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Bred to breed?! Implications of continuous mating on the emotional status of mouse offspring
Behavioural Brain Research 279 (2015) 155–165
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Research report
Bred to breed?! Implications of continuous mating on the emotional status of mouse offspring Sandra Lerch a,b , Christiane Brandwein b , Christof Dormann b , Peter Gass b , Sabine Chourbaji a,b,∗ a b
Interfaculty Biomedical Research Facility (IBF), University of Heidelberg, Heidelberg, Germany Central Institute of Mental Health (ZI) Mannheim, University of Heidelberg, Heidelberg, Germany
h i g h l i g h t s • Continuous mating affects the emotional behaviour of mouse offspring. • Females are more sensitive to disturbances in the perinatal environment. • Effects of the breeding environment should be included in the experimental design.
a r t i c l e
i n f o
Article history: Received 2 October 2014 Received in revised form 30 October 2014 Accepted 4 November 2014 Available online 13 November 2014 Keywords: Continuous mating Emotionality Stress Mouse behavior Depression
a b s t r a c t Working with mice represents a smart method to study pathophysiological mechanisms in vivo. However, using animals as model organisms also bears immense caveats. While many aspects in animal research are meanwhile standardized (e.g. nutrition, housing, health) the breeding environment remains unaddressed. Moreover, since the “production” of mice is mostly performed pragmatically, continuous mating (CM) represents a common method to boost the amount of offspring. This condition implies simultaneous pregnancy and lactation in presence of the male, which is associated with increased costs for the breeding dam. Facing the widely-accepted impact of perinatal conditions, our aim was to elucidate how CM affects emotional behaviour of mouse offspring. We therefore compared pregnant mice in CM with mice raising their pups without potentially disturbing influences. According to our hypothesis CM-deriving offspring should demonstrate increased anxiety and depression-like behaviour shaped by pre- and postnatal stress of the mother. Maternal care, i.e. nest building and pup retrieval, was analysed around delivery. To assess the emotional state of the offspring, males and females of either condition were exposed to a behavioural test battery for exploration, anxiety and fear, social and despair behaviour. In addition we analysed corticosterone as stressphysiological correlate. Our study demonstrates that CM affects the emotional phenotype regarding nearly all parameters addressed. These findings emphasize (i) the impact of the perinatal environment on stress-associated behaviour such as depression, and (ii) the need to imply perinatal conditions in the experimental design to decrease the risk of artefacts and increase the overall validity of animal studies. © 2014 Elsevier B.V. All rights reserved.
1. Introduction
Abbreviations: NC, Novel cage; NT, Nest test; OF, Openfield; NO, Novel object; DLB, Dark-Light Box; SR, Social Recognition Test; FST, Forced Swim test; h, hour; PND, postnatal day; m, male; f, female; CM, continuous mating; CON, control; g, gram. ∗ Corresponding author at: Interdisciplinary Biomedical Research Facility (IBF), University of Heidelberg, Im Neuenheimer Feld: 347, 69120 Heidelberg, Germany. Tel.: +49 0 6221 54 5723; fax: +49 0 6221 54 5735. E-mail address: [email protected] (S. Chourbaji). http://dx.doi.org/10.1016/j.bbr.2014.11.007 0166-4328/© 2014 Elsevier B.V. All rights reserved.
Without any doubt the mouse has become the most prominent model organism over the last decades [1,2]. This is due to the relatively easy handling, a great variability of in- and outbred strains with distinct properties and of course the high amount of offspring which can be generated within a conceivable time window [3]. Other arguments to work with mice include the great number of publications that are accessible to look up experimental protocols, to review and cite recent findings in this species.
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Working with mice nowadays is highly standardized. There are advices and guidelines for nearly all conditions, which could affect the experimental outcome. Recommendations of this kind imply the consideration of health state, e.g. SPF, housing conditions, nutrition, genetic background etc. While this is definitely essential to consider and in extension needs to be added to any data published, the breeding strategies of mice remain unaddressed. The reproduction of mice in the wild varies seasonally and – as in most mammals – long daily light phases in the circadian cycle support robust reproduction. Thus laboratory mice are permanently kept on an artificially long (12 h of light 12 h of dark) light cycle. Gestation in mice takes around 21 days. When the litter is delivered maternal care behaviour comprises nestbuilding, grooming and lactation, which may vary in different strains of mice [4–8]. What is known and described by recent literature is that the perinatal environment, including prenatal as well as neonatal stress evokes long-lasting effects, especially with regard to stresssensitive aspects [4,7,9–19]. A fortiori it is surprising that no official statements concerning breeding procedures exist are made by scientific societies. Moreover, in fact one has to realize that breeding of experimental animals is mostly motivated by the need of a great amount of offspring, be it on side of the commercial breeders or of scientists. Whether or not this is fruitful for the overall validity of the results is not questioned anywhere. One example of breeding strategies represents the continuous mating (CM) of mice. The intention of this method is to benefit from the postpartum oestrus as female mice present it in natural but also laboratory settings. Thus this strategy requires the permanent presence of a male counterpart that mating can take place directly after delivery of the litter. It also demands from the female to bear the costs—in the truest sense of the word [20,21]. While simultaneously nursing the actual litter with all energy available, the female is stressed by the upcoming pregnancy with all physical challenges that are associated with such a state. Additionally there is no way to cope, e.g. avoid the contact with the male, which could also be potentially dangerous for the youngborn pups [22]. Facing the unsatisfactory awareness that such perinatal conditions, we were interested in elucidating the following hypothesis: CM represents a sustainable form of perinatal stress that is induced by several challenging factors, (i) stress of the dam which is induced by an increase of costs and permanent presence of the male and (ii) stress of the offspring due to the limited energy the mother can raise when taking care of two generations at the same time. To follow up these thoughts our experimental design comprised two parts of investigations. First, maternal behaviour, i.e. nestbuilding, maternal exploration and pup retrieval were analysed. The second set of experiments assessed emotional behaviour of male and female offspring, which were analysed in a behavioural test battery. Emotional behaviour was examined by exploring changes in activity, anxiety, social behaviour as well as fear and depressivelinked aspects. All CM-induced behaviours were consecutively compared with those of dams maintained with one single litter and the behaviour of the offspring.
2. Materials and methods 2.1. Housing and breeding conditions Female mice at an age of 3–4 month were derived from Charles River Germany (Charles River, Sulzfeld) and were experienced in bringing up litters. All mice were housed in Macrolon cages type III (Tecniplast, Italy), which contained wooden chips (ABEDD LTE-001, Lab & Vet
Service, Vienna, Austria), tissue nesting material made of cellulose and food and water ad libitum (Rod16A, Lasvendi, Soest, Germany). Housing of the animals was standardized by 12:12 h dark–light cycle (dark phase: 9.00 a.m.–9.00 p.m.) at a room temperature of 22 ± 2 ◦ C. Humidity was set at 50%. The hygienic status was SPF according to recent FELASA recommendations [23]. Before first mating female mice were allowed to acclimate to the new housing room for 14 days. We examined two groups of mice: Group I (n = 7): control group (CON): mating was performed in Macrolon cages type III with one male (2–3 months) and two female mice (3–4 months). Females were separated from the male when pregnancy was ascertained by positive vaginal plug check and indicative weight changes. Females from then on were housed individually under same conditions outlined above. After delivery dams were left undisturbed till pup retrieval testing at postnatal day (PND) 7. Weaning took place at PND21, from then on brothers and sisters were group-housed in type III cages equipped with wooden bedding and tissue as nesting material and food and water ad libitum (Rod16A, Lasvendi, Soest, Germany). Group II (n = 5): continuous mating group (CM): mating was performed in Macrolon cages type III with one male (2–3 months) and one female mice (3–4 months). Males remained in breeding cages during pregnancy and lactation to generate the CM condition. All other housing conditions were comparable to those of the CON animals. The comparably smaller number of dams in Group CM was caused by cannibalism and disrupted pregnancy. 2.1.1. Maternal behaviour The assessment of maternal behaviour comprised: (i) pup retrieval behaviour (ii) maternal exploration and (iii) nest building. Maternal exploration and nest building performance was studied once a week (exceptionally during light phase between 8.00 and 9.00 a.m.) during pregnancy and during lactation. Pup retrieval testing was conducted unique at PND 7. If indicative weight changes could be observed one week after mating the first maternal care tests were conducted during pregnancy. Thus the maternal care assessment comprised two timeframes during pregnancy and during breeding. To avoid interference with test procedures with accompanying disturbance of dams and litter, tests were conducted within cage change procedures. 2.1.1.1. Pup retrieval behaviour: Pup retrieval test. On postnatal day (PND) 7 the pup retrieval behaviour was tested. Two pups were put into the far corners of the nest in the homecage. The latency until the dam retrieved her pups back into the nest was recorded [24]. The dams had a maximum of 300 s to perform the retrieval. 2.1.1.2. Maternal exploration: Novel cage test. In the context of cage changes once a week, the animals were placed into a new macrolon cage type III containing a layer of new bedding material and the latency as well as total number of rearings were measured for 5 min at the end of the light phase (during breeding period the pups are placed into new cage with the dam before measuring behaviour), (CM group: males were placed after novel cage testing into new cage). 2.1.1.3. Maternal nest building performance: Nest test. After novel cage test the dam with her pups (and for the CM group also the male) remained in the new cage. A nestlet (PLEXX, Arnheim) was introduced into new cage and nest building was analysed by scores of Deacon [25] after 5 and 24 h. Score 1: nestlet untouched, Score 2: nestlet partly picked to pieces, Score 3: nestlet completely picked to pieces, Score 4: recognizable nest, Score 5: complete covered nest.
