Daskalakis, N.
Citation
Daskalakis, N. (2011, December 8). Nurturing nature : testing the three-hit hypothesis of schizophrenia. Retrieved from https://hdl.handle.net/1887/18195
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Addendum
Environmental and tactile stimulation modulates the neonatal handling effect on adult rat spatial memory.
Nikolaos P. Daskalakis
1,2, Maria Kaperoni
1, Christos Koros
1, E. Ronald de Kloet
2, and Efthimia Kitraki
1,31
Laboratory of Histology & Embryology, University School of Medicine, University of Athens
2
Division of Medical Pharmacology, Leiden/ Amsterdam Center for Drug Research, Leiden University Medical Center, Leiden University
3
Department of Basic Sciences, School of Dentistry, University of Athens
Int J Dev Neurosci. 2009 Dec;27(8):747-55.
Abstract
Handling of rat pups promotes their adult cognitive performance. However, new data suggest that individual components of the handling procedure, like exposure to novelty or tactile stimulation, have distinct lasting effects on behaviour. In this study we examined the interaction of early novelty exposure with a varying amount of tactile stimulation on spatial recognition memory and corticosterone secretion of adult male and female rats. A split litter design was used and the experimental animals were also compared to animal facility reared controls.
The experiment was conducted in two phases. In the first phase, we examined the effect of novel or home environment during the 15-min of neonatal handling, following 10 back-strokes. Tactile stimulation of 10 back-strokes combined with novelty exposure, enhanced novel arm discrimination in a Y-maze task in adult rats of both sexes compared to their siblings that stayed at
home, as well as to the animal facility reared controls. In the second phase, additional back-stroking (total of 20 back-strokes) reduced the Y-maze performance of males neonatally exposed to novelty, while the same treatment enhanced the performance of their siblings that stayed at home. Basal corticosterone levels, determined one week post Y-maze, were significantly increased only in the novelty exposed/10 back-stroked females compared to same sex non-handled controls. In contrast, 10 back- strokes combined with the home cage environment increased corticosterone in males. Increase to 20 back- strokes reversed the impact of neonatal environment on corticosterone levels.
These data suggest that the nature and intensity of the individual components of a mild early-life manipulation, like handling, are critical in modifying aspects of adult memory performance and basal adrenocortical function.
1. Introduction
There is accumulating evidence that adult rats exposed as pups to early-life experience during the perinatal period show altered behaviour and neuroendocrine function [1, 2]. For instance, an adverse experience, like prolonged maternal separation of 24 hours, impairs later life cognitive performance and enhances adrenocortical and emotional reactivity. On the contrary, mild manipulations, like neonatal handling, can lead, via developmental programming, to animals with a better coping with stressful situations later in life [1, 3-7]. Such handled animals, which are separated from their mother daily for 15 minutes over the first three weeks of life, exhibit as adults reduced anxiety. In response to a mild stressor they have a lower output of corticosterone, prolactin and adrenaline [7-10]. On the other hand, handled animals have increased levels of glucocorticoid receptors in the hippocampus and frontal cortex [11-13].
Accordingly, their limbic - hypothalamic–pituitary–adrenal (LHPA) axis feedback
sensitivity to corticosterone is enhanced, providing a more efficient termination of the
stress response. The aforementioned features of handled animals have been linked to
A
their generally improved performance in cognitive tasks [14, 15].
Neonatal handling consists of at least three different manipulations: brief maternal separation (up to 15min), touching (tactile stimulation) by the experimenter, and exposure to novelty. Firstly, the short term maternal separation eliminates olfactory cues as well as the tactile stimulation by the mother, for which the latter compensates at reunion [16, 17]. Secondly, touching by the experimenter during the separation can, to some extent, mimic the maternal tactile stimulation, but the various protocols are different in this respect. Finally, the additional exposure to novelty offers new olfactory and visual stimulation (from 2nd week on). Although the effect of neonatal handling on the adult animal’s physiology and behavior has been extensively studied since the mid
‘50s [6], only few studies have examined the impact of its separate components.
