Chapter 2. Horm Behav Jul;60(2):

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1 Chater The newborn rat s stress system readily habituates to reeated and rolonged maternal searation, while continuing to resond to stressors in context deendent fashion. Nikolaos P. Daskalakis 1, Sanne E.F. Claessens 1, Jaser J.L. Laboyrie 1, Leo Enthoven 1, Melly S. Oitzl 1, Danielle L. Chamagne 1,, and E. Ronald de Kloet 1 1 Division of Medical Pharmacology, Leiden/ Amsterdam Center for Drug Research, Leiden University Medical Center, Leiden University Deartment of Integrative Zoology, Institute of Biology Leiden, Leiden University Horm Behav. 011 Jul;60():

2 5 Abstract Adrenal corticosterone secretion of newborn mice raidly desensitizes to reeated maternal absence. The resent study investigated the effects of novelty exosure, maternal care and genotye on this henomenon. Maternal searation (MS) took lace on ostnatal days (nd) 3-5. In Wistar rats, the degree of novelty in the MS-environment was varied by exosing us to: (i) home searation : us remained in the home cage; (ii) novel searation : us were laced individually in a novel cage. Maternal care was recorded on nd 1 to 4. To investigate the effect of genotye, we also examined Long Evans in the home searation condition. Basal and stress-induced ACTH and corticosterone levels were measured. Adrenal tyrosine hydroxylase (TH) and melanocortin recetor- (MCR-) roteins served as markers for adrenal function. We show, in both rat strains, that the rise in lasma corticosterone induced by a single 8h-MS on nd 5 was abolished, when this searation rocedure had also been erformed on nd 3 and 4. Habituation to maternal absence occurred irresective of housing conditions. However, us in the home searation condition received less maternal care uon reunion than those laced in the novel searation. These home searation us aeared more resonsive to a subsequent acute novelty-stressor, and their adrenal TH and MCR- were higher. Long Evans rats aeared more stress resonsive than the Wistars, in the home searation condition. In conclusion, searation environment, maternal care and genotye do not affect adrenal desensitization to reeated 8h-MS itself, but may modulate the adrenal stress-resonsiveness of searated us. 1. Introduction Aberrant HPA-axis activity and corticosterone (CORT) secretion induced by adverse early-life exeriences is considered a major risk factor for the develoment of sychiatric disorders in humans [1, ]. Therefore, rodents derived as us from maternal care have been widely used as a laboratory model for early adversity to study the underlying mechanism of CORT-enhanced vulnerabilities [3, 4]. Several adversity aradigms are currently used. One common aroach is a single eisode of 4h of maternal absence. Another aroach is reeated daily maternal searations (MS), which include reeated eriods of 3-8h absence of the dam during the first two ostnatal weeks. It has been roosed that us exeriencing reeated MS dislay as adult enhanced stress resonsiveness, increased anxiety, hellessness and anhedonia, deficits of sensorimotor gating and increased roensity for the intake of addictive drugs [4-8]. Interestingly, these rodent behaviors, rogrammed by early-life adversity, resemble clinical endohenotyes of deression and schizohrenia, hence roviding face and construct validity to these models [9]. However, the outcome of early-life adversity deends on strain and gender of the animals as well as the frequency, duration, age and time oint (within light cycle) of MS [10, 11]. Moreover, the environmental context

3 endocrine adatation to maternal absence 53 (housing in grous or in isolation, inside the home cage or in a novel environment) and the ambient temerature have an effect [11, 1]. Cumulatively, these observations have raised the question to what extent the HPA- axis is actually activated during reeated MS. Schmidt and colleagues [13, 14] found that, in mice, after 8h of a single MS the basal level of circulating CORT has slowly reached eak levels, while the stress hyoresonsive eriod (SHRP) has become disruted resulting in an enhanced resonsiveness of the adrenocortical secretion of CORT to mild stressors and exogenous ACTH administration [15, 16]. Enthoven and colleagues hyothesized that 3 consecutive daily 8h-MS from ostnatal (nd) 3-5 would amlify neuroendocrine resonses to both searation and novelty exosure. Surrisingly, they reorted instead that the increase in HPA-axis basal activity that is first observed after the initial 8h-MS was raidly abolished after reeated 8h-MS. This raid desensitization of the neonate s HPA-axis to reeated daily searations from the dam was not due to metabolic factors such as ghrelin, and also did not occur because of enhanced glucocorticoid negative feedback [17]. In site of this raid desensitization of the HPA-axis to the effect of daily searation, the SHRP remained disturbed because a subsequent novelty stressor triggered an enhanced lasma CORT and c-fos mrna resonse in the PVN [17]. This finding raised the question whether the environmental context exerienced by the u during MS can influence the outcome [17]. In the resent study we have extended these findings to the rat by examining the immediate effects of 3 daily reeated 8h-MS from nd 3 5. The objective of these exeriments was to investigate further the aarent desensitization of HPA-axis activity to reeated MS in different searation contexts in two rat strains. The degree of novelty was varied in the searation environment using: (i) home searation : the environment was the home cage and us remained groued together; (ii) novel searation : the environment did not contain any element of the home cage and us were additionally isolated from their littermates. The effect of the different MS rotocols was investigated on basal and novelty stress-induced ACTH and CORT levels on nd 5. Since in the study of Enthoven and colleagues [17], the aarent adrenal sensitivity to ACTH was altered dramatically during the reeated searations we also measured two biomarkers for adrenal function: tyrosine hydroxylase (TH) levels as index for adrenal medullary function and the level of melanocortin recetor (MCR- ) as an index of adrenal sensitivity to ACTH. Moreover, maternal care was measured for the first 4 ostnatal days to exlore its ossible imlication in the outcome of the different searation rocedures. Similar to what we observed in mice, we also found that the MS-induced CORT resonse is readily abolished in rats if the searations are reeated daily, but that the animals ability to resond to a novelty stressor deends on the searation context. Genotye and maternal care uon reunion did not affect the desensitization henomenon, but rather aeared to be associated with stress resonsiveness of the u to an acute novelty stressor.