S. Lerch et al. / Behavioural Brain Research 279 (2015) 155–165
The last maternal behaviour testing was conducted when pups aged 2 weeks. With 21 days pups were weaned and gender separated group housed under same conditions outlined above. 2.2. Offspring From weaning till 11th week of life offspring was weighed weekly. Two representative animals/litter (one female and one male) were chosen according to the median of all litter weights per gender. All experimental cohorts consisting of one pup/gender/litter to be representative and had gender-separated been randomized before testing to avoid “litter effects”. Testing (behaviour experiments) started at an age of 8 weeks. Testing last three weeks in total. 24 h after last experiment offspring was decapitated and samples of trunk blood were collected for corticosterone analysis. Behaviour testing was conducted during dark phase of the dark–light cycle to cover the active period of the animals. (number of offspring group CON: female: n = 7; male: n = 7; number of dams group CM: female: n = 5; male: n = 5) Our study complies with the actual regulations on animal experiments in Germany and was approved by local competent authorities (§15 Kommission, Regierungspräsidium Karlsruhe, Abteilung 3: Landwirtschaft, Ländlicher Raum, Veterinär- und Lebensmittelwesen, permit number: G-279/12) and the animal welfare committee of the Interfaculty Biomedical Research Facility of the University of Heidelberg. To minimize distress between the assessment of the relevant parameters, all animal care and testing was carried out by the same person. 2.2.1. Offspring behaviour 2.2.1.1. Weight monitoring. All offspring assigned to be analysed with regard to emotional behaviour in the testbattery was weight weekly when the cages were changed. 2.2.1.2. Behavioural testbattery. To avoid artefacts due to test–test interactions, the sequence of tests in the battery started with the
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least stressful and ended with the most invasive paradigm [26,27] as shown in Fig. 1. All behavioural analyses were carried out during the dark phase to assess behavioural performance in the active phase. Before each test the animals were acclimatized to the behavioural test room for 30 min—except nest test and novel cage test because these tests took place in housing room. 2.2.1.2.1. Nest test. The nest test is a valid procedure to assess this aspect of maternal care performance, but also detects inadequate behaviour in non-breeding mice [25], since nest-building is part of the natural behavioural repertoire [28]. For the analysis of nest quality the nesting material was removed from the home cage 1 h before dark phase (8 a.m.) and a standard nestlet (PLEXX, Arnheim) was introduced. The nest building performance was rated 5 and 24 h later with scores by Deacon [25]. 2.2.1.2.2. Novel cage. The novel cage test assesses the level of vertical exploration in a new environment. To reduce the handling of the mice, this test was performed after the weekly cage change. To make the results comparable to the maternal dams, as well as for practical reasons, the mouse was placed into a new cage type III (825 cm2 ) with clean bedding and the rearings as well as the latency to the first rearing were measured for 5 min. The mouse remained in the new cage and after testing the untested littermates were transferred to the fresh cage as well [29]. 2.2.1.2.3. Openfield/novel object. By means of the openfield test it is possible to detect changes with regard to activity, anxiety and exploration [30]. In our experiments the animal was placed into a 50 × 50 × 50 cm black openfield apparatus placed on an infrared light surface. Four mice are tracked simultaneously for 10 min with a light intensity of 25 lx. With a camera above the arena (Ikegami Digital) Ethovison 4.0 (Noldus Information Technology, Wageningen, The Netherlands) evaluates “distance moved”, “velocity” and the time spent in defined areas of the openfield, i.e. “centre time”. In addition the faecal boli were counted as indicator for emotional stress [31–33].
Fig. 1. Experimental design. Overview about the procedures for (I) control group (CON) and (II) continuous mating group (CM). The course of experiments comprises pre-and postpartal conditions of dams, i.e. conditions with the respective modification of the maternal environment. The course of experiments comprises also pre- and postnatal conditions of offspring in (I) control group (CON) and (II) continuous mating group (CM). Weaning took place at PND21 from when on the development of weight is examined. Behavioural analysis of offspring started at an age of approximately 8 weeks and lasted until the animals were sacrificed at an age of 10 weeks. Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).
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As an expansion of the openfield, a novel object test was conducted. After the first tracked 10 min of habituating to the empty openfield a falcon tube (50 ml) filled with water was placed into the middle of every test arena. The exploratory and neophobic behaviour, respectively, were measured for another 10 min by assessing the latency to approach the novel object up to two centimetres as well as the total number of approaches. Respective arenas were defined by using Ethovision 4.0. Between the different animals the apparatus was cleaned with 70% ethanol. 2.2.1.2.4. O-maze. The O-maze is an elevated circular arena with two open and two closed arms, which is comparable to the frequently used Elevated Plus Maze, and represents a test for anxiety. The O-maze apparatus in our experimental setup is made of rough high-grad steel. The outer diameter is 45 cm, the width of the arms 6 cm, and it is elevated 50 cm above ground. In our experiment anxiety was measured by counting the time on open arms, number of exits and full crosses (one circumnavigation of the Omaze). In total the test lasted 5 min and is performed at 25 lx [34]. Between the different animals the apparatus was cleaned with 70% ethanol. 2.2.1.2.5. Dark–light box. The dark–light box (54 × 22, 5 × 32 L/W/H) consists of a dark (22.5 cm) and a light compartment (31.5 cm) and is used to assess conflict-avoidance behaviour in mice [35]. The light compartment was illuminated by 600 lx. The mouse can switch between the compartments through a 5 × 5 cm aperture. The tests started by placing the mouse into the dark compartment. The latency, the time in, and the number of exits into in the light compartment were measured for 5 min [29]. Between the different animals the apparatus was cleaned with 70% ethanol. 2.2.1.2.6. Social recognition. The self-made Social Recognition test apparatus (50 × 50 × 50 cm) to assess socially induced motivation consists of three compartments (each compartment measures 16.5 cm) which are connected through apertures. The testing procedure comprised four phases: Phase 1: Five minutes of acclimatization with closed apertures (only the central compartment could be explored). Phase 2: The apertures were opened and the mouse could switch between all compartments for 5 min. The first two phases took place without behavioural tracking. Phase 3: A stranger mouse of the same gender was placed into a wire frame cage in the corner of one outer compartment. The opposite external compartment contained an empty wire frame cage. Tracking was conducted for 10 min. The analysis comprised the time spent within the centre and the external compartments, respectively. Additionally the time spent in close vicinity of the wire frame cages was measured. Phase 4: The empty wire cage was exchanged with a wire cage containing a novel stranger to observe the “social memory” of the animals. Additionally the compartments were changed, so the novel stranger (second stranger) was then placed into the compartment of the previously first stranger and conversely the first stranger was now in the compartment of the previously empty cage. Phase 4 lasted another 10 min. The entire test procedure was conducted at a light intensity of 25 lx. 2.2.1.2.7. Forced swim test. The forced swim test, originally established and validated by Porsolt [36], is a test to assess depressive-like behaviour in rodents. In our study, a mouse was placed into a 2000 ml beaker filled with 1000 ml 21 ◦ C cold water. Floating, i.e. complete lack of movement, and the latency to first display this behaviour interpreted as “giving-up”, was measured under redlight conditions by means of Ethovision Software for 6 min. Afterwards the mouse was placed back into its home cage and allowed to dry in front of a redlight for 10 min. After 24 h the mouse was retested under the same conditions [29].
2.2.1.2.8. Hotplate test. The Hotplate (Ugo Basile Srl) is used to determine pain threshold consists of a plate which is adjustable from 2 to 66 ◦ C and a glass cylinder to prevent the mouse from jumping off the plate. The temperature in our setup was 52 ◦ C. The test took place at 100–130 lx for max. The test took 45 s and latency till licking of hind paws was measured. When licking hindpaws was observed, the mouse was immediately removed from the Hotplate. The Hotplate test was performed to identify possible artefacts due to different pain thresholds induced by different perinatal stress conditions [37]. Between the different animals the apparatus was cleaned with 70% ethanol. 2.2.1.2.9. Emotional learning: Fear conditioning. For the analysis of hippocampus- and amygdala- associated emotional learning, the Fear Conditioning system was used (TSE systems, Bad Homburg, Germany). The paradigm applied in our study comprised three days of exposition. Day 1: shock: For initial conditioning on the first day, the mouse was placed into a cylinder onto a grid. After 120 s of habituation, followed by a 30 s long tone (conditioned stimulus, 2800 Hz, Intensity 67), a brief foot shock was applied (unconditioned stimulus, 2 s, 0.8 mA). Intertrial cleaning was performed with 70% Ethanol. Day 2: context: For context, the mouse was placed 24 h after Day 1 into the same experimental chamber onto the grid for 5 min without any stimulus. To analyze context conditioned freezing, i.e. complete lack of movements apart from respiration, freezing behavior was evaluated every 10 s and noted as yes or no (freezing). During this phase of the test, the apparatus was cleaned with 70% ethanol to ensure a comparable olfactoric context to day one. Day 3: cued: For the assessment of cued conditioning, freezing behaviour was analyzed in a different context. Here the mouse was placed in the same chamber but within another context into a different rectangular cylinder with a flat surface (to ensure an altered tactile context), which was cleaned with chamomille tea to additionally provide a different olfactoric context. After 180 s of habituation to the new context, the conditioned stimulus (2800 Hz, Intensity 67) was presented for 180 s. Freezing was evaluated as stated above. Score was calculated by means of percentage freezing/time of test duration. 2.2.1.2.10. Corticosterone analysis. For corticosterone analysis, trunk blood was obtained by conscious decapitation within 30 s of removing the animal from the cage. Anaesthesia was not used to avoid effects on corticosterone secretion. Samples were centrifuged for 5 min by 3500 rpm and plasm (supernatant) was collected and stored by −20 ◦ C till analysis. Samples were determined by radioimmunoassay (ICN Biomedicals, Eschwege, Germany); assay sensitivity was 12.5 ng/ml.
2.2.1.3. Statistical analysis. For statistical analysis the values of maternal behaviour were evaluated by one-way ANOVAS. Offspring behaviour was analyzed by means of two-way ANOVAS and subsequent Tukey Posthoc testing. Weight assessment was calculated by repeated measurement ANOVAS. Statistically significant effects were set at a p ≤ 0.05. To evaluate statistic differences the “InVivoStat” (invivostat.co.uk) statistical software was used.