The established handling paradigm [7] was modified by introducing a split litter design, for studying the impact of exposure to novelty separately [18]. In this model, all pups are experiencing daily a short term separation from their mother as well as the experimenter’s touching. However, only half of the pups in each litter are exposed individually to novelty, while the rest are left in their home cage. The adult male rats, exposed to novelty exposure as part of this handling protocol, exhibited as adults enhanced spatial and non-spatial memory [18, 19] and showed increased hippocampal LTP [20]. More recently, by using the same paradigm [21], it was shown that brief exposure of pups to an unfamiliar environment produced in adult males an attenuated fear and stress response, as well as diminished sexual behavior. Females have been scarcely studied in this paradigm. Juvenile and adult female rats, that had experienced as pups prolonged absence of the mother while being placed in a novel environment, were less reactive to stressors as compared to female pups that stayed in their nest during maternal absence [22]. However, others did not observe this difference using the same protocol [23].
During prolonged (24h) maternal separation, artificial tactile stimulation of male pups, especially in the anogenital region, has been shown to reverse the central effects of separation in rats, while additional feeding restored corticosterone secretion [24-26].
Tactile stimulation can also enhance the maturation of cortical neurons [27], improve passive avoidance response [28] and spatial working memory [29]. In paradigms of one hour maternal separation, tactile stimulation of the dorsal area has been shown to reverse the effect of separation on anxiety-like behavior and pain sensitivity in adult rats of both sexes [30]. The lasting behavioural effects of tactile stimulation during the handling paradigm have not been studied.
The aim of this study was to examine the impact and the interaction of early novelty
exposure with a varying amount of tactile stimulation on spatial recognition memory
and basal corticosterone secretion of adult male and female animals (Fig. 1A for an
overview).Therefore the experiment was conducted in two phases. We first examined
the effect of novel or home environment, following 10 back strokes, on the above
parameters. The outcome was further investigated with an additional amount of tactile
stimulation (total of 20 back-strokes) preceding the daily novelty or home exposure.
In both phases, a split litter design was used. The experimental animals were also compared to non-handled, animal facility reared (FR) controls.
2. Materials and Methods
2.1 Subjects
18 female and 6 male Wistar rats (obtained at 6 weeks of age from Hellenic Pasteur Institute, Athens, Greece) were used for breeding. After a habituation period of two weeks, 3 females were put in a cage with one male rat Type IV (macrolon-polycarbonate cages with wire lid; 60 x 38 x 20 cm; containing sawdust bedding). Pregnancy was determined by vaginal smears obtained every morning and pregnant females were transferred in individual cages (home cage: macrolon- polycarbonate type III cages with wire lid; 42.5 x 26.6 x 18.5 cm) containing sawdust. The day of birth was defined as postnatal day 0 (= PND 0). We accepted litter with a maximal excess of one male or female and our litter size was 7-9 pups. The pups of experimental groups were exposed to neonatal manipulations from PND1 to PND21. In the control litters, the pups remained undisturbed with their dams in the housing room until the time of behavioral testing, receiving only the animal facility rearing (change of cage once per week; starting at PND10). In total, offspring of 13 litters was used in this study.
All animals were weaned at PND22, group housed with the same sex littermates and received the normal animal facility rearing afterwards. At PND90 they were tested in the Y-maze task and one week later they were sacrificed under basal conditions. Estrus phase was detected in sacrificed females by vaginal smears. Rats were housed under a 12:12 h light/dark cycle (lights on at 07.00 h, illumination inside the cage: 20-30 lux, temperature: 22 ± 2 °C, relative humidity 60 ± 10%).
Food and tap water were provided ad libitum. All animal treatments were approved by the local ethical committee and have been carried out in agreement with ethical recommendation of the European Communities Council Directive of 24 November 1986 (86/609/EEC). All efforts were made to minimize the number of animals used and their suffering.
