# Further evidences that risk assessment and object exploration behaviours are useful to evaluate emotional reactivity in rodents

Vincent Roy, Pierre Chapillon

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Vincent Roy, Pierre Chapillon. Further evidences that risk assessment and object exploration behaviours are useful to evaluate emotional reactivity in rodents. Behavioural Brain Research, 2004, 154, pp.439 - 448. 10.1016/j.bbr.2004.03.010. hal-01576748

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Roy V, Chapillon P. Further evidences that risk assessment and object exploration behaviours are useful to evaluate emotional reactivity in rodents. **Behav Brain Res.** 2004 Oct 5;154(2):439-48.
doi: **10.1016/j.bbr.2004.03.010**. PMID: **15313032.**

Further evidences that risk assessment and object exploration behaviours are useful to evaluate
emotional reactivity in rodents

Vincent ROY *, Pierre CHAPILLON

UPRES PSY.CO, EA 1780, Equipe Neurobiologie de l'Apprentissage,
UFR des Sciences, Université de Rouen
76821 Mont-Saint-Aignan, FRANCE

* Corresponding author. E-mail: [vincent.roy@univ-rouen.fr](mailto:vincent.roy@univ-rouen.fr)

Abstract:

In this study, we tested the ability of risk assessment and exploration behaviours to emphasise PH effects. Indeed, postnatal handling (PH) decreases emotional reactivity in rats but inconsistent behavioural results can be observed and may be due to false negative (i.e. existing effects are not detected). Risk assessment behaviours were measured in the elevated plus maze, in the free exploration paradigm and in the open field. In addition, we measured object exploration behaviours towards familiar / new objects in the open field.

PH increased general activity in the elevated plus maze and in the free exploration paradigm and risk assessment behaviours allowed demonstrating that these effects were specific to emotional reactivity. In the open field, PH increased object exploration as early as first exposition while general activity was unaffected. PH also decreased behavioural inhibition in response to the introduction of a novel object.

On the whole, our results shows that risk assessment and object exploration behaviours are valuable
tools to measure more precisely emotional reactivity in rodents. This reinforces the idea that these
behaviours should be used more frequently in order to avoid false negative when emotional
reactivity changes are expected in unconditioned conflict tests.

**Keywords:** Emotional reactivity, Risk assessment, Object exploration, Elevated plus maze, Free exploration, Open field, Rats, Postnatal handling.
**Introduction**

Emotional reactivity changes in response to experimental treatments (e.g. pharmacological, lesional or early stimulation) are sometime difficult to emphasise in rodents' models of anxiety-like behaviours. In exploration models [1,4,33,61], the animal is exposed to a novel situation which is supposed to create a conflict between a motivation to explore the novel situation and an unconditioned fear of novelty. Behavioural parameters that “reflect” exploration such as locomotion or rearing are then used to evaluate emotional reactivity or “anxiety” with the subjacent idea that an animal that explores the novel situation is a non-emotive one. However, interpretation of these parameters is somehow difficult, particularly since animals are usually forced exposed to the unfamiliar apparatus [2]. In this case, the meaning of locomotion or rearing becomes uncertain since they may represent exploration (i.e. a non-emotive animal) but also tentative to escape from the apparatus (i.e. an emotive animal) [1,7]. For example, it appears now evident that classical parameters of “locomotion” are not easily distinguishable from emotional reactivity processes in the elevated plus maze [66].

To overcome this problem, the importance of a behavioural analysis that includes behaviours from the rodents' defensive repertory (i.e. ethoexperimental analysis of behaviour) has been emphasised [5,6,29]. These behaviours are fear-like behaviours (e.g. defensive threat, jump attack, vocalisation) and / or anxiety-like behaviours (e.g. risk assessment behaviours, approach / avoidance behaviours). In particular, risk assessment and avoidance behaviours seem to faithfully represent the approach / avoidance conflict in a novel situation and detailed ethological analyses have been recently applied to standard tests of emotional reactivity [10,49-53,66]. Some studies notably demonstrated the usefulness of risk assessment behaviours such as stretched attend postures (SAPs) and closed arm returns in the elevated plus maze [11,51,52] or SAPs, thigmotaxis and returns behaviours in the open field [10]. These behaviours are sensitive to pharmacological compounds [11,42,66] and their major contribution to the study of anxiety-like behaviours seems to lie in that they allow a better distinction between general activity and emotional reactivity processes [10,50].