3. Results Table 1 illustrates all tests conducted for maternal behaviour during pregnancy and breeding and illustrates significant and nonsignificant results. Table 2 shows all tests conducted in offspring with respectively significant and non-significant effects on offspring behaviour.
S. Lerch et al. / Behavioural Brain Research 279 (2015) 155–165 Table 1 Maternal behaviour: evaluation of “CM” in dams evaluation of the factor “CM” in all tests for maternal behaviour during pregnancy (prepartal conditions) and nursing (postpartal conditions). The order of the data reflects the order of the tests as they were conducted. Parameters not appearing in this table revealed non-significant results. Evaluation of “continuous mating”
Table 2 Offspring behaviour: evaluation of “CM” and “gender” effects in offspring Evaluation of the overall effects of “CM”, “gender” and interaction effects in all tests conducted in offspring. The order of the data presented reflects the order of all tests as they were conducted. Parameters within tests not appearing in this table revealed nonsignificant results. Evaluation of “continuous mating” and “gender” effect
Test
Timepoint
Parameter
NC NT NC NT PRT
1st Week of pregnancy 1st Week of pregnancy 2nd Week of pregnancy 2nd Week of pregnancy 1st Week of nursing (PND7) n.s. 1st Week of nursing 1st Week of nursing 2nd Week of nursing 2nd Week of nursing 2nd Week of nursing
n.s.
Littersize NC NT NC NT NT
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5 h [scores] Time till retrieval [seconds]
Effects
Test
Parameter
Effects/ interactions
Posthoc test (Tukey)
Weights
5th Week of life [g]
n.s.
NC
Rearings [number] * CM p = 0.001
*
T m CM ↓ m CON ↑ p = 0.086 n.s.
*
CM p = 0.048 n.s.
NT
5 h [scores]
n.s.
5 h [scores] 24 h [scores]
** *
24 h [scores]
CM p = 0.004 CM p = 0.035
n.s. = no significance; CM = continuous mating; NC = novel cage; NT = nest test; PRT = pup retrieval test. Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001). The order of the results reflects the order of the tests as they were conducted.
3.1. Maternal behaviour
3.2. Offspring behaviour 3.2.1. CM did not affect weight development Regular weekly weight development could be observed when pups of either gender were reared under CM conditions. Only a tendency in the 5th week of life within males could be identified (p = 0.086). There was a significant gender effect with increasing significance levels over the time [F1,24 = 50.09; p = 0.001] (see Fig. 3). 3.2.2. CM decreased exploration of male and female offspring in the novel cage test There was a visible overall “CM” effect on exploration behaviour (number of rearings) which occurred in the novel cage test [F1,24 = 5.05, p = 0.036]. The latency to start rearing was not affected (data not shown). 3.2.3. CM decreased nestbuilding performance in female and male offspring There was a significant overall effect of “CM” after 5 [F1,24 = 13.81; p = 0.001] and 24 h [F1,24 = 20.06; p = 0.001] in the nest
CM p = 0.001
OF
Fecal Boli [number]
NO
Visits [number]
OM DLB
3.1.1. CM increased nest building behaviour in second week of pregnancy The presence of a male mouse in the breeding cage affected maternal nest building quality in the nest test after 5 h in the second week of gestation. Pregnant CM dams presented an increased nest score of 3.8 in comparison to CON dams with a nest score of 3.5. [F0,12 = 5.44; p = 0.048] (see Fig. 2). 3.1.2. CM enhanced nest building behaviour in second week of nursing During the nursing period the quality of nests was increased after 5 [F0,12 = 14.63; p = 0.004] and 24 h [F0,12 = 6.14; p = 0.035]. Nursing CM dams presented a nest score of 4.6 after 5/24 h, CON dams demonstrated a nest score of 1.8 after 5 and 3.3 after 24 h (see Fig. 2). Maternal exploration as assessed in the novel cage test as well as pup retrieval were unaffected by CM conditions (see Table 1). The assessment of littersize of CM and CON group revealed no differences. The average number of mothers of CON group was 9.7 pups/litter and mothers of CM group gave birth to 8.6 pups/litter.
*
Latency 1st exit [s] Time spent outside [s] Phase 3
Phase 4
FST
Immobility day 1 [s] Immobility day 2 [s]
HP FC Stressphysiological analysis
n.s. * CM p = 0.02
*
Gender p = 0.044 Entries empty compartment [number]
Time in compartment stranger 1 compartment [s] Time at cage stranger 1 compartment [s] Time at cage stranger 2 compartment [s] * CM p = 0.001
*
CM p = 0.03
n.s. n.s. Corticosterone [ng/ml]
CM p = 0.036
f CM ↓ f CON ↑ * p = 0.025 m CM ↓ m CON ↑ ** p = 0.002 * CM p = 0.01
*
Gender p = 0.034
m CM ↑ m control ↓ * p = 0.029 n.s.
f CM ↑ f CON ↓ * p = 0.049 n.s. * Gender: CM p = 0.015
*
Gender p = 0.001 * Gender:CM p = 0.008 *
Gender p = 0.008 Gender:CM p = 0.037 * Gender p = 0.027 *
f CM ↓ m CM ↑ * p = 0.046 m CM ↑ m CON ↓ * p = 0.04 f CON ↓ m CON ↑ *** p = 0.001
f CON ↓ m CON ↑ ** p = 0.007 n.s.
f CM ↑ f CON ↓ ** p = 0.009 n.s.
*
Gender p = 0.025
f CON ↓ m CON ↑ * p = 0.049
Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001). The order of the results reflects the order of the tests as they were conducted.
test. Posthoc testing revealed significant nest building deficiencies after 5 h in CM females (p = 0.025) (Fig. 4a) and after 24 h in CM males (p = 0.002) (Fig. 4b). 3.2.4. CM increased the number of fecal boli in male offspring in the openfield The factor “CM” exerted an overall effect on “faecal boli” as an indicator for emotionality in openfield testing [F1,24 = 8.0; p = 0.01]. Posthoc testing revealed a significantly increased number of fecal boli in CM raised males (p = 0.029). Subsequent novel object testing showed an overall “gender” specific neophobic behaviour
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Fig. 2. Maternal behaviour/care. CM dams show better nest building performance after 5 h in 2nd week of pregnancy and after 5 and 24 h in 2nd week of nursing. Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).
Fig. 3. Weight development after weaning. Female offspring raised under CON conditions shows slightly higher weights compared to female CM offspring without statistical significance. Both, CON and CM demonstrate regular weight development over the time, however overall weight development was only affected by “gender”. Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).
[F1,24 = 5.22; p = 0.034] (Fig. 5). Other, i.e. activity-related parameters in the openfield were unaffected by CM. 3.2.5. CM enhanced anxiety of female offspring in the dark–light box While O-maze testing did not uncover any anxiety-related differences, dark–light box testing revealed an apparent overall effect of “CM” on the latency of first exit [F1,24 = 6.44; p = 0.02]. Posthoc testing showed a female-specific significant increase in the latency to enter the bright compartment when reared by CM dams (p = 0.049) (Fig. 6). Moreover the parameter “time spent outside” indicated a “gender” specific difference [F1,24 = 4.64; p = 0.044] (data not shown). The factor “CM” by itself did not affect this parameter.
3.2.6. CM decreases social interest and social recognition Entries into the empty compartment (Phase 3) indicating initial exploration, revealed an interaction of both factors, “gender” and “CM” [gender:CM: F1,24 = 7.09; p = 0.015] with significant Posthoc testing. Tukey Posthoc analysis here demonstrated decreased exploration in females when raised under CM conditions (p = 0.046). Contrary, male CM offspring presented more entries/increased exploration into the empty compartment (p = 0.04) (Fig. 7a). When an unfamiliar mouse was introduced for the first time (Phase 3, empty + Stranger 1) two overall effects occurred regarding “time in compartment Stranger 1”. There was an overall effect of “gender” [F1,24 = 13.55; p = 0.001]; and “gender:CM” [F1,24 = 8.7;
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Fig. 4. Nest test—(a) 5, (b) 24 h. (a) Female CON offspring shows higher nest building scores after 5 h than female CM offspring in the nest test. (b) CON male offspring shows higher Nest building scores after 24 h than male CM offspring. Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).
Fig. 5. Openfield-fecal boli Males reared under CM conditions demonstrate an increased number of fecal boli in contrast to males reared under CON conditions. Females present comparable numbers of fecal boli Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).
Fig. 6. Dark–light box—latency 1st exit. Female CM offspring demonstrate a higher latency to leave dark compartment of the Dark-light box compared to female CON offspring. Male mice do not show statistically significant differences. Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).
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Fig. 7. Social recognition—Phase 3: (a) Entries into empty compartment: male offspring raised under CM conditions shows increased exploration/more entries into compartment with empty cage, compared to CM females and males CON in Phase 3. (b) Time at compartment Stranger 1: male CON offspring shows an increased motivation to approach compartment of Stranger 1 in Phase 3. (c) Time at cage Stranger 1: in addition to (b) the “time at cage of Stranger 1” reveals an higher resting time of male CON offspring. Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).
p = 0.008]. Again Posthoc analysis indicated an increased motivation to approach compartment of Stranger 1 in male CON mice (p = 0.001) (Fig. 7b). As a consequence thereof the “time at cage stranger 1” (Phase 3) was influenced by “gender” [F1,24 = 8.62; p = 0.008] and there was an interaction between “gender” and “CM” [F1,24 = 5.01; p = 0.037] with significant Posthoc testing revealing increased interest to approach the newly introduced animal in male CON mice (p = 0.007) (Fig. 7c). When a second Stranger (Phase 4) was introduced, the parameter “time at cage stranger 2” was affected by “gender” [F1,24 = 5.68; p = 0.027]. There were no significances in the Posthoc tests (data not shown).
