergic signal in the olfactory circuit is mostly mediated by α1-adrenoceptors.

4.2. α1B-Adrenoceptor subtype and the prefrontal–limbicforebrain/midbrain network

To assess the specific role ofα1B-adrenoceptor subtype,we used [3H]prazosin labeling in presence of low concentration ofWB4101.With theexception of the hippocampus, the decrease ofα1- orα1B-adrenoceptorsubtypes was always found to affect different brain regions. Thus,while somatosensory and piriform cortices showed a decrease of α1-

in the hippocampus of adultmice. (D, E) The anti-apoptotic index calculated as the ratio ofssed as mean± S.E.M. (n=6). *p b 0.05 compared to control mice.

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adrenoceptor, the α1B-adrenoceptor subtypes were unaffected inthe same regions. Similarly, while α1-adrenoceptors were reduced inPC-MDM Thal, a decrease of α1B-adrenoceptor subtypes was observedonly in CM-IMD Thal. The same is true for the different susceptibilityof α1-adrenoceptor subtypes with regard to the amygdala or theaccumbens, where α1-adrenoceptors were reduced in the former butnot in the latter in which only the α1B-adrenoceptor binding sites weredecreased (Fig. 2B). An interesting exception is the CtxCG where briefor long maternal separation reduced NA levels (Arborelius and Eklund,2007) and in which we found a decrease of α1B-adrenoceptors witha possible compensatory increase of α1-adrenoceptor binding sites(Fig. 2).

CtxCG plays a relevant role in adverse events and reward processing.Here,we observed that adult animals that underwent early CB experienceexhibited anhedonic-like behavior in terms of reduced preference forsweetened fluids (i.e., sensitivity to reward). Together with amygdala,hippocampus, thalamus and accumbens, the CtxCG is an essentialcomponent of the prefrontal–limbic forebrain/limbic midbrain networkthat appears dysregulated in depressive disorder (Bennett, 2011;Morgane et al., 2005). Moreover, social stress affects CtxCG morphology(Bock et al., 2005; Murmu et al., 2006) and alterations in spine densityand dendritic morphology were also observed in hippocampal neurons,in amygdala and in accumbal cells of adult mice underwent neonatalmaternal separation (Martínez-Téllez et al., 2009; Monroy et al., 2010;Poeggel et al., 2003).

It is known that NA regulates arousal and adaptation toenvironmental stressors and the link between adversity and mooddisorders is corroborated by the role of NAergic transmission indepression. Indeed, drugs that antagonize α2 autoreceptors or inhibitNA transporter (NAT) are effective antidepressants (Haenisch andBönisch, 2011). Downregulation ofα1B-adrenoceptor subtypes secondaryto adverse events and increased rate of NA turnover are therefore possibleoutcomes of early social adversity. Our data provide the first evidencethat a perturbation of the early life olfactory communication can down-regulate the expression of α1B-adrenoceptor subtypes in a networkof brain regions (nucleus Acb, amygdala, CtxCG, Thal and hippocampus)that are critically involved in attachment learning and alter animals'responsiveness to rewarding stimuli in adult life.

4.3. α1-Adrenoceptor subtypes mRNA expression in mice underwentearly CB experience

Changes in density and properties of membrane receptors may resultfrom changes in gene expression or could be due to latermodifications. Inthis study, we investigated whether the changes in α1-adrenoceptorsubtypes induced by early CB experience do appear at the level of geneexpression. Analysis of the α1A-, α1B- and α1D-adrenoceptor mRNAsin the prefrontal cortex, thalamus and hippocampus showed that geneexpression was altered by early life event only in the hippocampus —

the brain structure importantly involved in the depression-relatedbehaviors and in long-term effects of early life experience (McEwen,2003). We found an increase of the α1-adrenergic receptor mRNAexpression in the α1A and α1B subtypes, while the α1D receptor mRNAlevel remained unaltered. Different changes in the expression of α1-adrenergic receptor subtypes may result not only from a differentregulation in response to NAergic transmission (Yang et al., 1999) butalso from othermodulators affecting brain function (e.g., corticosteroids),which most probably were involved in the early CB event and have beenshown to modulate mRNA of α1B and α1D-adrenoceptors (Day et al.,2008).