4 54. Materials and Methods.1 Animals Wistar rats (originally obtained from Harlan, Horst, The Netherlands) and Long Evans rats (originally obtained from Elevage Janvier, Le Genest- St- Isle, France) were used in this study and housed in our animal facility under a 11:13 h light/dark cycle (lights on at h, illumination inside the cage: 0-30 lux, temerature: 0 ± 1 C, relative humidity: 60 ± 10%) and low volume background noise (40 db[a]). Food (RM3, Secial Diet Services, Witham, Essex, UK) and water (containing 0.0% HCL) was ad libitum. Uon arrival males and females were housed in grous of or 3 in Tye IV macrolon-olycarbonate cages with wire lid; 60 x 38 x 0 cm; containing sawdust bedding and tissue, and used for breeding at least after a habituation eriod of one week. Animal exeriments were aroved by the Local Committee for Animal Health, Ethics and Research of Leiden University and carried out in accordance with Euroean Communities Council Directive 86/609/EEC.. Breeding Two or three females of F1 generation, which were grou housed for at least a week, were mated with a male in Tye IV macrolon-olycarbonate cages with wire lid. After 10 days, males were removed from the cage and regnant females were transferred individually to clean cages (Tye III macrolon-olycarbonate cages with wire lid; 4.5 x 6.6 x 18.5 cm) containing sawdust and two sheets of aer towels for nest material. Pregnant females were checked for litters daily at 19:30h starting from 0 days after the start of breeding. If litters were resent, the day of birth was defined as nd 0 for that litter. On the day after arturition, nd 1, each litter was culled to 8-10 healthy us (ratio males:females = 1:1) and remained undisturbed until used in the study..3 Maternal behaviour The maternal behavior of each dam was observed and scored for five 60min eriods er day during the first 4 days ost artum using a rocedure originally described before [18-0]. Observations were erformed at three eriods during the light hase (10:00, 13:30, and 17:00 h) and two eriods during the dark hase (07:30 and 19:30h; under x60 W red TLD-light). The behavior of each mother was scored every 3min (0 observations er eriod, 100 observations er day). We scored the following maternal behaviors: retrieval: the dam retrieves her us from either a location outside the nest to the nest or from inside the nest to a new location; maternal contact: the dam is in contact with the us but not nursing or licking; licking and grooming (LG): the dam is licking and grooming either the whole body or secifically the anogenital area of the u; assive nursing osture: the dam is in a assive osture (she is lying either on her back or side while the us are nursed); away from nest: there is no maternal contact; nest building: the dam gathers material to a nest side, redistributes nest material, creates nest or changes shae of nest; burying her us. Finally, arched-back nursing was also measured with a distinction of (i) (assive) low arch: dam in a assive osture where she lays over (some or all of) her us flat (blanket nursing) or with a low arch. (ii) (active) low arch: the dam is ositioned less assively over (some or all of) her us; her ossible other activities are licking us, moving nest material, self grooming, reositioning us in the nest, eating etc. (iii) Middle arch: in this osition the dam

5 endocrine adatation to maternal absence 55 shows a greater arch and her other activities are only u-oriented (licking us), (iv) High arch: in this osition the limbs of the dam are extended so the us have full access to her niles. We considered as (overall) assive nursing the sum of the assive nursing osture and the (assive) low arch back nursing scores. The other three nursing ostures (active low arch, middle arch, high arch were considered (overall) active nursing (AN). Other dam non-maternal care behaviors were also observed like eating, drinking water, chasing tail, self grooming, digging, and sleeing. Note that some behavioral categories were not mutually exclusive. For, examle, licking and grooming often occurs while the dam is nursing the us. Other litter conditions were noticed: slit litter (us divided over two ositions) and buried us. We analyzed the ercentage of observations in which: 1) the dams dislayed each behavior or ) litters were in a certain condition. In the result section, we reort frequencies of AN (as % of observations) and LG (as % of observations). Note that AN and LG frequencies both include instances where both of the behaviours occurred simultaneously and those instances are quite frequent..4 Maternal Searation (MS; Fig. 1) MS occurred at either nd 3, 4 and 5 or only nd 5, lasting 8h each. Litters were randomly distributed over exerimental conditions..4.1 Dams transfer from the litter ( Dam out ) At 9:00h, dams selected for MS were removed from their cage ( home cage), laced in a cage of the same tye and transferred to an adjacent room ( dams room). In the dams room, the environmental conditions were the same excet the lighting intensity was higher (illumination inside the cage: lux)..4. Searation rocedure After the dam was relocated to a new cage, litters were ket without any food or water available for 8h (9:00 to 17:00h). The home cage was laced on heating ads (33 38 C; TM, Beurer, Ulm, Germany) to maintain the body temerature of the us. To acquire the desired temerature, heating ads were turned on 30 min rior use. We used two following searation contexts: - Home searation (HOME SEP; Fig. 1A). The us remained in their familiar environment (housing room, home cage) together with their littermates. - Novel searation (NOVEL SEP; Fig. 1B). The us were moved to an adjacent unfamiliar room, with similar conditions as the housing room. Pus were ut individually in new clean cages (Tye II macrolon-olycarbonate, which were divided in comartments of 18 x 0 x 14 cm, containing fresh sawdust bedding) and laced on heating ads. The searated us housed in this unfamiliar novel context, exerienced the absence of their dam, the home cage environment and roximal contact with their littermates (isolation)..4.3 Reunion ( Dam back ) At 17:00h, the us were returned to their home cage followed by their dams. Dams of searated us in home and novel contexts were reunited with their litter at the same time..4.4 Control litters Non-searated (NON SEP) litters remained undisturbed with their dams in the housing room until the time of testing.