3.2.7. CM promoted depressive-like behaviour in the forced swim test in females Day 1: The “Immobility” on Day 1 was affected by an overall effect of “CM” [F1,24 = 15.57; p = 0.001]. Tukey Posthoc Test showed an increased immobility of females (p = 0.009) (Fig. 8). The latency to start floating was unchanged. Day 2: “Immobilty” on Day 2 was influenced by “CM” [F1,24 = 5.48; p = 0.03] but there were no significances in Posthoc tests (data not shown). The latency to start floating was also unchanged.
3.2.8. Corticosterone determination did not reveal any digestible results Determination by radioimmunoassay showed significant results regarding the overall effect of “gender:CM” [F1,15 = 5.47; p = 0.039]. Due to a small number of samples which were detectable in the radioimmunoassay we didn’t pursue such findings. 4. Discussion This study was conducted to determine whether specific breeding conditions affect (i) maternal behaviour and (ii) the emotional/depressive-like phenotype of male and female offspring. To address this question we kept mouse mothers and their litters under two different conditions: (I) control (CON) conditions, i.e. one single-housed mother raising one litter or (II) continuous mating (CM) conditions, which comprised pregnancy, lactation of the previous litter and presence of the sire at the same time. In a first step we assessed maternal exploration and nest building behaviour. Later on we exposed the adult offspring to a behavioural test battery to analyze the emotional and depressionassociated status. Our data indicate that the breeding environment indeed plays a role at both levels—maternal care and offspring performance. Most interestingly not only basal behaviours, but also depressive-like features were affected in a significant manner with
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Fig. 8. Forced swim test. Female CM offspring show higher depressive-like behaviour, i.e. increased immobility on Day 1 of the forced swim test compared to female CON offspring. Males do not show alterations with regard to immobility. Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).
females presenting an increased immobility in the forced swim test, a fact that is rather alarming. This could, however, not be referred to a generally decreased performance of the mother since CM dams exhibited even increased nest building performance during pregnancy and nursing. Such a finding is somehow surprising since we expected that CM represent a stressful environment rather compromising maternal behaviour. What may be postulated is an increased motivation to provide shelter to the newborns or that males by themselves build the nests, which is part of their behavioural repertoire [25,28,38,39]. Maternal exploration and pup retrieval were similar in both groups indicating that CM did not exert broad effects, at least with regard to the parameters examined here. Interestingly and in line with our hypothesis nearly all tests beside our depression-paradigm revealed significant findings in mouse offspring. When the animals were exposed to a novel cage, “CM” affected exploration of the novel environment. In the nest test effects could be found after 5 (females) and 24 h (males). CM females herby built poorer nests after 5 h and CM-males built poorer nests after 24 h. This is fairly interesting since the decreased motivation of nest building again cannot be related to a decreased functioning of the mothers. What could be speculated therefore is, that housing of mouse pairs increases nest building performance. Alternatively one could assume a decreased welfare considering recent literature, e.g. by Gaskill et al. who identified nest building as indicator for the animals’ wellbeing [40], but it can also be speculated that other facets in maternal care exerted respective impairments [8]. When behaviour of CM and CON offspring was investigated in the openfield, “CM” exerted an overall and a male-specific effect on the number of fecal boli, a measure for enhanced emotionality [32,41]. This effect was not present in females. In the dark–light box CM females were affected regarding the latency to 1st exit into the bright compartment presenting increased anxiety and a greater variance within the group. The “gender” effect considering the time the mice spent outside in the bright compartment is not specifically surprising since it is known that male and female mice respond differently with regard to anxiety-related features [42]. With regard to social recognition our analyses demonstrate a generally decreased (social) exploration in females. This decreased investigation when it comes to an unfamiliar animal was, however, not affected by “CM”, but seems to be rather a gender-specific issue.
From our point of view and as stated at the beginning of the discussion the most exciting finding of our study was found in the forced swim test. Here both days of testing revealed a significant overall effect of “CM” on floating behaviour, i.e. immobility a correlate for despair-behaviour. Again in this paradigm it were the females exhibiting a significantly enhanced depressive-like behaviour. Keeping in mind that research in the field of experimental psychiatry still explores valid animal models in which a number of depressive-like features occur together to closely mimic a depressive symptom in humans, these results are rather worrying. If breeding conditions per se lead to changes in emotionality and depressive-like features this bears an immense impact with regard to both, valid scientific results and the animal welfare thought. To examine a stress-physiological correlate, we also assessed corticosterone from blood plasma. While we expected an increase of corticosterone indicating a dysregulation of the hypothalmus pituitary adrenal (HPA) system there were no such effects. Also of interest for our study was the fact whether emotional learning was influenced by early perinatal conditions. This was however not the fact when tested in context and cued conditioning in the Fear Conditioning paradigm. Since pain sensitivity was not affected, neither by the factor “CM” not “gender” artefacts were excluded. The overall validity of our testbattery and potential test interactions were considered by starting with less stressful analyses while more stressful tests were set to the end [26,27]. A very interesting finding beside the general effect of CM on most and especially depression-associated parameters was the obviously increased sensitivity of females. Female offspring raised in a CM environment had impairments in nest building, an essential feature especially in this gender, increased anxiety and presented a depressive-like phenotype in the forced swim test. While this could be very attractive for research groups dealing with depression models, it also has to be considered in the context of animal welfare. When animals are used for scientific purposes it is obligatory to provide best possible conditions to the animals and minimize stress and discomfort [43]. Continuous mating is a method of breeding in which due to postpartum oestrus high numbers of animals can be bred. However if side effects are likely to take an influence on the results because of increased emotionality, this should definitely taken into account and requires a well-considered appreciation of values. What anyway becomes clear is that it is
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immensely important to explicitly report on how animal studies were conducted and imply all background information, including breeding conditions, e.g. according to the ARRIVE guidelines [44–47]. Only the entire contemplation of a model organism allows valid and reproducible results and interpretation of data for sustainable research and human conditions for animals used for all disciplines of science. 5. Conclusion Our study demonstrates that the maternal environment in common breeding conditions exerts long-lasting effects on the emotional status of male and female offspring. While this may be suggested as attractive approach when embedded in neurobiological research it may on the other hand threat studies in which stress is unwanted and could cause artefacts. With regard to a long-range perspective a healthy life relies upon early experiences and adaptations can be made adequately [48,49]. With disadvantaged early conditions like postulated by our CM paradigm there is an increased risk of stress and maladaptation. Therefore these findings need to be likewise considered by researchers, animal facilities and commercial breeders to ensure high quality science as well as responsible implementation of justifiable animal welfare. Acknowledgements We thank Charles River (Sulzfeld, Germany) for providing us with the mice used in this study as well as the animal caretakers of the clinic experimental facility of the IBF for their support. References [1] Rosenthal N, Brown S. The mouse ascending: perspectives for human-disease models. Nat Cell Biol 2007;9:993–9. [2] Hardouin SN, Nagy A. Mouse models for human disease. Clin Genet 2000;57:237–44. [3] Festing MF. Inbred strains should replace outbred stocks in toxicology, safety testing, and drug development. Toxicol Pathol 2010;38:681–90. [4] Caldji C, Diorio J, Meaney MJ. Variations in maternal care in infancy regulate the development of stress reactivity. Biol Psychiatry 2000;48:1164–74. [5] Chourbaji S, Hoyer C, Richter SH, Brandwein C, Pfeiffer N, Vogt MA, et al. Differences in mouse maternal care behavior—is there a genetic impact of the glucocorticoid receptor? PLoS One 2011;6:e19218. [6] Catalani A, Marinelli M, Scaccianoce S, Nicolai R, Muscolo LA, Porcu A, et al. Progeny of mothers drinking corticosterone during lactation has lower stressinduced corticosterone secretion and better cognitive performance. Brain Res 1993;624:209–15. [7] Coutellier L, Friedrich AC, Failing K, Wurbel H. Variations in the postnatal maternal environment in mice: effects on maternal behaviour and behavioural and endocrine responses in the adult offspring. Physiol Behav 2008;93:395–407. [8] Cox KH, So NL, Rissman EF. Foster dams rear fighters: strain-specific effects of within-strain fostering on aggressive behavior in male mice. PLoS One 2013;8:e75037. [9] Anisman H, Zaharia MD, Meaney MJ, Merali Z. Do early-life events permanently alter behavioral and hormonal responses to stressors. Int J Dev Neurosci 1998;16:149–64. [10] Branchi I, D’Andrea I, Fiore M, Di Fausto V, Aloe L, Alleva E. Early social enrichment shapes social behavior and nerve growth factor and brainderived neurotrophic factor levels in the adult mouse brain. Biol Psychiatry 2006;60:690–6. [11] Casolini P, Cigliana G, Alema GS, Ruggieri V, Angelucci L, Catalani A. Effect of increased maternal corticosterone during lactation on hippocampal corticosteroid receptors, stress response and learning in offspring in the early stages of life. Neuroscience 1997;79:1005–12. [12] Catalani A, Casolini P, Cigliana G, Scaccianoce S, Consoli C, Cinque C, et al. Maternal corticosterone influences behavior, stress response and corticosteroid receptors in the female rat. Pharmacol Biochem Behav 2002;73:105–14. [13] D’Amato FR, Zanettini C, Lampis V, Coccurello R, Pascucci T, Ventura R, et al. Unstable maternal environment, separation anxiety, and heightened CO2 sensitivity induced by gene-by-environment interplay. PLoS One 2011;6:e18637. [14] Franklin TB, Linder N, Russig H, Thony B, Mansuy IM. Influence of early stress on social abilities and serotonergic functions across generations in mice. PLoS One 2011;6:e21842. [15] Gheorghe CP, Goyal R, Mittal A, Longo LD. Gene expression in the placenta: maternal stress and epigenetic responses. Int J Dev Biol 2010;54:507–23.