2.2 Early-life manipulations
Early-life manipulations consisted of 4 consecutive steps (see in Fig. 1B a detailed description):
i. Dams’ transfer out of the litter. Between 14:00 and 15:00h, dams were removed from their cage and placed in a same type cage, in the same room, at a distance from the home cage.
ii. Back-painting (tactile stimulation): In Experimental Phase I, back-painting, lasted 1min per pup with 10 passages of the marker (10 strokes, 10S). In Experimental Phase II, back- painting lasted 1min per pup with 20 passages of the marker (20 strokes, 20S).
iii. Novelty exposure (environmental stimulation) of 3min daily. Exposure to novelty was performed according to Tang [18]. Home and Novel pups within each experimental phase (see following section) were daily experiencing the brief maternal separation and the same amount of tactile stimulation. Novel pups were additionally exposed to a different context, compared to home pups, namely the novel environment in isolation from their littermates.
iv. Dams’ reunion.
All animals with the exception of AFR were back-painted. From day 2 to 21, the animals were
A
identified (on step 2) by the experimenter by the different colour in the back that was renewed daily. The two markers used had the same odour.
2.3 Experimental design
To study the impact of early novelty exposure on spatial recognition memory and basal corticosterone secretion of adult male and female animals we used a (treatment) x (sex) design and the treatment was comprised of three levels with a control group (animal facility rearing), a home group (back-stroked in the home cage), and a novel group (back-stroked and exposed to novelty).
To study, additionally, the interaction of early novelty exposure with a varying amount of tactile stimulation we conducted the experiment in two phases:
- In Phase I, 21 male and 21 female pups from 6 different litters received, on their back, 10 strokes (10S) during back-painting, with or without novelty exposure (‘10S-Novel’ and ‘10S-Home’ groups, respectively). Maternal separation lasted in total 15min.
- In Phase II, 16 male and 13 female pups from 4 different litters received on their back 20 strokes (20S) during back-painting, with or without novelty exposure (‘20S-Novel’ and ‘20S-Home’ groups, respectively). Maternal separation lasted in total 15min.
Early life manipulations Weaning Decapitation
in basal conditions Y- maze test
PND97 PND90
PND1-21 PND22
N N N N H H H H DAM
DAM
N N N N H H H H DAM N N
N N
H H H H ii. 1-min Back-painting
H H H H
TIME
i. Dam out iv. Dam back
N N N N
N N N N H H H H
iii. 3-min Novelty exposure
min0 1
min 14
min 15
10 min min
Figure 1. Experimental methods. (A) Time line of longitudinal study. (B) Sequential steps of early-life manipulations. (i) “Dam out”. The dams were transferred out of the litter. This step took approximately 1min (as indicated in the clock). (ii) Back-painting (tactile stimulation): Back-stroking, consisting of passages of a marker tip over the pups’ back, was used as daily tactile stimulation;
started 1min after the dam was removed and lasted 1min per animal. For all the pups of a litter, this step took approximately 9min (as indicated in the clock). (iii) Novelty exposure (environmental stimulation): Exposure to novelty was performed according to Tang [18] and started 10min after the dam was removed. A split-litter design was used so that half of the animals in each litter
(painted with a certain color during stroking) were put individually in “novel” cages (macrolon- polycarbonate 18 x 20 x 14 cm; containing fresh sawdust bedding), under heating lamps for 3min (Novel animals). The other half of the litter (painted with another color during stroking) remained for 3min in the home cage under heating lamps (Home animals). After 3min the Novel pups were transferred back to their home cage. This step took approximately 4min (as indicated in the clock).
During the transfer of Novel (back and forth), the home pups were also equally touched. (iv)
“Dams’ back” (dams’ reunion): Finally, the dams were returned in the home cage. This step took approximately 1min (as indicated in the clock).
A
B
In both phases of the experiment, the experimental animals were also compared to AFR controls.