6 56 A Home searation DAM i H BED ii DAM H BED DAM H BED B 8 hours Novel searation ii iii DAM i N BED iv DAM H BED DAM H BED Figure 1. Sequential stes of early-life maniulations: (A) Home searation: i. Dam is taken out of the nest, but us ( ) stay altogether in the home cage on to of their home bedding ( H BED ) for 8 hours; ii. Dam is reunited with the us. 8 hours (B) Novel searation: i. Dam is taken out of the nest; ii. Pus ( ) laced in novel context for 8 hours (other room, isolated from the littermates, novel bedding: N BED ); iii. Pus are brought back to their home cage. iv Dam is reunited with the us..5 Testing: novelty exosure (nd 5) We determined the HPA-axis resonsiveness to a mild stressor at 17:00h on nd 5. Pus were removed from their searation environment (home or novel) and either sacrificed immediately by decaitation or laced individually in new clean cages (same tye as in the novel searation), containing fresh sawdust. Novelty exosure was carried out in a searate room, the novelty exosure room, under similar environmental conditions as the housing room. The cages were laced on heating ads (33 38 C) to maintain the body temerature of the us. After 30min from the onset of the stressor, the us were sacrificed. Note that this maniulation is different for us of the different grous: for the HOME SEP us (1 st HOME SEP, 3 rd HOME SEP), it is the first time they exerience a novel and unfamiliar cage, for the NOVEL SEP us it is a relatively familiar maniulation since they are coming from a similar environment. Therefore, the erceived degree of novelty may be different..6 Exerimental design (Fig. ) Litters for the endocrine exeriments: in order to minimize inter-litter differences, every treatment grou consisted of 4-7 litters and, within each litter, we distributed the us equally in terms of

7 endocrine adatation to maternal absence 57 A Exeriment I nd 0 light dark B C 1 st HOME SEP 3 rd HOME SEP 3 rd NOVEL SEP Exeriment II nd 0 light dark st HOME SEP 3 rd HOME SEP Exeriment III nd 0 light dark st HOME SEP 3 rd HOME SEP basal searated novelty basal searated novelty basal searated novelty basal searated basal searated basal searated novelty basal searated novelty Figure. Grahical reresentation of the three exeriments for endocrine measures roviding an overview of the time lines of different treatment. First is the time bar from ostnatal day (nd) 0 to 5; within each nd in white is the light eriod of the light cycle and in black the dark eriod. Each big white bar under the time bar reresents different conditions of sacrifice. Within every white bar there are boxes indicating the exerimental maniulations; gray box reresents 8h of maternal searation in home context, gray box with black hatched stries reresents 8h of maternal searation in novel context and black box reresents 30min of novelty exosure. Pus were Table 1 Synosis of the number of litters used sacrificed on nd 5 measured in basal conditions (basal), after 8h of maternal searation (searated) or 8h of maternal searation with an additional 30min of exosure to novelty (novelty). Treatment grous: first searation (1 st HOME SEP) had no revious history of treatments, third searation animals were exosed to 8h of maternal searation on nd 3 and 4 in a home (3 rd HOME SEP) or novel context (3 rd NOVEL SEP). Note that novelty exosure is different for us of the different grous: for the HOME SEP (1 st HOME SEP, 3 rd HOME SEP) us, it is the first time they exerience a novel and unfamiliar cage, for the 3 rd NOVEL SEP us it is a relatively familiar maniulation since they are coming from a similar environment. Ex. Grou Exeriments I-III 1 st HOME SEP 3 rd HOME SEP 3 rd NOVEL SEP Maternal care observations Ex. I Ex. II Ex. III NON SEP HOME SEP NOVEL SEP Maternal care observations 4+6=10 4+5=9 4+5=9 Note. First searation (1 st HOME SEP) or Non searated us (NON SEP) had no revious history of treatments nd 1 to 4, searated us were exosed to 8h of maternal searation on nd 3 and 4 in a home (3 rd HOME SEP/ HOME SEP) or novel context (3 rd NOVEL SEP/ NOVEL SEP).

8 58 time oint of sacrifice and sex. Number of animals er time oint in every treatment grou was See Table 1 for a synosis of litters used in the different exerimental grous..6.1 Exeriment I (Fig. A): to determine the effects of reeated searations in different searation context on body growth, ACTH & CORT secretion and maternal care, litters were divided into three treatment grous: single home searation (1 st HOME SEP), reeated home searation (3 rd HOME SEP), and reeated novel searation (3 rd NOVEL SEP). We sacrificed rat us in three different testing conditions: basal levels (basal), 8h of searation (searated), 8h of searation + 30min of novelty (novelty). A total of 1 litters were used (4 litters for each grou with 8 us in every time oint)..6. Exeriment II (Fig. B): to determine the effects of reeated searations in home context on adrenal activity, litters were divided into two treatment grous: single home searation (1 st HOME SEP) and reeated home searation (3 rd HOME SEP). We sacrificed rat us in two different testing conditions: basal levels (basal) and 8h of searation (searated). A total of 8 litters were used (4 litters for each grou with 8 us in every time oint)..6.3 Exeriment III (Fig. C): to determine the effects of reeated searation in home context on ACTH and CORT secretion of another rat strain (Long Evans), litters were divided into two treatment grous: single home searation (1 st HOME SEP) and reeated home searation (3 rd HOME SEP). We sacrificed rat us in three different testing conditions: basal levels (basal), 8h of searation (searated), 8h of searation + 30-min of novelty (novelty). A total of 13 litters were used (6 litters for the 1 st HOME SEP grou with 16 us in every time oint, 7 litters for the 3 rd HOME SEP grou with 18 us in every time oint)..6.4 Litters for maternal care observations: maternal care measurements were erformed for the litters of Exeriment I together with some extra litters that were not used in the endocrine exeriments: 6 non searated litters, 5 reeatedly searated litters on nd 3 and 4 in a home (HOME SEP) or novel context (NOVEL SEP). On nd 3 & 4, for the 3 rd HOME SEP/ HOME SEP and 3 rd NOVEL SEP/ NOVEL SEP grous, dams were not in contact with the us at two time oints (10:00 and 13:30) because they were searated, and, therefore, we could not collect maternal care data..7 Collection of blood lasma and adrenals At the designated time oint, the us were sacrificed by decaitation. Trunk blood from all us was collected individually in 1.5 ml EDTA-coated microcentrifuge tubes. All blood samles were ket on ice and later centrifuged for 15min at rm at 4 C. Plasma was transferred to clean 1.5 ml microcentrifuge tubes. All lasma samles were stored frozen at 0 C until the determination of ACTH and CORT. After decaitation, adrenals were dissected and sna frozen in isoentane on dry ice and stored at -80 o C until used for Western blotting..8 Measurements.8.1 Body weight (gr) was measured just before every exerimental maniulation with an electronic recision scale (MXX-001, Denver Instrument, Göttingen Germany; readability 0.1 g, linearity 0. g). Since the grous were different in birth weight (nd 1 weight), we calculated the ratio (in %) of the body weight measurements to birth weight..8. ACTH (g/ml) was measured by radioimmunoassay (MP Biomedicals, LLC, NY, USA; sensitivity 10 g/ml, intra-assay variation 4.1%, interassay variation 4.4%). Samles were determined in a 50% dilution, starting with 5μl blood lasma. All samles were analysed in one assay to exclude