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Effect of predatory stress on sucrose intake and behavior on the plus-maze in male mice. Physiol Behav 1999;67:189–96. [42] Navarro-Frances CI, Arenas MC. Influence of trait anxiety on the effects of acute stress on learning and retention of the passive avoidance task in male and female mice. Behav Processes 2014;105:6–14. [43] Baumans V. Science-based assessment of animal welfare: laboratory animals. Rev Sci Tech 2005;24:503–13. [44] Baker D, Lidster K, Sottomayor A, Amor S. Two years later: journals are not yet enforcing the ARRIVE guidelines on reporting standards for pre-clinical animal studies. PLoS Biol 2014;12:e1001756. [45] Blomme EA. The ARRIVE guidelines: a resource for authors and reviewers to ensure that submissions to The Veterinary Journal meet minimal expectations of completeness, accuracy and transparency. Vet J 2011;189:237–8.
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Research report
Bred to breed?! Implications of continuous mating on the emotional status of mouse offspring Sandra Lerch a,b , Christiane Brandwein b , Christof Dormann b , Peter Gass b , Sabine Chourbaji a,b,∗ a b
Interfaculty Biomedical Research Facility (IBF), University of Heidelberg, Heidelberg, Germany Central Institute of Mental Health (ZI) Mannheim, University of Heidelberg, Heidelberg, Germany
h i g h l i g h t s • Continuous mating affects the emotional behaviour of mouse offspring. • Females are more sensitive to disturbances in the perinatal environment. • Effects of the breeding environment should be included in the experimental design.
a r t i c l e
i n f o
Article history: Received 2 October 2014 Received in revised form 30 October 2014 Accepted 4 November 2014 Available online 13 November 2014 Keywords: Continuous mating Emotionality Stress Mouse behavior Depression
a b s t r a c t Working with mice represents a smart method to study pathophysiological mechanisms in vivo. However, using animals as model organisms also bears immense caveats. While many aspects in animal research are meanwhile standardized (e.g. nutrition, housing, health) the breeding environment remains unaddressed. Moreover, since the “production” of mice is mostly performed pragmatically, continuous mating (CM) represents a common method to boost the amount of offspring. This condition implies simultaneous pregnancy and lactation in presence of the male, which is associated with increased costs for the breeding dam. Facing the widely-accepted impact of perinatal conditions, our aim was to elucidate how CM affects emotional behaviour of mouse offspring. We therefore compared pregnant mice in CM with mice raising their pups without potentially disturbing influences. According to our hypothesis CM-deriving offspring should demonstrate increased anxiety and depression-like behaviour shaped by pre- and postnatal stress of the mother. Maternal care, i.e. nest building and pup retrieval, was analysed around delivery. To assess the emotional state of the offspring, males and females of either condition were exposed to a behavioural test battery for exploration, anxiety and fear, social and despair behaviour. In addition we analysed corticosterone as stressphysiological correlate. Our study demonstrates that CM affects the emotional phenotype regarding nearly all parameters addressed. These findings emphasize (i) the impact of the perinatal environment on stress-associated behaviour such as depression, and (ii) the need to imply perinatal conditions in the experimental design to decrease the risk of artefacts and increase the overall validity of animal studies. © 2014 Elsevier B.V. All rights reserved.
1. Introduction
Abbreviations: NC, Novel cage; NT, Nest test; OF, Openfield; NO, Novel object; DLB, Dark-Light Box; SR, Social Recognition Test; FST, Forced Swim test; h, hour; PND, postnatal day; m, male; f, female; CM, continuous mating; CON, control; g, gram. ∗ Corresponding author at: Interdisciplinary Biomedical Research Facility (IBF), University of Heidelberg, Im Neuenheimer Feld: 347, 69120 Heidelberg, Germany. Tel.: +49 0 6221 54 5723; fax: +49 0 6221 54 5735. E-mail address: [email protected] (S. Chourbaji). http://dx.doi.org/10.1016/j.bbr.2014.11.007 0166-4328/© 2014 Elsevier B.V. All rights reserved.
Without any doubt the mouse has become the most prominent model organism over the last decades [1,2]. This is due to the relatively easy handling, a great variability of in- and outbred strains with distinct properties and of course the high amount of offspring which can be generated within a conceivable time window [3]. Other arguments to work with mice include the great number of publications that are accessible to look up experimental protocols, to review and cite recent findings in this species.
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Working with mice nowadays is highly standardized. There are advices and guidelines for nearly all conditions, which could affect the experimental outcome. Recommendations of this kind imply the consideration of health state, e.g. SPF, housing conditions, nutrition, genetic background etc. While this is definitely essential to consider and in extension needs to be added to any data published, the breeding strategies of mice remain unaddressed. The reproduction of mice in the wild varies seasonally and – as in most mammals – long daily light phases in the circadian cycle support robust reproduction. Thus laboratory mice are permanently kept on an artificially long (12 h of light 12 h of dark) light cycle. Gestation in mice takes around 21 days. When the litter is delivered maternal care behaviour comprises nestbuilding, grooming and lactation, which may vary in different strains of mice [4–8]. What is known and described by recent literature is that the perinatal environment, including prenatal as well as neonatal stress evokes long-lasting effects, especially with regard to stresssensitive aspects [4,7,9–19]. A fortiori it is surprising that no official statements concerning breeding procedures exist are made by scientific societies. Moreover, in fact one has to realize that breeding of experimental animals is mostly motivated by the need of a great amount of offspring, be it on side of the commercial breeders or of scientists. Whether or not this is fruitful for the overall validity of the results is not questioned anywhere. One example of breeding strategies represents the continuous mating (CM) of mice. The intention of this method is to benefit from the postpartum oestrus as female mice present it in natural but also laboratory settings. Thus this strategy requires the permanent presence of a male counterpart that mating can take place directly after delivery of the litter. It also demands from the female to bear the costs—in the truest sense of the word [20,21]. While simultaneously nursing the actual litter with all energy available, the female is stressed by the upcoming pregnancy with all physical challenges that are associated with such a state. Additionally there is no way to cope, e.g. avoid the contact with the male, which could also be potentially dangerous for the youngborn pups [22]. Facing the unsatisfactory awareness that such perinatal conditions, we were interested in elucidating the following hypothesis: CM represents a sustainable form of perinatal stress that is induced by several challenging factors, (i) stress of the dam which is induced by an increase of costs and permanent presence of the male and (ii) stress of the offspring due to the limited energy the mother can raise when taking care of two generations at the same time. To follow up these thoughts our experimental design comprised two parts of investigations. First, maternal behaviour, i.e. nestbuilding, maternal exploration and pup retrieval were analysed. The second set of experiments assessed emotional behaviour of male and female offspring, which were analysed in a behavioural test battery. Emotional behaviour was examined by exploring changes in activity, anxiety, social behaviour as well as fear and depressivelinked aspects. All CM-induced behaviours were consecutively compared with those of dams maintained with one single litter and the behaviour of the offspring.
2. Materials and methods 2.1. Housing and breeding conditions Female mice at an age of 3–4 month were derived from Charles River Germany (Charles River, Sulzfeld) and were experienced in bringing up litters. All mice were housed in Macrolon cages type III (Tecniplast, Italy), which contained wooden chips (ABEDD LTE-001, Lab & Vet
Service, Vienna, Austria), tissue nesting material made of cellulose and food and water ad libitum (Rod16A, Lasvendi, Soest, Germany). Housing of the animals was standardized by 12:12 h dark–light cycle (dark phase: 9.00 a.m.–9.00 p.m.) at a room temperature of 22 ± 2 ◦ C. Humidity was set at 50%. The hygienic status was SPF according to recent FELASA recommendations [23]. Before first mating female mice were allowed to acclimate to the new housing room for 14 days. We examined two groups of mice: Group I (n = 7): control group (CON): mating was performed in Macrolon cages type III with one male (2–3 months) and two female mice (3–4 months). Females were separated from the male when pregnancy was ascertained by positive vaginal plug check and indicative weight changes. Females from then on were housed individually under same conditions outlined above. After delivery dams were left undisturbed till pup retrieval testing at postnatal day (PND) 7. Weaning took place at PND21, from then on brothers and sisters were group-housed in type III cages equipped with wooden bedding and tissue as nesting material and food and water ad libitum (Rod16A, Lasvendi, Soest, Germany). Group II (n = 5): continuous mating group (CM): mating was performed in Macrolon cages type III with one male (2–3 months) and one female mice (3–4 months). Males remained in breeding cages during pregnancy and lactation to generate the CM condition. All other housing conditions were comparable to those of the CON animals. The comparably smaller number of dams in Group CM was caused by cannibalism and disrupted pregnancy. 2.1.1. Maternal behaviour The assessment of maternal behaviour comprised: (i) pup retrieval behaviour (ii) maternal exploration and (iii) nest building. Maternal exploration and nest building performance was studied once a week (exceptionally during light phase between 8.00 and 9.00 a.m.) during pregnancy and during lactation. Pup retrieval testing was conducted unique at PND 7. If indicative weight changes could be observed one week after mating the first maternal care tests were conducted during pregnancy. Thus the maternal care assessment comprised two timeframes during pregnancy and during breeding. To avoid interference with test procedures with accompanying disturbance of dams and litter, tests were conducted within cage change procedures. 2.1.1.1. Pup retrieval behaviour: Pup retrieval test. On postnatal day (PND) 7 the pup retrieval behaviour was tested. Two pups were put into the far corners of the nest in the homecage. The latency until the dam retrieved her pups back into the nest was recorded [24]. The dams had a maximum of 300 s to perform the retrieval. 2.1.1.2. Maternal exploration: Novel cage test. In the context of cage changes once a week, the animals were placed into a new macrolon cage type III containing a layer of new bedding material and the latency as well as total number of rearings were measured for 5 min at the end of the light phase (during breeding period the pups are placed into new cage with the dam before measuring behaviour), (CM group: males were placed after novel cage testing into new cage). 2.1.1.3. Maternal nest building performance: Nest test. After novel cage test the dam with her pups (and for the CM group also the male) remained in the new cage. A nestlet (PLEXX, Arnheim) was introduced into new cage and nest building was analysed by scores of Deacon [25] after 5 and 24 h. Score 1: nestlet untouched, Score 2: nestlet partly picked to pieces, Score 3: nestlet completely picked to pieces, Score 4: recognizable nest, Score 5: complete covered nest.