The importance of NA in the region-dependent regulationof α1-adrenoceptors' expression is also supported by our earlierstudies (Kreiner et al., 2011; Nalepa et al., 2002), in which weshowed that antidepressant treatment that enhances the availabilityof synaptic NA also elevated the density of α1A-adrenoceptor proteinin the hippocampuswithout changing the level of itsmRNA expression.

On this basis, the elevated hippocampal expression of genes coding forα1A- and α1B-adrenoceptor mRNAs (Fig. 4) may represent an adaptiveresponse to the decrease in the receptors' density in the same brainregion, thus suggesting a post-transcriptional reduction ofα1-AR densitythat is not necessarily mirrored by mRNA changes. Moreover,it is plausible that the opposite changes in the receptors' density(a decrease) and their mRNA expression (an increase) observed in thestudy may also reflect an enhancement in the receptors' turnover.

On the other hand, the lack of changes in other brain structurestested are not unusual and may be due to the fact that the brain is nothomogenous and processes in various brain areas may differ. There isextensive evidence that changes in monoaminergic systems inducedby brief maternal separation are often area specific and limited toparticular brain structures (e.g., see for review, Arborelius and Eklund,2007; Vetulani, in press).

4.4. Early CB experience, Hsp72 and Hsp90A mRNA expression

To our knowledge, this is the first study reporting that an earlydisruption of olfactory information processing can inhibit the brainexpression of HSPs in adult mice. Hsp72 is the major inducible formof HSP-70 that suppresses several apoptotic signaling pathways(e.g., caspase activity). A number of studies have underlined thelink between cell death, inflammatory cytokines and mood disorders(Maes et al., 2009; Wager-Smith and Markou, 2011). Antidepressantefficacy is related to the reduction of proinflammatory cytokines andthis effect appears associated to the activation of HSP-70 (Pae et al.,2007).

The Hsp72-mediated upregulation of cytokines (e.g., IL-6, TNF-α) is part of the immune-neuroendocrine system that stimulatesglucocorticoid (GC) release, thus triggering the anti-inflammatoryresponse (Asea et al., 2000). Notably, CG release can induce Hsp72expression during the exposure to inescapable physical stress andpromote recovery from inflammation (Campisi and Fleshner, 2003).A viable GC receptor (GR) signaling requires the presence of achaperone complex that is associated with cytosolic GR and includestheHsp90 (Hsp90-GR heterocomplex). Thus, Hsp90 regulates the activityof GR and inhibition of Hsp90 or alteration of assembly may alter GRsignaling (Kovacs et al., 2005; Murphy et al., 2005) and contribute todevelopment of depressive disorders (McEwen, 2005).

Apoptosis in hippocampal neurons and neuroinflammation can betriggered by psychosocial stress (Kubera et al., 2011) and increasedexpression of Hsp72 in the hippocampus has been shown to decreasestress-induced neuronal apoptosis (Yao et al., 2007). Moreover, Hsp72plasma levels are increased following exposure to different stressorsamong which predatory stress and noise exposure, thus underlyingthe role of adrenergic system (Fleshner et al., 2004). This view isfurther supported by the data reporting that α1-adrenoceptorsstimulation increases HSP70 plasma levels that can be preventedby α1-adrenoceptor pharmacological blockade (Johnson et al.,2005). Accordingly, the abnormal increase in pro-inflammatorycytokines observed in depressed patients is probably involvedin the alteration of noradrenergic transmission. In this regard, there isevidence that noradrenaline reuptake inhibitors can exert a potentanti-inflammatory action in the rat cortex (O'Sullivan et al., 2009).Notably, the stimulation of pro-inflammatory cytokine secretion afterexposure to the bacterial lipopolysaccharide (a model of depression)has been shown to decrease themotivation to obtain a saccharin solution(Yirmiya, 1996). Hence, the α1-adrenoceptor downregulation foundin the CtxPIR or in the CtxCG (α1B-adrenoceptor) of mice underwentCB exposure might have blunted the expression of prefrontal Hsp72,both factors concurring to the expression of the depressive-like behavior.