The aim of our work was to extend the usefulness of risk assessment behaviours to the evaluation of early stimulation and more specifically to the evaluation of postnatal handling (PH) effects. Indeed, PH is generally admitted to decrease emotional reactivity in rats [18,20] along with numerous brain changes [8,34,36], but the behavioural effects are sometimes difficult to emphasise or to separate from motor responses changes. In several studies that demonstrated clear PH effects on the whole, the expected effects were missing or inconsistent in respect to behavioural parameters in one particular test. This was reported in the open field [38,58,63], the elevated plus maze [15,28,64] or in emergence tests [21,43]. Genetic factors have been advanced to explain some results [15] but can not be used in studies that found PH effects when the same animals were tested in other devices. In this situation, a valuable explanation could be testing conditions and / or behavioural analysis. Indeed, ceiling effects can occur since the testing situation must be stressful enough (but not too much) in order to discriminate control animals from experimental ones. In addition, we believe that a behavioural evaluation based exclusively on standard parameters related to general activity can also lead to the non-appearance of existing PH effects (i.e. false negative).

In this study, we tested DA/Han rats in three unconditioned conflict tests that are extensively used in the field of behavioural neuroscience and that can possibly lead to difficulties in interpreting results about PH effects (see above): the elevated plus maze [32,46] that is currently the most popular device for testing anxiety-like behaviours, the free exploration paradigm [3,22] which represent an alternative to forced exposition paradigms and the open field [2,48] that was the first unconditioned response test used to evaluate the effects of PH on emotional reactivity [23,31]. In each of these paradigms, we measured both parameters related to general activity and parameters related to risk assessment / avoidance. Since the absence of a clear approach / avoidance conflict in the open field, thus leading to reduced risk assessment behaviours, we detailed our behavioural analysis by adding familiar and novel objects to our open field procedure. The measurement of the
behaviours towards these objects was taken as a supplementary tool to discuss emotional reactivity [40].

# Material and Methods

## Animals

DA/Han rats (also refereed as Dark Agouti) were used in this study. This strain has been described as an anxious strain in other teams [30,37] and previous works in our laboratory demonstrated prenatal and postnatal stress effects in DA/Han rats [9,35]. This makes the strain a relevant one for the study of early stimulation effects and, as expected, we previously reported effects of postnatal handling on emotional reactivity in DA/Han rats [55,56].

A total of 91 DA/Han male rats from our own breeding colony were tested. Throughout the experiment, the animals were maintained in an air-conditioned room (21 ± 2°C) under a 12 hour light-dark cycle (lights on at 00h00). Standard polycarbonate cages (40 × 28 × 18 cm) were used with food and water freely available. The research reported in this paper was conducted in accordance with the guidelines for care and use of laboratory animals established by the National Institute of Health of the United States of America [27].

## Handling procedure

When obviously pregnant, multiparous dams were isolated. On the date of birth (day 0), litters from each dam were randomly assigned to the control (C) or the handled (H) group. PH sessions took place once a day between 7h00 and 11h00 from day 1 to day 21. Sessions consisted of first removing the dam from the home cage and then placing the pups individually in plastic cages lined with paper towel. During the 15 min session, each pup was gently handled for three periods of five seconds at 5, 10 and 15 minutes. Immediately after the third period, the pups were replaced in their home cage, just before the dam was. In the same time, C animals remained in their home cages and were manipulated once a week for cage cleaning. This procedure is based on the works of other teams [19,24,57] and previously showed significant effects in DA/Han rats in our laboratory [55,56]. At day 22, the pups were sexed and weaned. Males from the same condition were housed by groups of 4 (± 1) and left undisturbed until behavioural testing.

## Behavioural testing

Animals were tested at the age of 8 / 10 weeks, during the first part of the dark phase (between 14h00 and 18h00). For each test, animals were habituated to the testing room for a period of at least 30 min. In addition, all tests were video recorded and analysed with Etholog 2.25 [45].

## Elevated Plus Maze

Fourteen C and 16 H rats, each obtained from 8 different litters were tested in the elevated plus maze. The maze was made of clear painted wood. The arms were 50 cm long and 10 cm width and the apparatus was elevated at a height of 50 cm. The closed arms were surrounded by a 50 cm wall while open arms had 0.5 cm edges in order to maximize open arms entries [62]. White light (250 lux on open arms and centre, 160 lux on closed arms extremities) was chosen to provide mild anxiogenic conditions and to allow a better discrimination between rats from the two rearing conditions. Closed arms and open arms were divided into a proximal and a distal part to allow a better measurement of locomotion within the maze [13]. The test was 10 min long and began with the placement of a rat in the centre of the maze, with its head facing an enclosed arm. Time and number of entries in the different parts of the maze and numbers of rears were taken as classically recorded measured. A four-paw criterion validated an entrance into one arm and the animal was considered out of the arm when two paws had left the arm. Ethological measures included closed
arm returns [53], head scans (the rat points its head in the open arm but its body is still in the centre or in a closed arm of the maze), head dipping and stretched attended postures (SAPs). These three latter behaviours were measured according to the place they were displayed (i.e. protected or unprotected parts) [12].