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The last maternal behaviour testing was conducted when pups aged 2 weeks. With 21 days pups were weaned and gender separated group housed under same conditions outlined above. 2.2. Offspring From weaning till 11th week of life offspring was weighed weekly. Two representative animals/litter (one female and one male) were chosen according to the median of all litter weights per gender. All experimental cohorts consisting of one pup/gender/litter to be representative and had gender-separated been randomized before testing to avoid “litter effects”. Testing (behaviour experiments) started at an age of 8 weeks. Testing last three weeks in total. 24 h after last experiment offspring was decapitated and samples of trunk blood were collected for corticosterone analysis. Behaviour testing was conducted during dark phase of the dark–light cycle to cover the active period of the animals. (number of offspring group CON: female: n = 7; male: n = 7; number of dams group CM: female: n = 5; male: n = 5) Our study complies with the actual regulations on animal experiments in Germany and was approved by local competent authorities (§15 Kommission, Regierungspräsidium Karlsruhe, Abteilung 3: Landwirtschaft, Ländlicher Raum, Veterinär- und Lebensmittelwesen, permit number: G-279/12) and the animal welfare committee of the Interfaculty Biomedical Research Facility of the University of Heidelberg. To minimize distress between the assessment of the relevant parameters, all animal care and testing was carried out by the same person. 2.2.1. Offspring behaviour 2.2.1.1. Weight monitoring. All offspring assigned to be analysed with regard to emotional behaviour in the testbattery was weight weekly when the cages were changed. 2.2.1.2. Behavioural testbattery. To avoid artefacts due to test–test interactions, the sequence of tests in the battery started with the
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least stressful and ended with the most invasive paradigm [26,27] as shown in Fig. 1. All behavioural analyses were carried out during the dark phase to assess behavioural performance in the active phase. Before each test the animals were acclimatized to the behavioural test room for 30 min—except nest test and novel cage test because these tests took place in housing room. 2.2.1.2.1. Nest test. The nest test is a valid procedure to assess this aspect of maternal care performance, but also detects inadequate behaviour in non-breeding mice [25], since nest-building is part of the natural behavioural repertoire [28]. For the analysis of nest quality the nesting material was removed from the home cage 1 h before dark phase (8 a.m.) and a standard nestlet (PLEXX, Arnheim) was introduced. The nest building performance was rated 5 and 24 h later with scores by Deacon [25]. 2.2.1.2.2. Novel cage. The novel cage test assesses the level of vertical exploration in a new environment. To reduce the handling of the mice, this test was performed after the weekly cage change. To make the results comparable to the maternal dams, as well as for practical reasons, the mouse was placed into a new cage type III (825 cm2 ) with clean bedding and the rearings as well as the latency to the first rearing were measured for 5 min. The mouse remained in the new cage and after testing the untested littermates were transferred to the fresh cage as well [29]. 2.2.1.2.3. Openfield/novel object. By means of the openfield test it is possible to detect changes with regard to activity, anxiety and exploration [30]. In our experiments the animal was placed into a 50 × 50 × 50 cm black openfield apparatus placed on an infrared light surface. Four mice are tracked simultaneously for 10 min with a light intensity of 25 lx. With a camera above the arena (Ikegami Digital) Ethovison 4.0 (Noldus Information Technology, Wageningen, The Netherlands) evaluates “distance moved”, “velocity” and the time spent in defined areas of the openfield, i.e. “centre time”. In addition the faecal boli were counted as indicator for emotional stress [31–33].
Fig. 1. Experimental design. Overview about the procedures for (I) control group (CON) and (II) continuous mating group (CM). The course of experiments comprises pre-and postpartal conditions of dams, i.e. conditions with the respective modification of the maternal environment. The course of experiments comprises also pre- and postnatal conditions of offspring in (I) control group (CON) and (II) continuous mating group (CM). Weaning took place at PND21 from when on the development of weight is examined. Behavioural analysis of offspring started at an age of approximately 8 weeks and lasted until the animals were sacrificed at an age of 10 weeks. Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).
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As an expansion of the openfield, a novel object test was conducted. After the first tracked 10 min of habituating to the empty openfield a falcon tube (50 ml) filled with water was placed into the middle of every test arena. The exploratory and neophobic behaviour, respectively, were measured for another 10 min by assessing the latency to approach the novel object up to two centimetres as well as the total number of approaches. Respective arenas were defined by using Ethovision 4.0. Between the different animals the apparatus was cleaned with 70% ethanol. 2.2.1.2.4. O-maze. The O-maze is an elevated circular arena with two open and two closed arms, which is comparable to the frequently used Elevated Plus Maze, and represents a test for anxiety. The O-maze apparatus in our experimental setup is made of rough high-grad steel. The outer diameter is 45 cm, the width of the arms 6 cm, and it is elevated 50 cm above ground. In our experiment anxiety was measured by counting the time on open arms, number of exits and full crosses (one circumnavigation of the Omaze). In total the test lasted 5 min and is performed at 25 lx [34]. Between the different animals the apparatus was cleaned with 70% ethanol. 2.2.1.2.5. Dark–light box. The dark–light box (54 × 22, 5 × 32 L/W/H) consists of a dark (22.5 cm) and a light compartment (31.5 cm) and is used to assess conflict-avoidance behaviour in mice [35]. The light compartment was illuminated by 600 lx. The mouse can switch between the compartments through a 5 × 5 cm aperture. The tests started by placing the mouse into the dark compartment. The latency, the time in, and the number of exits into in the light compartment were measured for 5 min [29]. Between the different animals the apparatus was cleaned with 70% ethanol. 2.2.1.2.6. Social recognition. The self-made Social Recognition test apparatus (50 × 50 × 50 cm) to assess socially induced motivation consists of three compartments (each compartment measures 16.5 cm) which are connected through apertures. The testing procedure comprised four phases: Phase 1: Five minutes of acclimatization with closed apertures (only the central compartment could be explored). Phase 2: The apertures were opened and the mouse could switch between all compartments for 5 min. The first two phases took place without behavioural tracking. Phase 3: A stranger mouse of the same gender was placed into a wire frame cage in the corner of one outer compartment. The opposite external compartment contained an empty wire frame cage. Tracking was conducted for 10 min. The analysis comprised the time spent within the centre and the external compartments, respectively. Additionally the time spent in close vicinity of the wire frame cages was measured. Phase 4: The empty wire cage was exchanged with a wire cage containing a novel stranger to observe the “social memory” of the animals. Additionally the compartments were changed, so the novel stranger (second stranger) was then placed into the compartment of the previously first stranger and conversely the first stranger was now in the compartment of the previously empty cage. Phase 4 lasted another 10 min. The entire test procedure was conducted at a light intensity of 25 lx. 2.2.1.2.7. Forced swim test. The forced swim test, originally established and validated by Porsolt [36], is a test to assess depressive-like behaviour in rodents. In our study, a mouse was placed into a 2000 ml beaker filled with 1000 ml 21 ◦ C cold water. Floating, i.e. complete lack of movement, and the latency to first display this behaviour interpreted as “giving-up”, was measured under redlight conditions by means of Ethovision Software for 6 min. Afterwards the mouse was placed back into its home cage and allowed to dry in front of a redlight for 10 min. After 24 h the mouse was retested under the same conditions [29].
2.2.1.2.8. Hotplate test. The Hotplate (Ugo Basile Srl) is used to determine pain threshold consists of a plate which is adjustable from 2 to 66 ◦ C and a glass cylinder to prevent the mouse from jumping off the plate. The temperature in our setup was 52 ◦ C. The test took place at 100–130 lx for max. The test took 45 s and latency till licking of hind paws was measured. When licking hindpaws was observed, the mouse was immediately removed from the Hotplate. The Hotplate test was performed to identify possible artefacts due to different pain thresholds induced by different perinatal stress conditions [37]. Between the different animals the apparatus was cleaned with 70% ethanol. 2.2.1.2.9. Emotional learning: Fear conditioning. For the analysis of hippocampus- and amygdala- associated emotional learning, the Fear Conditioning system was used (TSE systems, Bad Homburg, Germany). The paradigm applied in our study comprised three days of exposition. Day 1: shock: For initial conditioning on the first day, the mouse was placed into a cylinder onto a grid. After 120 s of habituation, followed by a 30 s long tone (conditioned stimulus, 2800 Hz, Intensity 67), a brief foot shock was applied (unconditioned stimulus, 2 s, 0.8 mA). Intertrial cleaning was performed with 70% Ethanol. Day 2: context: For context, the mouse was placed 24 h after Day 1 into the same experimental chamber onto the grid for 5 min without any stimulus. To analyze context conditioned freezing, i.e. complete lack of movements apart from respiration, freezing behavior was evaluated every 10 s and noted as yes or no (freezing). During this phase of the test, the apparatus was cleaned with 70% ethanol to ensure a comparable olfactoric context to day one. Day 3: cued: For the assessment of cued conditioning, freezing behaviour was analyzed in a different context. Here the mouse was placed in the same chamber but within another context into a different rectangular cylinder with a flat surface (to ensure an altered tactile context), which was cleaned with chamomille tea to additionally provide a different olfactoric context. After 180 s of habituation to the new context, the conditioned stimulus (2800 Hz, Intensity 67) was presented for 180 s. Freezing was evaluated as stated above. Score was calculated by means of percentage freezing/time of test duration. 2.2.1.2.10. Corticosterone analysis. For corticosterone analysis, trunk blood was obtained by conscious decapitation within 30 s of removing the animal from the cage. Anaesthesia was not used to avoid effects on corticosterone secretion. Samples were centrifuged for 5 min by 3500 rpm and plasm (supernatant) was collected and stored by −20 ◦ C till analysis. Samples were determined by radioimmunoassay (ICN Biomedicals, Eschwege, Germany); assay sensitivity was 12.5 ng/ml.