The fact that this reduced expression was found in adult animalsemphasizes the possibility that a brief postnatal alteration of odor-based mother–infant communication may have enduring effects on thevulnerability of prefrontal neurons towards neuroinflammatory signals.

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Although we do not know how the reduced expression of antiapoptoticHSP70 and HSP90 can affect reward-oriented behavior, the existenceof proinflammatory cytokines and altered GC signaling in thepathogenesis of mood disorders appears to support the possible linkbetween anhedonia in CB mice and α1-adrenoceptor downregulation.

4.5. Early CB experience and rise of antiapoptotic Bcl-2 proteins

Abnormalities in hippocampal neuronal architecture are frequentlyreported in depressive disorder (Sapolsky, 2004). Bcl-2 proteins areknown to confer protection against multiplemechanisms (e.g., oxidativestress, reduced mitochondrial redox capacity, neurite loss) involvedin the apoptotic death cascade. Considering neuroinflammatory factorsin mood disorders and the importance of psychosocial environmentin depression-like behaviors (Kubera et al., 2011; Maes et al., 2011),the link between adverse life events, disease onset (e.g., depression)and variation in Bcl-2 protein expression appears well supported(Hunsberger et al., 2011). Antidepressant treatment has been shownto counteract both the downregulation of Bcl-xL mRNA expression andthe increase of proapoptotic Bax levels observed in the hippocampusafter exposure of adult animals to repeated unpredictable stress (Kostenet al., 2008).

In our study, adult mice that underwent neonatal CB experienceshowed hippocampal increase of the antiapoptotic Bcl-2 expression anda tendency towards the increase of Bcl-xL mRNA levels. In parallel,proapoptotic Bax did not change and both Bcl-2/Bax andBcl-xL/Bax ratioswere significantly increased. Thus, the increase of the antiapoptotic Bcl-2in lack of changes of Bax expression supports the idea that the riseof antiapoptotic/proapoptotic ratio may confer resilience againstproinflammatory signaling and cell death in oxidation-sensitivehippocampal neurons during the adult life. The hypothesis connectingstress resilience to the elevation of Bcl-xL and Bcl-2 mRNA is alsocorroborated by the increase of hippocampal Bcl-2 protein expressionand no effects on Bax levels after acute restraint in mice (Morettiet al., 2013) or by the negative correlation between depression-likebehaviors and hippocampal Bcl-xL/Bax ratio (Shishkina et al., 2010).In agreement, the increase of Bcl-2 may confer neuroprotection andrepresent a defensive and compensatory mechanism induced by theearly CB experience during postnatal development of hippocampalneurons.

5. Conclusions

This study provides evidence that an early alteration of mother–infant olfactory communication in the form of CB experience producedanhedonic-like behavior in adult life and enduring downregulationof α1-adrenoceptors in brain circuitries involved in the encoding ofolfactory cues and odor learning. Apoptotic mechanisms contributed todepression-like behaviors and α1-adrenoceptors downregulation is apossible factor underlying the blunted expression of prefrontal Hsp72,while inhibition of Hsp90A expression may contribute to deregulationof GR signaling. However, mice experiencing brief neonatal maternalseparation appear less vulnerable to proapoptotic signaling in the hippo-campal complex. Our study shows that early CB experience can produceboth deleterious and beneficial consequences by downregulatingprefrontal HSPs or priming hippocampal circuits, thus conferringneuroprotection against cell death via an increase of Bcl-2 mediatedsignaling.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.pnpbp.2013.10.004.

Acknowledgments

This paper originated in the frame of cooperation between theItalian National Research Council and the Polish Academy of Sciences.The work was partly supported by the statutory funds of the Institute

of Pharmacology, Polish Academy of Sciences and by POIG.01.01.02-12-004/09-00 financed by the European Regional Development Fund.

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