## Free Exploration

The free exploration paradigm is based on the exploration of a novel place from a familiar place [3,22]. We tested 12 C and 12 H rats in this paradigm. Animals from each condition were obtained from 4 litters. Our apparatus was composed of two identical compartments (40 × 40 × 30 cm) linked together by a tunnel (10 × 8 × 8 cm). The compartments were made of clear painted wood. One of the two compartments, named the familiarisation compartment, contained some food and water and its floor was covered by 2 cm of sawdust. In the second compartment, named the new compartment, there was no sawdust on the floor but a blue glass cup was placed in front of its back wall. The day before the test, rats were isolated into familiarisation compartments for a 20 hours period. A removable door blocked the access to the tunnel, and subsequently to the new compartment. A grid was placed on the familiarisation compartment to prevent that a rat escaped. The next day, the grid was removed 30 minutes before the beginning of the test and red light was set on above the apparatus (10 lux in each compartment). The test was 10 minutes long and began with the removal of the door that gives access to the tunnel. Latency to enter the tunnel and latency to first entrance into the new compartment (four paws criterion) were noted. In addition to these classically measured parameters, the number of SAPs, the number of head scans into the new compartment and the number of returns into the familiarisation compartment before first entrance were taken.

## Open field

Nineteen C and 18 H rats, respectively obtained from 7 and 6 litters, were tested in the open field. The apparatus consisted of a yellow plastic circular arena (100 cm in diameter) surrounded by a 50 cm wall. The apparatus was lit by white light (260 lux on the centre) and divided into 1 central and 6 peripheral parts of equal surface. At the beginning of the test, the rats were placed in a peripheral part of the open field. Three identical objects (white glass incubation tray) were placed in the opposite, clockwise and anti-clockwise quadrants. The procedure consisted in 1 daily sessions of 5 minutes, on 4 consecutive days. On the first three sessions, all animals were tested with three identical objects. On session 4, the object in the opposite quadrant was replaced by a blue glass cup for 9 C and 9 H randomly chosen rats. Other animals were tested with the three familiar objects. The following parameters were recorded: latency to leave the starting place, total number of entries into peripheral and central parts, number of rears, motionless time, number of SAPs and time of contact with the objects. The behaviours "rearing against an object" and "passing head into an object" were also noted. Behaviours directed towards objects were pooled. However, on day 4, behaviours towards the object in the opposite quadrant (replaced or not) were measured separately from the behaviours towards the two familiar objects as to allow a correct evaluation of reaction to novelty. The differences between session 4 and session 3 for the above-mentioned parameters were then calculated and analysed. This procedure was chosen in order to suppress any possible difference between groups on day 3 and therefore to focus on the effect of novelty [59].

## Statistics

All statistics were run under Statistica software (5.1). Student t tests were employed for comparisons in the elevated plus maze. Mann-Whitney U tests were used in the free exploration paradigm since homogeneity of variance was not respected for a majority of the parameters. Open field data were analysed with a two-way analysis of variance (ANOVA) with the rearing condition factor (C or PH) and the session factor (repeated-measures for sessions 1 to 3). A two-way ANOVA
with the rearing condition factor (C or PH) and the object replacement factor (familiar or novel object) was performed for the differences between session 4 and session 3. Post hoc comparisons were realised with Tuckey's HSD tests.

## Results

### Elevated Plus Maze

Results from the elevated plus maze session are shown in Table 1. H rats spent significantly more time than C rats in the open arms [$t_{28} = 2.23, p < 0.05$] and made more entries into these arms, though not significantly. H rats also spent more time in the distal parts of the open arms and in the centre of the maze, even if the differences did not reach significance. On the contrary, C rats spent more time in the closed arms of the maze, but once again the difference was not significant [$t_{28} = 1.93, p = 0.06$].

Total number of arm entries was significantly higher in H rats [$t_{28} = 2.18, p < 0.05$], as well as total number of sector crossing [$t_{28} = 2.79, p < 0.01$]. A small increase was observable in H rats for the numbers of open arm and closed arm entries but there was no significant difference (Figure 1). However, closed arm returns were more frequent in C rats [$t_{28} = 2.82, p < 0.01$] (Figure 1).

Therefore, when the number of closed arm entries was corrected by subtraction of closed arms returns, the difference between C and H rats became significant [$t_{28} = 3.22, p < 0.01$]. H rats made fewer protected SAPs than C rats but the difference was not significant. Rears were more frequent in H rats [$t_{28} = 2.26, p < 0.05$], as well as head scans [$t_{28} = 2.23, p < 0.05$] and head dips [$t_{28} = 4.34, p < 0.01$].

### Free Exploration

Results are shown in Table 2. Five C rats out of 12 entered the new compartment whereas all 12 H rats did. A comparison of these proportions by the Fisher exact probability leads to a significant difference [$p < 0.01$].