Control litters were the same group of animals in both phases and consisted of 10 male and 13 female pups that derived from 3 different litters. For a synopsis of the different experimental groups according to the early-life manipulations see Table 1.
2.4 Behavioral Testing – Y-maze test
The animals’ ability for spatial recognition memory was assessed at PND 90 by using the Y-maze test [31, 32]. In brief, a black, wooden Y-maze apparatus, having three identical arms (50 x 16 x 32 cm) was used. Extra-maze only, visual cues (posters and laboratory glassware equipment) were placed around the maze and above the top of the black wooden sides. No visual cues were placed inside the maze, and the extra-maze cues were not equally visible from all points within the maze, thus enabling spatial orientation. The investigator was not visible by the rat. Rats were tested during the light phase of their cycle (between 9:00-13:00 h). The testing room was next to animals’ room. The floor of the maze was covered with sawdust bedding that was renewed for each animal using a mixture of sawdust that differed for males and females. The task consisted of two trials having a 4h intertrial interval. The same arm was always designated as a start arm, where the animal was introduced facing the wall of the arm. The other two arms were alternating as “Novel” and “Other” arm between rats to reduce arm-bias effects. In Trial 1, the novel arm was blocked with a black wooden guillotine. At the start of testing, a rat was placed in the start arm and the rat was allowed to explore the start and other arms for 15min. At the end of Trial 1, the rat was returned to its home cage and the bedding inside the maze was mixed, to reduce the possibility of using odors as a cue. In Trial 2, all arms were accessible and the rat was allowed to explore them for 5min. The animals were recorded with a video-camera adjusted on the ceiling of the room. The videos were analyzed blindly by two independent observers by using a computer software [33]. The dependent variables measured in the 1
stand the whole 5min period of Trial 2 were: a) number of total entries b) % entries in each arm, c) % duration in each arm and in the center of the apparatus. Entry into an arm was counted when the head and two front paws were inside the arm, and the duration of an arm visit ended when the head and two front paws were outside of the arm again.
2.5 Corticosterone measurement
One week following Y-maze testing, the animals were decapitated under basal conditions between 13.00-15.00 h. Trunk blood was collected in heparinized tubes on ice and centrifuged at 14.000 rpm, for 5min, at 4 °C. Plasma was collected and kept at -80
oC until use. Corticosterone levels were
ELM
Group name
CON 10S-HOME 10S-NOVEL 10S-HOME 10S-NOVEL
Experimental Phase I Experimental Phase II
Tactile Stimulation PND 2-21 Novelty exposure PND 2-21 Number of animals per group
Animal Facility
Rearing 10 back-strokes
daily 10 back-strokes
daily 20 back-strokes
daily 20 back-strokes daily
- - + - +
20 (10♂13♀) 21 (10♂11♀) 20 (11♂9♀) 15 (8♂7♀) 14 (8♂6♀)
Table 1
Synopsis of the experimental groups according to the different early-life manipulations (ELM)
Note: PND, postnatal day
A
determined by using a standard radioimmunoassay kit for small rodents (MP Biomedicals, LLC, NY, USA; sensitivity 1.25 ng/ml, intra-assay variation, 4.4%, interassay variation 6.5%).
2.6 Data analysis
Animals’ behaviour in Y-maze was analyzed in the 1
stmin and in the whole-5min- period of Trial 2, as it has been reported that animals might gradually loose their interest during a testing period [32]. The following behavioural measures were analysed:
- General locomotion and exploration behaviour during Y-maze test assessed by the total number of entries in all arms, during Trial 2.
- Novel arm discrimination during Y-maze test. To measure novel arm discrimination in the Y-maze test, a ‘Difference score’ was calculated by subtracting the % entries in Other arm from the % entries in Novel arm for each animal. Additionally the % entries in Other and Novel arms were compared.
- Strategy in Y-maze performance. Differences in the strategy of exploration were examined by analyzing the % of entries in the start arm and the % duration of stay in the center of the maze during the 1
stmin of Trial 2.