2.2.1.3. Statistical analysis. For statistical analysis the values of maternal behaviour were evaluated by one-way ANOVAS. Offspring behaviour was analyzed by means of two-way ANOVAS and subsequent Tukey Posthoc testing. Weight assessment was calculated by repeated measurement ANOVAS. Statistically significant effects were set at a p ≤ 0.05. To evaluate statistic differences the “InVivoStat” (invivostat.co.uk) statistical software was used.
3. Results Table 1 illustrates all tests conducted for maternal behaviour during pregnancy and breeding and illustrates significant and nonsignificant results. Table 2 shows all tests conducted in offspring with respectively significant and non-significant effects on offspring behaviour.
S. Lerch et al. / Behavioural Brain Research 279 (2015) 155–165 Table 1 Maternal behaviour: evaluation of “CM” in dams evaluation of the factor “CM” in all tests for maternal behaviour during pregnancy (prepartal conditions) and nursing (postpartal conditions). The order of the data reflects the order of the tests as they were conducted. Parameters not appearing in this table revealed non-significant results. Evaluation of “continuous mating”
Table 2 Offspring behaviour: evaluation of “CM” and “gender” effects in offspring Evaluation of the overall effects of “CM”, “gender” and interaction effects in all tests conducted in offspring. The order of the data presented reflects the order of all tests as they were conducted. Parameters within tests not appearing in this table revealed nonsignificant results. Evaluation of “continuous mating” and “gender” effect
Test
Timepoint
Parameter
NC NT NC NT PRT
1st Week of pregnancy 1st Week of pregnancy 2nd Week of pregnancy 2nd Week of pregnancy 1st Week of nursing (PND7) n.s. 1st Week of nursing 1st Week of nursing 2nd Week of nursing 2nd Week of nursing 2nd Week of nursing
n.s.
Littersize NC NT NC NT NT
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5 h [scores] Time till retrieval [seconds]
Effects
Test
Parameter
Effects/ interactions
Posthoc test (Tukey)
Weights
5th Week of life [g]
n.s.
NC
Rearings [number] * CM p = 0.001
*
T m CM ↓ m CON ↑ p = 0.086 n.s.
*
CM p = 0.048 n.s.
NT
5 h [scores]
n.s.
5 h [scores] 24 h [scores]
** *
24 h [scores]
CM p = 0.004 CM p = 0.035
n.s. = no significance; CM = continuous mating; NC = novel cage; NT = nest test; PRT = pup retrieval test. Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001). The order of the results reflects the order of the tests as they were conducted.
3.1. Maternal behaviour
3.2. Offspring behaviour 3.2.1. CM did not affect weight development Regular weekly weight development could be observed when pups of either gender were reared under CM conditions. Only a tendency in the 5th week of life within males could be identified (p = 0.086). There was a significant gender effect with increasing significance levels over the time [F1,24 = 50.09; p = 0.001] (see Fig. 3). 3.2.2. CM decreased exploration of male and female offspring in the novel cage test There was a visible overall “CM” effect on exploration behaviour (number of rearings) which occurred in the novel cage test [F1,24 = 5.05, p = 0.036]. The latency to start rearing was not affected (data not shown). 3.2.3. CM decreased nestbuilding performance in female and male offspring There was a significant overall effect of “CM” after 5 [F1,24 = 13.81; p = 0.001] and 24 h [F1,24 = 20.06; p = 0.001] in the nest
CM p = 0.001
OF
Fecal Boli [number]
NO
Visits [number]
OM DLB
3.1.1. CM increased nest building behaviour in second week of pregnancy The presence of a male mouse in the breeding cage affected maternal nest building quality in the nest test after 5 h in the second week of gestation. Pregnant CM dams presented an increased nest score of 3.8 in comparison to CON dams with a nest score of 3.5. [F0,12 = 5.44; p = 0.048] (see Fig. 2). 3.1.2. CM enhanced nest building behaviour in second week of nursing During the nursing period the quality of nests was increased after 5 [F0,12 = 14.63; p = 0.004] and 24 h [F0,12 = 6.14; p = 0.035]. Nursing CM dams presented a nest score of 4.6 after 5/24 h, CON dams demonstrated a nest score of 1.8 after 5 and 3.3 after 24 h (see Fig. 2). Maternal exploration as assessed in the novel cage test as well as pup retrieval were unaffected by CM conditions (see Table 1). The assessment of littersize of CM and CON group revealed no differences. The average number of mothers of CON group was 9.7 pups/litter and mothers of CM group gave birth to 8.6 pups/litter.
*
Latency 1st exit [s] Time spent outside [s] Phase 3
Phase 4
FST
Immobility day 1 [s] Immobility day 2 [s]
HP FC Stressphysiological analysis
n.s. * CM p = 0.02
*
Gender p = 0.044 Entries empty compartment [number]
Time in compartment stranger 1 compartment [s] Time at cage stranger 1 compartment [s] Time at cage stranger 2 compartment [s] * CM p = 0.001
*
CM p = 0.03
n.s. n.s. Corticosterone [ng/ml]
CM p = 0.036
f CM ↓ f CON ↑ * p = 0.025 m CM ↓ m CON ↑ ** p = 0.002 * CM p = 0.01
*
Gender p = 0.034
m CM ↑ m control ↓ * p = 0.029 n.s.
f CM ↑ f CON ↓ * p = 0.049 n.s. * Gender: CM p = 0.015
*
Gender p = 0.001 * Gender:CM p = 0.008 *
Gender p = 0.008 Gender:CM p = 0.037 * Gender p = 0.027 *
f CM ↓ m CM ↑ * p = 0.046 m CM ↑ m CON ↓ * p = 0.04 f CON ↓ m CON ↑ *** p = 0.001
f CON ↓ m CON ↑ ** p = 0.007 n.s.
f CM ↑ f CON ↓ ** p = 0.009 n.s.
*
Gender p = 0.025
f CON ↓ m CON ↑ * p = 0.049
Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001). The order of the results reflects the order of the tests as they were conducted.
test. Posthoc testing revealed significant nest building deficiencies after 5 h in CM females (p = 0.025) (Fig. 4a) and after 24 h in CM males (p = 0.002) (Fig. 4b). 3.2.4. CM increased the number of fecal boli in male offspring in the openfield The factor “CM” exerted an overall effect on “faecal boli” as an indicator for emotionality in openfield testing [F1,24 = 8.0; p = 0.01]. Posthoc testing revealed a significantly increased number of fecal boli in CM raised males (p = 0.029). Subsequent novel object testing showed an overall “gender” specific neophobic behaviour
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Fig. 2. Maternal behaviour/care. CM dams show better nest building performance after 5 h in 2nd week of pregnancy and after 5 and 24 h in 2nd week of nursing. Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).
Fig. 3. Weight development after weaning. Female offspring raised under CON conditions shows slightly higher weights compared to female CM offspring without statistical significance. Both, CON and CM demonstrate regular weight development over the time, however overall weight development was only affected by “gender”. Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).
[F1,24 = 5.22; p = 0.034] (Fig. 5). Other, i.e. activity-related parameters in the openfield were unaffected by CM. 3.2.5. CM enhanced anxiety of female offspring in the dark–light box While O-maze testing did not uncover any anxiety-related differences, dark–light box testing revealed an apparent overall effect of “CM” on the latency of first exit [F1,24 = 6.44; p = 0.02]. Posthoc testing showed a female-specific significant increase in the latency to enter the bright compartment when reared by CM dams (p = 0.049) (Fig. 6). Moreover the parameter “time spent outside” indicated a “gender” specific difference [F1,24 = 4.64; p = 0.044] (data not shown). The factor “CM” by itself did not affect this parameter.
3.2.6. CM decreases social interest and social recognition Entries into the empty compartment (Phase 3) indicating initial exploration, revealed an interaction of both factors, “gender” and “CM” [gender:CM: F1,24 = 7.09; p = 0.015] with significant Posthoc testing. Tukey Posthoc analysis here demonstrated decreased exploration in females when raised under CM conditions (p = 0.046). Contrary, male CM offspring presented more entries/increased exploration into the empty compartment (p = 0.04) (Fig. 7a). When an unfamiliar mouse was introduced for the first time (Phase 3, empty + Stranger 1) two overall effects occurred regarding “time in compartment Stranger 1”. There was an overall effect of “gender” [F1,24 = 13.55; p = 0.001]; and “gender:CM” [F1,24 = 8.7;
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Fig. 4. Nest test—(a) 5, (b) 24 h. (a) Female CON offspring shows higher nest building scores after 5 h than female CM offspring in the nest test. (b) CON male offspring shows higher Nest building scores after 24 h than male CM offspring. Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).
Fig. 5. Openfield-fecal boli Males reared under CM conditions demonstrate an increased number of fecal boli in contrast to males reared under CON conditions. Females present comparable numbers of fecal boli Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).
Fig. 6. Dark–light box—latency 1st exit. Female CM offspring demonstrate a higher latency to leave dark compartment of the Dark-light box compared to female CON offspring. Male mice do not show statistically significant differences. Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).
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Fig. 7. Social recognition—Phase 3: (a) Entries into empty compartment: male offspring raised under CM conditions shows increased exploration/more entries into compartment with empty cage, compared to CM females and males CON in Phase 3. (b) Time at compartment Stranger 1: male CON offspring shows an increased motivation to approach compartment of Stranger 1 in Phase 3. (c) Time at cage Stranger 1: in addition to (b) the “time at cage of Stranger 1” reveals an higher resting time of male CON offspring. Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).
p = 0.008]. Again Posthoc analysis indicated an increased motivation to approach compartment of Stranger 1 in male CON mice (p = 0.001) (Fig. 7b). As a consequence thereof the “time at cage stranger 1” (Phase 3) was influenced by “gender” [F1,24 = 8.62; p = 0.008] and there was an interaction between “gender” and “CM” [F1,24 = 5.01; p = 0.037] with significant Posthoc testing revealing increased interest to approach the newly introduced animal in male CON mice (p = 0.007) (Fig. 7c). When a second Stranger (Phase 4) was introduced, the parameter “time at cage stranger 2” was affected by “gender” [F1,24 = 5.68; p = 0.027]. There were no significances in the Posthoc tests (data not shown).