9 endocrine adatation to maternal absence 59 inter-assay variability..8.3 CORT (ng/ml) was measured by radioimmunoassay (MP Biomedicals, LLC, NY, USA; sensitivity 1.5 ng/ml, intra-assay variation, 4.4%, interassay variation 6.5%;). Concentrations were determined in dulicate from an extended standard curve (0, 6.5, 1.5, 5, 50, 100, 50, 500 and 1000ng corticosterone/ml), since we noted that the lower boundary rovided by the kit was not sensitive enough to measure neonatal basal lasma concentrations. All samles were analysed in one assay to exclude inter-assay variability..8.4 Tyrosine hydroxylase (TH) & Melanocortin recetor tye (MCR-) rotein levels. Adrenals were homogenized in 400µl lysis buffer (Triethanolamine, NaCl, DOC, SDS, triton-x-100) and rotease inhibitor was added to inhibit roteins degradation. This lysate was sun down and suernatant was ket and stored in -0 C. Concentration of roteins resent in the suernatant was determined using a Thermo Scientific Pierce BCA Protein Assay. Therefore, a calibration curve (Bovine Serum Albumin in 5 dilutions) was done. Western blotting was erformed, according to a reviously described method [1], in dulicate on the suernatant of the homogenized adrenals to measure TH and MCR- rotein levels. Each samle suernatant was loaded in a concentration of 1mg/ml (by varying the amount of H O added to the samle). The samles also included a standard volume of samle buffer and were denaturized at 95 C (5min) and subjected to SDS PAGE. After electrohoresis, the roteins were transferred to a membrane (blotting) overnight (4 C, 15mA). The day after, the blots were blocked in 10mM Tris-HCl (H 8.0), 150mM NaCl, and 0.05% Tween 0 containing 5% non-fat dried milk owder and, then, incubated with the rimary antibody and the secondary antibody consecutively. For TH, the rimary antibody used was rabbit anti Tyrosine Hydroxylase (TH) (AB15) ordered from Milliore in a 1:1000 concentration. The secondary antibody used was Goat-anti-rabbit IgG-HRP in a 1:5000 concentration. For MCR-, the rimary antibody used was mouse anti Melanocortin Recetor- (MCR-) ordered from Chemicon in a 1:1000 concentration. The secondary antibody used was goat anti mouse IgG-HRP in a 1:5000 concentration. For TH, we used liver tissue as negative control and adult rat adrenal tissue as ositive control. For MCR-, we used human skin samles as ositive control and water as negative control. Samles were also tested on their α-tubulin levels, as well, to correct for the total amount of rotein. The rimary antibody used was anti-mouse α-tubulin in a 1:5000 concentration and the secondary antibody used was goat anti mouse IgG-HRP in a 1:10000 concentration. After washing of the antibodies, blots were incubated with eroxidase-conjugated antibodies (1:10.000; Jackson ImmunoResearch Laboratories, West Grove, PA). Immunoreactive bands were visualized by enhanced chemiluminescence and the blots were exosed to films. The autoradiograhs (films) were scanned and otical density (OD) of the TH, MCR- and α-tubulin bands were determined using Image J software. The TH and MCR- values of the samles were corrected for total rotein (α-tubulin); therefore, the ratios between the TH and α-tubulin levels and MCR- and α-tubulin levels were calculated. In order to comare samles ran in different gels we used also one samle ( standard samle ) that was loaded in all gels. Grou size n=8 adrenals (from 8 searate animals) er time oint of each treatment grou were used for the measurements..9 Statistical analysis The results were analyzed by analysis of variance (ANOVA) with the level of significance set at

10 60 < Where aroriate, simle and interaction main effects were investigated further with subsequent ost-hoc comarisons (by Tukey test). The statistical analysis was adjusted for nonequivalent grous when needed. The initial analysis of us measurements included sex as a factor; once it was determined that sex was not a significant factor, data from males and females were ooled. Data are resented as mean ± SEM. The level of significance was set at Results 3.1 Exeriment I Wistar us were exosed to reeated MS under different searation contexts with the goal to examine the desensitization of the endocrine resonses Body Weight (data not shown) Reeated measures ANOVA, for the ratio of body weight to birth weight, revealed main effects of time (F 1,45 = ; 0.001) and the interaction of time and treatment (F 1,45 = 34.36; 0.001). The treatment effect was significant in all time oints ( 0.001). On nd 5, HOME SEP and NOVEL SEP were not different, but both were lighter than the NON SEP ( 0.001). HOME SEP vs. NOVEL SEP: The two reeatedly searated grous were followed for body growth in more time oints (nd 3 and nd 4) than the controls. Therefore, we erformed a searate statistical analysis for their comarison. Reeated measures ANOVA revealed main effects of time (F 6,180 = ; 0.001), but not of treatment or the interaction of time and treatment. The two grous (HOME SEP and NOVEL SEP) were not different in weight at any time oint ACTH (data not shown) ACTH basal levels for naïve Wistars on nd 5 were ± (g/ml). Two-way ANOVA did not reveal any main effects of treatment, time or their interaction. ACTH is not different after a single event of 8h of MS on nd 5. Rat us also did not resond to 30min novelty if it was imlemented immediately after the first or third MS eriod. There was no effect of treatment on ACTH. However, if reeatedly searated grous were comared searately, ACTH levels of NOVEL SEP us, after the third MS, were lower than the ones of the HOME SEP us (=0.046) CORT (Fig. 3) Two-way ANOVA revealed effects of treatment (F,63 = 51.54; 0.001), time (F,63 = 17.50; 0.001) and their interaction (F 4,63 = 10.97; 0.001). 1 st HOME SEP: Naïve rat us resonded on nd 5 with a three-fold increase of CORT to 8h-MS ( 0.001) or to the combination of MS with novelty exosure ( 0.001). The novelty exosure did not, however, create an additional increase over the MS levels. 1 st HOME SEP vs. 3 rd HOME SEP/ 3rd NOVEL SEP: If on nd 3 and 4 us were searated