- First choice. As first choice was considered the first arm visited.
All the above variables, along with corticosterone measurements, were statistically evaluated by two-way analysis of variance (ANOVA) with ‘treatment’ and ‘sex’ as the independent variables.
When appropriate, Bonferroni or Least Significant Difference (LSD) posthoc analysis was applied (as specified in the legends). The preference for Novel versus Other arm was also analyzed by repeated two-way ANOVA with ‘arm’ as the within-subject factor. In this case, paired t-tests within the groups were applied for further comparisons of % entries between these two arms.
Experimental Phases I and II were analyzed separately. Choice of first arm entered after leaving the start was compared to chance (50%) by a binomial 2-tailed test [32, 34].
According to the vaginal smears obtained at sacrifice, females comprised a mixed population as to the estrus cycle in all groups. Since they had a comparable distribution of the phases among groups, we have pooled the data within each female group for all the parameters measured. The same non-treated animals were used as the control group in statistical analysis of Experimental Phases I and II. The level of significance was set at p<0.05. The statistical analysis was adjusted for non-equivalent groups.
3. Results
3.1 Effect of neonatal exposure to novelty following 10 back-strokes on Y-maze performance (Experimental Phase I)
3.1.1 General locomotion and exploration behaviour during Y-maze test
All animals showed the same motivation and ability to explore the Y-maze apparatus
during Trial 2, as assessed by the total number of entries in all arms that did not differ
significantly among groups (Fig. 2).
3.1.2 Novel arm discrimination during Y-maze test
Early-life exposure to novelty following 10 back-strokes significantly enhanced the adult animals’ performance in the Y-maze memory test (Fig. 3). Analysis of the difference score for the 1 st min and 5min of Trial 2 (Fig. 3A,B), gave an overall effect of treatment [1st min: F(2,53) = 5.00, p = 0.011; 5 min: F(2,54) = 9.33, p = 0.001], sex [1st min: F(1,53) = 4.59, p = 0.037; 5min: non significant] and treatment-sex interaction[
F(2,53) = 3.62, p = 0.034; 5min: non significant]. In general, Novel animals (10S-NOVEL) showed increased ability to discriminate the novel arm in the Y-maze, as depicted by their higher difference scores. Novel males had a higher score in the 1st and 5min of Trial 2, compared to controls (p = 0.004 and p = 0.029 respectively), but they did not differ significantly from the Home (10S-HOME) males (p = 0.084 and p = 0.22 for the 1st and 5min respectively). On the other hand, Novel females were superior to both Home females (p = 0.014 and p = 0.008 for the 1 st and 5min of Trial 2, respectively; Fig.
3A, B) and control females in the 5min period (p = 0.045; Fig. 3B). Home females were impaired, as compared to control females (p = 0.017) and Home males (p = 0.011) in the 1st min of Trial 2 (Fig. 3A).
Novel animals of both sexes entered more often the novel than the other arm, compared to Home animals and controls (Fig. 3C, D). More specifically, during the 1 st
0 2 4 6
8 MALE FEMALE
ENTRIES
Total Entries (Trial 2) 1 min
0 5 10 15 20
25
MALE FEMALE
ENTRIES
5 min
CON 10S-HOME 10S-NOVEL CON 10S-HOME 10S-NOVEL CON 10S-HOME 10S-NOVEL CON 10S-HOME 10S-NOVEL
Figure 2. General locomotion and exploration behaviour during Y-maze test in Experimental Phase I. Mean ± SEM of entries in all arms of the Y-maze during the 1
stand whole 5min period of Trial 2. CON, animal facility reared controls;
10S-HOME, animals neonatally 10 times back- stroked in the home cage; 10S-NOVEL, animals neonatally 10 times back-stroked and exposed to novelty. No significant differences were observed (2-way ANOVA; significance accepted for p < 0.05).