3.2.7. CM promoted depressive-like behaviour in the forced swim test in females Day 1: The “Immobility” on Day 1 was affected by an overall effect of “CM” [F1,24 = 15.57; p = 0.001]. Tukey Posthoc Test showed an increased immobility of females (p = 0.009) (Fig. 8). The latency to start floating was unchanged. Day 2: “Immobilty” on Day 2 was influenced by “CM” [F1,24 = 5.48; p = 0.03] but there were no significances in Posthoc tests (data not shown). The latency to start floating was also unchanged.
3.2.8. Corticosterone determination did not reveal any digestible results Determination by radioimmunoassay showed significant results regarding the overall effect of “gender:CM” [F1,15 = 5.47; p = 0.039]. Due to a small number of samples which were detectable in the radioimmunoassay we didn’t pursue such findings. 4. Discussion This study was conducted to determine whether specific breeding conditions affect (i) maternal behaviour and (ii) the emotional/depressive-like phenotype of male and female offspring. To address this question we kept mouse mothers and their litters under two different conditions: (I) control (CON) conditions, i.e. one single-housed mother raising one litter or (II) continuous mating (CM) conditions, which comprised pregnancy, lactation of the previous litter and presence of the sire at the same time. In a first step we assessed maternal exploration and nest building behaviour. Later on we exposed the adult offspring to a behavioural test battery to analyze the emotional and depressionassociated status. Our data indicate that the breeding environment indeed plays a role at both levels—maternal care and offspring performance. Most interestingly not only basal behaviours, but also depressive-like features were affected in a significant manner with
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Fig. 8. Forced swim test. Female CM offspring show higher depressive-like behaviour, i.e. increased immobility on Day 1 of the forced swim test compared to female CON offspring. Males do not show alterations with regard to immobility. Asterisks indicate the level of significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).
females presenting an increased immobility in the forced swim test, a fact that is rather alarming. This could, however, not be referred to a generally decreased performance of the mother since CM dams exhibited even increased nest building performance during pregnancy and nursing. Such a finding is somehow surprising since we expected that CM represent a stressful environment rather compromising maternal behaviour. What may be postulated is an increased motivation to provide shelter to the newborns or that males by themselves build the nests, which is part of their behavioural repertoire [25,28,38,39]. Maternal exploration and pup retrieval were similar in both groups indicating that CM did not exert broad effects, at least with regard to the parameters examined here. Interestingly and in line with our hypothesis nearly all tests beside our depression-paradigm revealed significant findings in mouse offspring. When the animals were exposed to a novel cage, “CM” affected exploration of the novel environment. In the nest test effects could be found after 5 (females) and 24 h (males). CM females herby built poorer nests after 5 h and CM-males built poorer nests after 24 h. This is fairly interesting since the decreased motivation of nest building again cannot be related to a decreased functioning of the mothers. What could be speculated therefore is, that housing of mouse pairs increases nest building performance. Alternatively one could assume a decreased welfare considering recent literature, e.g. by Gaskill et al. who identified nest building as indicator for the animals’ wellbeing [40], but it can also be speculated that other facets in maternal care exerted respective impairments [8]. When behaviour of CM and CON offspring was investigated in the openfield, “CM” exerted an overall and a male-specific effect on the number of fecal boli, a measure for enhanced emotionality [32,41]. This effect was not present in females. In the dark–light box CM females were affected regarding the latency to 1st exit into the bright compartment presenting increased anxiety and a greater variance within the group. The “gender” effect considering the time the mice spent outside in the bright compartment is not specifically surprising since it is known that male and female mice respond differently with regard to anxiety-related features [42]. With regard to social recognition our analyses demonstrate a generally decreased (social) exploration in females. This decreased investigation when it comes to an unfamiliar animal was, however, not affected by “CM”, but seems to be rather a gender-specific issue.
From our point of view and as stated at the beginning of the discussion the most exciting finding of our study was found in the forced swim test. Here both days of testing revealed a significant overall effect of “CM” on floating behaviour, i.e. immobility a correlate for despair-behaviour. Again in this paradigm it were the females exhibiting a significantly enhanced depressive-like behaviour. Keeping in mind that research in the field of experimental psychiatry still explores valid animal models in which a number of depressive-like features occur together to closely mimic a depressive symptom in humans, these results are rather worrying. If breeding conditions per se lead to changes in emotionality and depressive-like features this bears an immense impact with regard to both, valid scientific results and the animal welfare thought. To examine a stress-physiological correlate, we also assessed corticosterone from blood plasma. While we expected an increase of corticosterone indicating a dysregulation of the hypothalmus pituitary adrenal (HPA) system there were no such effects. Also of interest for our study was the fact whether emotional learning was influenced by early perinatal conditions. This was however not the fact when tested in context and cued conditioning in the Fear Conditioning paradigm. Since pain sensitivity was not affected, neither by the factor “CM” not “gender” artefacts were excluded. The overall validity of our testbattery and potential test interactions were considered by starting with less stressful analyses while more stressful tests were set to the end [26,27]. A very interesting finding beside the general effect of CM on most and especially depression-associated parameters was the obviously increased sensitivity of females. Female offspring raised in a CM environment had impairments in nest building, an essential feature especially in this gender, increased anxiety and presented a depressive-like phenotype in the forced swim test. While this could be very attractive for research groups dealing with depression models, it also has to be considered in the context of animal welfare. When animals are used for scientific purposes it is obligatory to provide best possible conditions to the animals and minimize stress and discomfort [43]. Continuous mating is a method of breeding in which due to postpartum oestrus high numbers of animals can be bred. However if side effects are likely to take an influence on the results because of increased emotionality, this should definitely taken into account and requires a well-considered appreciation of values. What anyway becomes clear is that it is
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immensely important to explicitly report on how animal studies were conducted and imply all background information, including breeding conditions, e.g. according to the ARRIVE guidelines [44–47]. Only the entire contemplation of a model organism allows valid and reproducible results and interpretation of data for sustainable research and human conditions for animals used for all disciplines of science. 5. Conclusion Our study demonstrates that the maternal environment in common breeding conditions exerts long-lasting effects on the emotional status of male and female offspring. While this may be suggested as attractive approach when embedded in neurobiological research it may on the other hand threat studies in which stress is unwanted and could cause artefacts. With regard to a long-range perspective a healthy life relies upon early experiences and adaptations can be made adequately [48,49]. With disadvantaged early conditions like postulated by our CM paradigm there is an increased risk of stress and maladaptation. Therefore these findings need to be likewise considered by researchers, animal facilities and commercial breeders to ensure high quality science as well as responsible implementation of justifiable animal welfare. Acknowledgements We thank Charles River (Sulzfeld, Germany) for providing us with the mice used in this study as well as the animal caretakers of the clinic experimental facility of the IBF for their support. References [1] Rosenthal N, Brown S. The mouse ascending: perspectives for human-disease models. Nat Cell Biol 2007;9:993–9. [2] Hardouin SN, Nagy A. Mouse models for human disease. Clin Genet 2000;57:237–44. [3] Festing MF. Inbred strains should replace outbred stocks in toxicology, safety testing, and drug development. Toxicol Pathol 2010;38:681–90. [4] Caldji C, Diorio J, Meaney MJ. Variations in maternal care in infancy regulate the development of stress reactivity. Biol Psychiatry 2000;48:1164–74. [5] Chourbaji S, Hoyer C, Richter SH, Brandwein C, Pfeiffer N, Vogt MA, et al. Differences in mouse maternal care behavior—is there a genetic impact of the glucocorticoid receptor? PLoS One 2011;6:e19218. [6] Catalani A, Marinelli M, Scaccianoce S, Nicolai R, Muscolo LA, Porcu A, et al. Progeny of mothers drinking corticosterone during lactation has lower stressinduced corticosterone secretion and better cognitive performance. Brain Res 1993;624:209–15. [7] Coutellier L, Friedrich AC, Failing K, Wurbel H. Variations in the postnatal maternal environment in mice: effects on maternal behaviour and behavioural and endocrine responses in the adult offspring. Physiol Behav 2008;93:395–407. [8] Cox KH, So NL, Rissman EF. Foster dams rear fighters: strain-specific effects of within-strain fostering on aggressive behavior in male mice. PLoS One 2013;8:e75037. [9] Anisman H, Zaharia MD, Meaney MJ, Merali Z. Do early-life events permanently alter behavioral and hormonal responses to stressors. Int J Dev Neurosci 1998;16:149–64. [10] Branchi I, D’Andrea I, Fiore M, Di Fausto V, Aloe L, Alleva E. Early social enrichment shapes social behavior and nerve growth factor and brainderived neurotrophic factor levels in the adult mouse brain. Biol Psychiatry 2006;60:690–6. [11] Casolini P, Cigliana G, Alema GS, Ruggieri V, Angelucci L, Catalani A. Effect of increased maternal corticosterone during lactation on hippocampal corticosteroid receptors, stress response and learning in offspring in the early stages of life. Neuroscience 1997;79:1005–12. [12] Catalani A, Casolini P, Cigliana G, Scaccianoce S, Consoli C, Cinque C, et al. Maternal corticosterone influences behavior, stress response and corticosteroid receptors in the female rat. Pharmacol Biochem Behav 2002;73:105–14. [13] D’Amato FR, Zanettini C, Lampis V, Coccurello R, Pascucci T, Ventura R, et al. Unstable maternal environment, separation anxiety, and heightened CO2 sensitivity induced by gene-by-environment interplay. PLoS One 2011;6:e18637. [14] Franklin TB, Linder N, Russig H, Thony B, Mansuy IM. Influence of early stress on social abilities and serotonergic functions across generations in mice. PLoS One 2011;6:e21842. [15] Gheorghe CP, Goyal R, Mittal A, Longo LD. Gene expression in the placenta: maternal stress and epigenetic responses. Int J Dev Biol 2010;54:507–23.
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