11 endocrine adatation to maternal absence 61 from their mother, on nd 5, CORT levels, in resonse to the third eriod of MS, was not altered regardless if the MS haened in a home or a novel context ( searated vs. basal levels in 3 rd HOME SEP or in 3 rd NOVEL SEP). CORT levels after MS or after the combination of MS and novelty ( searated and novelty levels) were lower for the reeatedly searated us in both contexts ( 0.001) comared to single MS us. 3 rd HOME SEP vs. 3 rd NOVEL SEP: However, when exosed additionally to novelty after the third MS, the 3 rd HOME SEP us dislayed an increase in CORT levels (comared to basal levels: =0.03, 50% increase, but not over searated levels: =0.065). The small increase of CORT when novelty is combined with MS is absent when MS occurs in the novel context. 0 Wistar, nd 5 basal searated novelty Corticosterone (ng/ml) * * * 0 Figure 3. Corticosterone (ng/ml) blood lasma levels measured at basal conditions (basal; white bars), after 8h of maternal searation [searated; home searated (HOME SEP): grey bars/ novel searated (NOVEL SEP): grey bars with black hatched stries] or 8h of maternal searation with an additional 30 min of exosure to novelty (novelty; black bars). First searation (1 st HOME SEP) had no revious history of treatments. Third searation animals were exosed to 8h of maternal searation on nd 3 and 4 in a home (3 rd HOME SEP) or novel context (3 rd NOVEL SEP). 1 st HOME SEP 3 rd HOME SEP 3 rd NOVEL SEP Note that novelty exosure is different for us of the different grous: for the HOME SEP (1 st HOME SEP, 3 rd HOME SEP) us, it is the first time they exerience a novel and unfamiliar cage, for the 3 rd NOVEL SEP us it is a relatively familiar maniulation since they are coming from a similar environment. Data reresented MEAN±SEM. Significance level was set at * vs. basal, # vs. searated levels of the same treatment grou, vs. corresondent value of 1 st HOME SEP, vs. corresondent value of 3 rd HOME SEP. n=8 er time oint of each treatment grou Maternal Care In order to investigate whether the raid desensitization of the CORT resonse to reeated MS was related to mother-u interaction uon reunion we measured several comonents of maternal behavior on nd Licking & Grooming (Fig. 4). Reeated measures ANOVA revealed main effects of time (F 19,475 = 1.94; 0.001) and the interaction of time and treatment (F 38,475 = 3.7; 0.001) on LG (Fig. 4A), but overall mean of LG across the first ostnatal days (Fig. 4B) was not significantly different between treatment grous (NON SEP: 6.08 ± 0.59, HOME SEP: 5.17 ± 0.5, NOVEL SEP: 5.4 ± 0.51). The time effect was not significant for the dams of the NON SEP us, but it was

12 6 significant for the dams of the HOME SEP ( 0.001) and NOVEL SEP us ( 0.001). Post hoc analysis revealed that the differences were mainly in the ost searation hour observation eriod on nd 3 and nd 4. On nd 3 (Fig. 4B), the dams of searated us dislayed increased LG levels uon reunion after the first 8h-MS from their us as comared to before searation levels (for the HOME SEP dams: =0.01, for the NOVEL SEP dams: 0.001). Between the treatment grous, before first MS, there was no difference in LG levels. After the searation, only the dams of the NOVEL SEP us dislayed higher levels of maternal care at this time oint comared to both NON SEP ( 0.001) and HOME SEP grous (=0.043). A Licking & Grooming Time course nd 1-4 τ τ NON SEP(n=10) HOME SEP (n=9) NOVEL SEP(n=9) :00 13:30 17:00 19:30 7:30 10:00 13:30 17:00 19:30 7:30 10:00 13:30 17:00 19:30 7:30 10:00 13:30 17:00 19:30 % observations 7:30 nd 1 nd nd 3 nd 4 B % observations Reunion nd 3 7:30-8:30 17:00-18: st searation * * C % observations Reunion nd 4 7:30-8:30 17:00-18:00 30 nd searation 5 * 0 * NON SEP HOME SEP NOVEL SEP NON SEP HOME SEP NOVEL SEP Figure 4. Licking & Grooming (LG). (A) Time course during nd 1-4: Non searated us (NON SEP; emty circles with discontinuous black connecting line) had no revious history of treatments. Searated us had exerienced times 8h of maternal searation on nd 3 and 4 in home (HOME SEP: home searated; grey squares with grey connecting line) or novel context (NOVEL SEP: novel searated; triangles with black connecting line). On nd 3 & 4, for the HOME SEP and NOVEL SEP grous, dams were not in contact with the us at the time oints 10:00 and 13:30. Therefore in the time course grah, they don t have data oints and this is highlighted with a symbol:. (B&C): Maternal care at reunion on nd 3 & 4 resectively: LG measured for an hour at the researation time eriod 7:30-8:30 (white bars) or one hour during the ost searation time eriod at 17:00-18:00 (white bars with black horizontal stries). Data reresented MEAN±SEM. Significance level was set at <0.05. τ denotes overall time effect in this grou, * vs. re-searation levels, vs. corresondent value of NON SEP, vs. corresondent value of HOME SEP. n=9-10 er treatment grou.