A
B
A
min (Fig. 3C) of testing Novel and control animals entered more often the novel than the other arm (p = 0.006 for Novel males; p = 0.045 for control males; p = 0.036 for Novel females and p = 0.049 for control females). In the whole 5min period (Fig. 3D), only Novel animals still showed preference for the novel arm (p = 0.019 for Novel males and p = 0.001 for Novel females). Home animals did not show any arm preference for novel or other arm during the test.
3.1.3 Strategy in Y-maze performance
Differences in exploring the start arm and the center of the apparatus were detected
1 min
0 20 40
60 OTHER
NOVEL
MALE FEMALE
33,3
% ENTRIES
5 min
0 20 40 60
OTHER NOVEL
MALE FEMALE
33,3
% ENTRIES
-5 5 15 25
35 MALE FEMALE
Difference score
ac ab
b
a c$
ab 1 min
Novel arm discrimination
-5 5 15
25 MALE FEMALE
a ab
b
a a
b 5 min
Difference score
CON 10S-HOME 10S-NOVEL CON 10S-HOME 10S-NOVEL
CON 10S-HOME 10S-NOVEL CON 10S-HOME 10S-NOVEL
CON 10S-HOME 10S-NOVEL CON 10S-HOME 10S-NOVEL
CON 10S-HOME10S-NOVEL CON 10S-HOME 10S-NOVEL
Figure 3. Novel arm discrimination during Y-maze test in Experimental Phase I. Mean ± SEM of (A, B) Difference score (% entries in Novel arm - % entries in Other arm) and (C, D) of % entries in the Other and Novel arm during the 1
stand whole 5min period of Trial 2. CON, animal facility reared controls, 10S-HOME, animals neonatally 10 times back-stroked in the home cage; 10S-NOVEL, animals neonatally 10 times back-stroked and
exposed to novelty. In (A, B), different letters (one or combination of two) over the bars denote significantly different groups within the same sex (2-way ANOVA and LSD posthoc test). In (C, D), stars over horizontal lines denote significant differences between arms for each animal group (repeated 2-way ANOVA and paired t-tests). Doted lines define chance level; significance accepted for p < 0.05.
A
B
C
D
among groups in the 1 st min [ANOVA treatment effect: F(2,51):4.23, p=0.021, sex:
F(1,51):4.44, p=0.041, treatment - sex F(2,51):7.71, p=0.001 ; Fig. 4A ], but not the 5min period of Trial 2. Home males visited the start arm significantly less than the Novel (p
= 0.024) and control (p = 0.001) male groups and the female Home group (p = 0.001).
No significant differences were detected among female groups. Concerning the time spent in the center during the 1 st min [ANOVA treatment effect: F(2,55):3.357, p=0.43 ; Fig. 4B], both Home and Novel males spent less time in the center than controls (p = 0.001 and p = 0.029, respectively) (Fig. 4B). No difference in this parameter was detected in female groups.
3.1.4 First choice
Only male rats of the Novel group showed a trend (p=0.065) in entering first the novel than the other arm (Table 2), but the same trend was found towards the right arm compared to the left one (p=0.065; Table 2). Control males entered first more the right arm (p=0.021) compared to the left one, independently of its novelty (Table 2). In the females’ first choice there was not a specific novel-other or right-left preference.
0 20 40
60 MALE FEMALE
b
a a$
a a
33,3
ab
% E N TR IE S
START ARM (1 min)
0 10 20
30 MALE FEMALE
b b
a
% D U R AT IO N
CENTER (1 min)
CON 10S-HOME 10S-NOVEL CON 10S-HOME 10S-NOVEL
CON 10S-HOME 10S-NOVEL CON 10S-HOME 10S-NOVEL
Figure 4. Strategy in Y-maze performance for animals of Experimental Phase I. Mean ± SEM of (A)
% entries in the Start arm and (B) % duration in the Center of the apparatus during the 1st min of Trial 2. CON, animal facility reared controls, 10S-HOME, animals neonatally 10 times back-stroked in the home cage; 10S-NOVEL, animals neonatally
10 times back-stroked and exposed to novelty.