13 endocrine adatation to maternal absence 63 On nd 4 (Fig. 4C), the dams of searated us increase their LG levels uon reunion after 8 h-ms for the second time (for the dams of HOME SEP: =0.003, for the dams of NOVEL SEP: 0.001) ), as comared to before searation levels. Interestingly, the effect of MS on maternal care did not habituate. Before the second MS the dams of NOVEL SEP us dislayed the least LG (=0.011 vs. NON SEP). When they were reunited with their us, after the second MS, dams of searated us in both contexts dislayed higher maternal care comared to the controls (for the dams of HOME SEP: =0.038, for the dams of NOVEL SEP: =0.005) Active Nursing (Fig. 5). Reeated measures ANOVA revealed main effects of time (F 19,475 = 16.0; 0.001) and the interaction of time and treatment (F 38,475 = 7.37; 0.001) on AN (Fig. 5A), and of treatment (F,5 = 3.6; =0.041) on the overall mean of AN across the first ostnatal days (Fig. 5B). The time effect was significant for all grous (for the dams of NON SEP us =0.008, of HOME SEP us and of NOVEL SEP us 0.001). Dams of HOME SEP us, comared to the controls (NON SEP), dislayed different levels of AN across the individual time oints (=0.04) and endedu with less overall mean across nd 1-4 (=0.039). We conducted further ost hoc analysis for the ost searation hour observation eriod on nd 3 and nd 4. On both days, there was no treatment effect before or after the first 8 h-ms indicating there was no ost-reunion increase of AN in both days. A % observations Time course nd 1-4 NON SEP (n=10) τ HOME SEP (n=9) τ NOVEL SEP (n=9) τ 10:00 13:30 17:00 19:30 7:30 10:00 13:30 17:00 19:30 17:00 19:30 nd 1 nd nd 3 nd 4 Figure 5. Active Nursing (AN). (A) time course during nd 1-4: Non searated us (NON SEP; emty circles with discontinuous black connecting line) had no revious history of treatments. Searated us had exerienced 3 times 8h of maternal searation on nd 3and 4 in home (HOME SEP: home searated; grey squares with grey connecting line) or novel context (NOVEL SEP: novel searated; triangles with black connecting line). On nd 3 & 4, for the HOME SEP and NOVEL 7:30 10:00 13:30 Active Nursing 7:30 10:00 13:30 17:00 19:30 7:30 B Overall mean nd 1-4 NON SEP HOME SEP NOVEL SEP SEP grous, dams were not in contact with the us at the time oints 10:00 and 13:30. Therefore in the time course grah, they don t have data oints and this is highlighted with a symbol:. (B) Overall mean across the first four ostnatal days. NON SEP: white bars, HOME SEP: grey bars, NOVEL SEP: grey bars with black hatched stries. Data reresented MEAN±SEM. Significance level was set at <0.05. τ denotes overall time effect in this grou, vs. corresondent value of NON SEP. n=9-10 er treatment grou % observations 3. Exeriment II Because the endocrine blood levels imlied changes in adrenal sensitivity to

14 64 subsequent stressors uon reeated MS in the home context we have examined two biomarkers that may give an indication how adrenal function is affected TH (Fig. 6A) Two-way ANOVA revealed effects of time (F 1,31 = 5.43; =0.07) and the interaction of treatment and time (F,31 = 4.5; =0.009). If, on nd 3 and 4, us were searated from their mother in a home context, on nd 5, a 65% reduction in TH level is dislayed comared to naïve us (basal levels 1 st HOME SEP vs. 3 rd HOME SEP; =0.014). The reduction was followed by a four-fold increase in resonse to the third eriod of MS (=0.006) ( searated vs. basal levels in 3 rd HOME SEP). 3.. MCR- (Fig. 6B) Two-way ANOVA revealed effect of the interaction of treatment and time (F,31 = 6.09; =0.00). If, on nd 3 and 4, us were searated from their mother in a home context, on nd 5 recetor levels dislay a 50% reduction comared to naïve us (basal levels 1 st HOME SEP vs. 3 rd HOME SEP), but it was not significant (=0.055). The reduction was followed by an increase (three-fold) in resonse to the third eriod of MS (=0.047) ( searated vs. basal levels in 3 rd HOME SEP). A Wistar, nd 5 basal searated B 5 Wistar, nd 5 basal searated Adrenal TH/ a-tubulin normalised 1 * Adrenal MC- R/ a-tubulin normalised * 0 1 st HOME SEP 3 rd HOME SEP 0 1 st HOME SEP 3 rd HOME SEP Figure 6. Adrenal TH (A) & MC recetor (B) rotein levels measured at basal conditions (basal; white bars), or after 8h of maternal searation [searated; home searated (HOME SEP): grey bars]. First searation (1 st HOME SEP) had no revious history of treatments. Third searation animals were exosed to 8h of maternal searation on nd 3 and 4 in a home context (3 rd HOME SEP). Data reresented MEAN±SEM. Significance level was set at 0.05.* vs. basal, vs. corresondent value of 1 st HOME SEP. n=8 adrenals (from searate us) er time oint of each treatment grou; the Westerns were erformed in dulicate. 3.3 Exeriment III In order to assess ossible strain differences on the effect of reeated MS in home context, Long Evans rats were used ACTH (data not shown) ACTH basal levels for naïve Long Evans on nd 5 were ± 1.96 (g/ml). Twoway ANOVA revealed effects of treatment (F 1,110 = 80.51; 0.001), time (F,110 = 8.5;