Dotted line in (A) defines chance level. Different letters over the bars denote significantly different groups within the same sex (2-way ANOVA and Bonferroni posthoc test). Significance accepted for p < 0.05.
A
B
A
3.2 Effect of neonatal exposure to novelty following 20 back-strokes on Y-maze performance (Experimental Phase II)
3.2.1 General locomotion and exploration behaviour during Y-maze test
All animals showed the same motivation and ability to explore the Y-maze apparatus during the 1st min of Trial 2 (Fig. 5). An overall effect of ‘treatment’ on the total number of entries was obtained for the whole 5min period of Trial 2 [F(2,43) = 5.65, p = 0.007].
Further analysis per sex showed that the effect of treatment was marginally non- significant in males [F(2,21) = 3.47, p = 0.052], with 20 back-strokes Novel (20S-NOVEL) males showing a trend for less total entries versus controls (p = 0.055). No significant differences were observed between Novel and Home males. In females, an analogous trend was observed towards reduced exploration versus control, which did not reach significance.
3.2.2 Novel arm discrimination during Y-maze test
Early novelty or home exposure in combination with 20 back-strokes significantly modified the animals’ ability for spatial recognition memory in adulthood (Fig. 6).
Analysis of the difference score for the 1 st min and 5min of Trial 2, gave an overall effect of treatment [1st min: F(2,47) = 3.29, p = 0.047; 5min: F(2,45) = 6.22, p = 0.004 ]. In the 1st min, there was a trend of 20 back-stroked Home (20S-HOME) animals to outperform Novel (20S-NOVEL) animals, which however did not reach significance when further analyzed per sex (Fig. 6A). In the 5min period (Fig. 6B), males 20S-Home significantly outperformed 20S-Novel (p = 0.021) and control (p = 0.019) animals of the same sex (F(2,24) = 4.52, p = 0.023). An analogous trend in 20S-Home females did not reach significance (p = 0.079).
Home animals of both sexes, as well as controls entered more often the novel
Experimental group Novel arm as 1st choice Right arm as 1st choice
CON male 10S-HOME male 10S-NOVEL male CON female 10S-HOME female 10S-NOVEL female
60.0% (6/10) 80.0% (8/10) 90.9% (10/11)b 46.2% (6/13) 66.7% (7/11) 44.4% (4/9)
90.0% (9/10)a 20.0% (2/10) 81.8% (9/11)b 61.5% (8/13) 33.3% (4/11) 77.8% (7/9) Note. Given are percentages of rats entering the
novel arm or the right arm first after leaving the start arm. First number in parentheses indicates the number of rats that entered the novel or right arm first and the second number indicates the total number of rats tested.
aP<0.05, compared
to chance (50%);
bP=0.065, compared to chance (50%). CON, animal facility reared controls;
20S-HOME, animals neonatally 20 times back- stroked in the home cage; 20S-NOVEL, animals neonatally 20 times back-stroked and exposed to novelty.
Table 2
First choice in Experimental Phase I
than the other arm during the 1st min of Trial 2 (p = 0.042 , 0.045, 0.030 and 0.049 for the comparisons between Other and Novel arm in 20S-HOME males, control males, 20S-HOME females and control females, respectively) (Fig. 6C). At the end of 5min, 20S-Home males were still showing a preference for visiting novel versus other arm (p
= 0.044, Fig. 6D), while this preference was lost over time in the female 20S-Home and control groups. 20S-Novel animals of both sexes showed no preference for novel versus other arm in either time point.
3.2.3 Strategy in Y-maze performance
No differences in exploring the start arm were observed among the groups of Phase II (data not shown). Treatment [F(2,43) = 4.19, p = 0.023] and sex [F(1,43) = 10.02, p = 0.003] significantly affected the time spent in the center of the maze during the 1 st but not the whole 5min period of Trial 2 (Fig. 7). Further analysis showed that 20S-Home females spent more time in the center compared to 20S-Novel females (p = 0.046). In addition, 20S-Novel females spent less time in the center compared to 20S-Novel males (p = 0.033).