15 endocrine adatation to maternal absence 65 =0.00) and their interaction (F,110 = 9.5; 0.001). 1 st HOME SEP: Naïve rat us on nd 5 resonded with an ACTH increase to the combination of MS with novelty exosure ( 0.001; 50% increase). The novelty exosure created an additional increase over the MS levels (=0.030). 3 rd HOME SEP: If on nd 3 and 4 rat us were searated from their mother, on nd 5 ACTH levels, in resonse to the third eriod of MS, were decreased ( searated vs. basal levels in 3rd HOME SEP; = 0.038) and they even became lower than basal levels of naïve us of nd 5 (=0.00; 35% decrease). 1 st HOME SEP vs. 3rd HOME SEP: ACTH levels, either basal, after MS or after combination of MS and novelty levels of reeatedly searated us were reduced comared to the resective values of the singly searated us (=0.031 for basal values, for searated values, for novelty values) CORT (Fig. 7) Two-way ANOVA revealed effects of treatment (F 1,110 = ; 0.001), time (F,110 = 86.1; 0.001) and their interaction (F,110 = 37.76; 0.001). 1 st HOME SEP: Naïve rat us on nd 5 resonded with a CORT increase to 8h- MS ( 0.001; four-fold increase) or to the combination of MS with novelty exosure ( 0.001; five-fold increase). The novelty exosure resulted in an additional increase over the MS levels (=0.004). 3 rd HOME SEP: If, on nd 3 and 4, rat us were searated from their mother, CORT levels were not altered on nd 5, in resonse to the third eriod of MS ( searated vs. basal levels in 3 rd HOME SEP). The HOME SEP us dislayed an increase in CORT over basal levels on nd 5 if they were additionally exosed to novelty after MS ( 0.001; two-fold increase), over searated levels (=0.015) and over CORT levels of naïve us ( 0.001). 50 Long Evans, nd 5 basal searated novelty Corticosterone (ng/ml) * * # * # 0 1 st HOME SEP 3 rd HOME SEP Figure 7. Corticosterone (ng/ml) blood lasma levels measured at basal conditions (basal; white bars), after 8h of maternal searation [searated; home searated (HOME SEP): grey bars] or 8h of maternal searation with an additional 30min of exosure to novelty (novelty; black bars). First searation (1 st HOME SEP) had no revious history of treatments. Third searation animals were exosed to 8h of maternal searation on nd 3 and 4 in a home context (3 rd HOME SEP). Data reresented MEAN±SEM. Significance level was set at <0.05. * vs. basal, # vs. searated levels of the same treatment grou, vs. basal of 1 st HOME SEP, vs. corresondent value of 1 st HOME SEP. n=14-16 er time oint of each treatment grou.

16 66 1 st HOME SEP vs. 3 rd HOME SEP: CORT levels after MS or after combination of MS and novelty ( searated and novelty levels) were reduced in the reeatedly searated us comared to the resective values of the singly searated us ( for both comarisons). 4. Discussion The resent study was designed to extend revious data on the immediate outcome of reeated daily maternal searations on the HPA-axis from the mouse to the rat. Our revious studies had revealed the remarkable henomenon that these reeated daily maternal searations in mice resulted in a desensitization of the CORT-resonse to the searation rocedure itself, while the us continued to resond to an acute novelty stressor [17]. In this study we demonstrate that also the rat readily adats to reeated daily searations. Secifically, we show that after 8h of maternal searation CORT levels increased markedly in the 5 day old Wistar and Long Evans rat u. However, if the us had been exosed also the two receding days to 8h-MS this rise in CORT was abolished. The adrenal desensitization induced by the homotyic daily reeated 8h-MS was not affected by genotye, searation environment or maternal care uon reunion. However, reeatedly home searated us show a subtle enhancement of the adrenal CORT resonse to a heterotyic acute novelty exosure. Additionally, the adrenal TH and MCR- rotein content was higher than observed in us exosed to solely a single 8h-MS. This increased stress resonse to novelty after reeated eisodes of home rather than novel 8h-MS was observed in both genotyes, with the Long Evans us dislaying a relatively higher stress-resonse comared to the Wistar us. 4.1 Differential ACTH and CORT time course during maternal absence In line with exectations, CORT levels following a single 8h-MS on nd 5 were significantly increased relative to basal levels. Both in rats and mice, a single rolonged eriod of maternal searation of 8h during the SHRP is needed for a large increase of CORT [14, 16, 17,, 3]. As far as ACTH levels are concerned, in both rat strains, they were not increased after 8h of maternal absence. However, in mice it is clearly shown by Schmidt et al. that during the course of a 4h-MS, ACTH levels are increased between 8 and 1h from the onset of searation [14, 17]. Hence, the lack of ACTH resonse in rats observed in the resent study is robably a rat-mouse difference [4], since our ACTH findings are actually in agreement with other studies using rats. Walker et al. [3] showed already at 30min of maternal absence a rise in ACTH level, which then returned to baseline after 8h of searation ossibly by either deletion of the ituitary ACTH stores or glucocorticoid feedback inhibition of ACTH release. That the feedback inhibition already oerates in newborn rats at nd 5 was shown by injecting CORT or dexamethasone to naïve, derived or adrenalectomized us [5-8]. Accordingly, a single 8h-MS eriod ermits a robust CORT increase, while at that time the ACTH

17 endocrine adatation to maternal absence 67 resonse is suressed. Our findings (in Long Evans) argue against the deletion of ACTH from the ituitary stores since we observed increased ACTH levels in resonse to 30min of novelty following a single 8h-MS. 4. Desensitisation of CORT resonse to maternal absence: a robust henomenon The interesting novel asect of our revious studies with mice was that uon reeated searations the u readily adats to maternal absence and as a result the searation-induced increase in CORT does not occur any longer. We reort here that also in the newborn rat the basal CORT resonse to daily reeated 8h-MS is abolished, both in Wistar and Long Evans 5 day old rat us while their adrenals are able to resond to a single 8h-MS. This habituation or adatation of the u to the exerience of reeated MS was not due to a shift in the time course of the MS-induced CORT resonse [17]. Also, reeated MS in a home context does not result in a cumulative CORT resonse irresective of the duration (from 15 min to 8h) of maternal absence [16, 9]. It should be noted, however, that these authors did not reort desensitization of the adrenal resonse to reeated maternal absence as was shown in Enthoven s study for the mouse, and here in two rat strains. However, the current study differs from the mouse study in one asect. While Enthoven et al. found a ersistent decrease in basal CORT release after two daily searations; we did not see this decrease consistently in the two rat strains. Possibly this difference could be exlained by small circadian fluctuation resent already in that age [17]. Enthoven measured basal levels at 9:00 h (16h after revious searation) and we did at 17:00h (4h after the end of the revious8h-ms). However, the circadian fluctuation of CORT and the otential influence of early-life exerience has not been reorted in neonate rodents so far earlier than the third week of life [30]. In the resent study it was shown that if the us are housed in a novel environment isolated from their littermates and derived from all familiar cues during the 8h-MS, the desensitization of the adrenal still haens. In the rat, comarable studies have been erformed, however with variable outcomes. Several studies using shorter eriods of maternal absence in a novel environment (1h x 8 days) showed sensitization of the adrenal resonse to this rocedure [31, 3]. Other studies (15min x 8 days, 1h x 3 days) did not [3, 33]. Thus, taken together, the duration and frequency of the daily searations might influence the outcome. Overall, the fact that adrenal corticosterone secretion under two widely different conditions dislayed desensitization to maternal absence demonstrated the robustness of this henomenon. 4.3 Enhanced adrenal sensitivity to stress after reeated maternal absence While it is now well-established that the adrenal readily desensitizes to homotyic reeated maternal searations, it is also well-established that MS causes increased adrenal sensitivity to heterotyic stressors and exogenous ACTH. This enhanced adrenal sensitivity to a heterotyic stressor needs at least 8h-MS to develo and is

18 68 rofound after 4h of searation [15, 34]. Previously, it was reorted that desite the raid adatation of the HPA-axis to daily reeated maternal absence, the CD1 mouse u stayed on alert and retained the ability to resond to stressors with an increase in ACTH and CORT levels [17], which indicates a large steroid roduction caacity and argues against ACTH deletion. Wistar us, when exosed to novelty for 30min immediately after the searation, show no ACTH resonse irresective of whether the searation was the first (the Long Evans did) or the third. Also it did not matter whether the searation context was familiar (home cage) or unfamiliar (novel cage). However, a subtle CORT resonse to the novelty stressor still occurred desite desensitization to the homotyic reeated maternal searation both in Wistar and Long Evans us. Interestingly, in Wistars, reeated home searation vs. novel searation had a different outcome on the resonse to the subsequent 30min novelty stressor. Rat us searated for 8h on 3 consecutive days in a novel environment aarently adated to this condition because the additional 30min of novelty stress (which in this grou could be considered a homotyic stressor) could not trigger a resonse anymore. Aarently, the revious exerience of maternal absence in a novel environment not only did reare the us for maternal absence but also for the exerience of novelty itself. Both Wistar and Long Evans us, in the home searation condition, still show a CORT resonse. Moreover, Enthoven et al. reorted that a daily reeated exosure to a combination of 8h home searation + additional 30min novelty was not able to attenuate the resonse to a subsequent 30min novelty exosure itself [17]. This suggests that only if the environment, in which the novelty stress is exerienced, is intrinsic to the housing conditions during searation, then the novelty stressor can be considered homotyic and the resonse to this stressor is abolished. These observations underscore revious research showing that the neonatal adrenal function is altered after maternal absence [34]. To further examine the altered adrenal function we measured ACTH recetors (MCR-) in the neonate adrenals in an attemt to exlain why adrenal sensitivity to the novelty stressor was increased in the face of unaltered circulating ACTH levels. We reort that MCR- rotein content was reduced 4h after the second home searation, but enhanced after a third searation interval. This would redict enhanced resonsiveness to exogenous ACTH as had been shown before, immediately after rolonged 4h maternal searation [34, 35]. We also measured TH rotein level as an indirect measure of medullary-catecholamine resonse to maternal absence [34]. We show that the third home searation induced an increase of TH rotein levels over the basal levels (which were reduced 4h after the second eriod of MS). Aarently, the increase in adrenal activity develos after reeated 8h eisodes of maternal absence (in our study) or after rolonged 4h of maternal absence (Okimoto study) and not after a single 8h eisode. These exeriments rovide some insights into the mechanistic underinning of enhanced adrenal sensitivity towards heterotyic stressors, which are in line with the elegant exeriments of singly searated us subjected to chemical symathectomy [36].

19 endocrine adatation to maternal absence Maternal care uon reunion First, we would like to underline a ossible methodological constraint in all the above studies (including the resent). AN nd 1-4 (alone or together with LG) in our study was 56 % (of the observations), which is in the same range of revious reorts on nd 1-4 [49% [37], 60% [38]] and higher than reviously reorted values taking into account longer eriods (nd 1-8: 40%; [37], 47% [19]; (Long Evans), 43% [38]]. For LG we reorted a mean of 6 % (of the observations), which is aroximately fold lower than reviously reorted [14% [37], 1% [39]]. Strain differences, culling of us, litter size and litter s sex ratio could be imortant factors influencing baseline levels of LG in the various studies [10]. However, even the same research grou, using the same strain of rats with small changes in the exerimental rocedure, reorted large differences in the nd 1-4 LG scores [aroximate mean values: 11 % [40], 1% [39] 14% [41], 15% [19]]. The question raised in this study was to investigate to what extent maternal care the us received, after reunion with the dam, could exlain the effect of reeated searations on the HPA-axis. It is believed that maternal absence can affect adrenocortical cell differentiation and function [4] and this effect seems related to feeding rather than tactile stimulation rovided by the dam. Feeding acts as an inhibitory factor to the neonate s basal and stress-induced adrenal activity [13, 6, 43]. Therefore, one other mechanism for the CORT desensitization to maternal absence could be related to the attern of tactile stimulation (in the form of LG) or food intake (nursing) exerienced by the us after reunion with their dam. In the current study we observed the following: (i) home and novel searated grous received overall similar care which, in the case of AN, was reduced comared to that of the controls; (ii) ost searation bouts of LG were higher for novel searated than for home searated us, (iii) searation did not induce an increase in AN uon reunion. These findings suggest that for some maternal behaviors (LG) the dam comensates uon reunion. However, for others (AN) she does not comensate, resulting in a lack of care. This altered attern of care after MS might have led to a greater suression of the HPA-axis and result in blunted stress activation after the third 8h-MS. However, differences in maternal care for us exeriencing MS in familiar or unfamiliar contexts, were not reflected in the CORT-resonse to reeated absence. However, those differences in maternal care may exlain the differences between searation contexts in the resonse to a heterotyic stressor. Plotsky and colleagues have argued that maternal care is the major factor driving the effects of MS. Their arguments were based on exeriments showing that dam s exosure to foster litters while her us where in MS, did not lead, in adulthood, to enhanced HPA-axis resonsiveness for the searated animals [44]. In contrast, other investigators, using a similar design, observed that dams are actually able to distinguish the searated us from non searated us, by their altered vocalization behavior,