0 2 4 6 8
ENTRIES
1 min
MALE FEMALE
Total Entries (Trial 2)
0 5 10 15 20 25
ENTRIES
5 min
MALE FEMALE
CON 20S-HOME 20S-NOVEL CON 20S-HOME 20S-NOVEL
CON 20S-HOME 20S-NOVEL CON 20S-HOME 20S-NOVEL
Figure 5. General locomotion and exploration behaviour during Y-maze test in Experimental Phase II. Mean ± SEM of entries in all arms of the Y-maze during the 1st and whole 5 min period of Trial 2. CON, animal facility reared controls, 20S-HOME, animals neonatally 20 times back-
stroked in the home cage; 20S-NOVEL, animals neonatally 20 times back-stroked and exposed to novelty. No significant differences were observed (2-way ANOVA and Bonferroni posthoc test;
significance accepted for p < 0.05).
A
B
A
3.2.4 First choice
No specific novel-other or right-left preference was observed for the 20 back- stroked male groups (Table 3). Control males, as mentioned in Experimental Phase I, entered first the right arm (p=0.021) compared to the left one, independently of its novelty. In female rats, the 20 back-stroked Novel animals showed a preference for entering first
-5 5 15 25
35
1 min
MALE FEMALE
Difference score
Novel arm discrimination
-5 5 15 25 35
b
a
a
MALE FEMALE
5 min
Difference score
1 min
0 20 40
60 OTHER
NOVEL 33,3
MALE FEMALE
% ENTRIES
5 min
0 20 40
60 OTHER
NOVEL 33,3
MALE FEMALE
% ENTRIES
CON 20S-HOME 20S-NOVEL CON 20S-HOME20S-NOVEL CON 20S-HOME 20S-NOVEL CON 20S-HOME20S-NOVEL CON 20S-HOME 20S-NOVEL CON 20S-HOME 20S-NOVEL CON 10S-HOME 10S-NOVEL CON 10S-HOME 10S-NOVEL
Figure 6. Novel arm discrimination during Y-maze test in Experimental Phase II. Mean ± SEM of (A,B) Difference score (% entries in Novel arm - % entries in Other arm) and (C,D) of % entries in the Other and Novel arm during the 1st and whole 5 min period of Trial 2. CON, animal facility reared controls; 20S-HOME, animals neonatally 20 times back-stroked in the home cage; 20S-NOVEL, animals neonatally 20 times back-stroked and exposed to novelty. In (A,B), different letters (one
or combination of two) over the bars denote significantly different groups within the same sex (2-way ANOVA and LSD posthoc test). No significant differences were observed in the 1st min period for both sexes and in the 5min period for females. In (C,D), stars over horizontal lines denote significant differences between arms for each animal group (repeated 2-way ANOVA and paired t-tests). Doted lines define chance level;
Significance accepted for p < 0.05.
A
B
C
D
the right arm (p=0.031) compared to the left (Table 3), while no specific preference was observed for the other female groups.
3.3 Basal corticosterone levels (Experimental Phase I & II)
Early-life exposure to novelty following 10 back-strokes (Phase I) significantly affected the animals’ basal corticosterone levels in adulthood in a sexually dimorphic manner (ANOVA treatment effect: F(2,53):6.15, p=0.010, sex effect: F(1,53):32.44, p=0.001; Fig. 8A). Home (10S-HOME) males and Novel (10S-NOVEL) females had elevated corticosterone levels compared to same sex controls (p = 0.043 and 0.007, respectively).
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% DURATION a
ac bc$
CENTER (1 min)
MALE FEMALE
CON 20S-HOME 20S-NOVEL CON 20S-HOME 20S-NOVEL