H rats entered more rapidly the corridor than C rats [$U = 32.5, p < 0.05$] and their latency to enter the new compartment was also significantly shorter [$U = 17, p < 0.01$]. In addition, H rats made fewer returns towards the familiar compartment [$U = 31.5, p < 0.01$] and fewer numbers of SAPs [not significant] before first entry in the new compartment (Figure 2). The number of head scans directed towards the new compartment was also lower in H rats but not significantly. Behaviours directed towards the object in the new compartment were not analysed since too few C rats entered this compartment.

### Open field

#### Session 1 to 3

The data analysis for session 1 to 3 in the open field revealed several significant differences (Table 3). A significant main effect of the session factor was observed for locomotion in the peripheral parts [$F_{2,70} = 5.37, p < 0.01$], as well as a significant interaction between the session and PH [$F_{2,70} = 3.26, p < 0.05$]. Post hoc comparisons show that locomotion during session 3 was lower than locomotion during session 1 [$p < 0.01$]. The interaction was significant since the previous observation was only significant in C rats when separate analyses were run ($p < 0.01$, session 1 vs. session 3 in C rats]. Consequently, locomotion in peripheral parts during session 3 was higher in H rats than in C rats [$p < 0.05$]. There was no difference between C and H rats on locomotion in the central part of the arena. Indeed, animals made few entries in the centre of the apparatus, about one per session, and this score was stable during sessions. As a result, there was no significant effect of PH for total locomotion. However, a significant main effect of the session factor was observed [$F_{2,70}$
p < 0.01] and explained by a lower locomotion during session 3 [p < 0.01 vs. session 1]. The interaction between PH and sessions was closed to significance [F<sub>2,70</sub> = 2.87, p = 0.064]. Motionless time during sessions 1 to 3 is represented by Figure 3. The analysis showed significant effects for the PH factor [F<sub>1,35</sub> = 9.95, p < 0.01], the session factor [F<sub>2,70</sub> = 19.63, p < 0.001] and their interaction [F<sub>2,70</sub> = 7.35, p < 0.01]. These effects were explained by a general augmentation of motionless time [respectively p < 0.01 and p < 0.001 for comparison between session 1 and sessions 2 and 3] and a significantly lower motionless time in H rats during session 3 [p < 0.001]. The number of rearing did not differ between groups but decreased during sessions [F<sub>2,70</sub> = 5.88, p < 0.01]. Indeed, rears were less frequent during sessions 2 and 3 in comparison to session 1 [p < 0.05 for each comparison]. In addition, the numbers of SAPs and the latency to leave the starting place did not differ between groups and did not evolve significantly during the three sessions of test. As shown by Figure 4, the time in contact with the objects was significantly higher in H rats [F<sub>1,35</sub> = 8.40, p < 0.01]. Moreover, it decreased among sessions [F<sub>2,70</sub> = 22.15, p < 0.001], with a significant difference between sessions 1 and 3 [p < 0.001]. A similar trend was observed for behaviours directed towards objects. Climbing onto one object was more frequent in the H group [F<sub>1,35</sub> = 4.66, p < 0.05] as well as passing the head into one object [F<sub>1,35</sub> = 8.35, p < 0.01]. However, while the first behaviour did not evolve during sessions, the frequency of passing the head into one object increased during sessions [F<sub>2,70</sub> = 4.24, p < 0.05], with a significantly higher frequency during session 3 than during session 1 [p < 0.05].

## Reactions to object replacement

Analyses for the differences between session 4 and 3 (Table 4) revealed significant effects of interaction between PH and object replacement on locomotion in peripheral parts [F<sub>1,33</sub> = 11.48, p < 0.01] and total locomotion [F<sub>1,33</sub> = 8.75, p < 0.01] (Figure 5A). Post hoc comparisons show that these effects were due to a significant difference between the positive difference in novelty-exposed H rats and the slightly negative difference in novelty-exposed C rats [p < 0.05 for total locomotion and locomotion in peripheral parts]. A significant interaction was also obtained for the difference in motionless time between session 4 and 3 [F<sub>1,33</sub> = 8.05, p < 0.01] (Figure 5B). The post hoc analysis shows that the difference for H rats exposed to a novel object was positive and significantly higher than the negative one of C rats exposed a novel object [p < 0.05]. There was also a significant difference between C rats from the familiar object group and those exposed to a novel object [p < 0.05].

Lastly, similar analyses were realised on behaviours directed towards objects. Analyses were conducted separately for behaviours directed towards the two familiar objects never replaced and for behaviours towards the object that was replaced or not. Analyses from the former situation did not yield any significant difference while, in the latter, revealed several differences. Effects of object replacement [F<sub>1,33</sub> = 4.44, p < 0.05], PH [F<sub>1,33</sub> = 20.16, p < 0.001] and interaction between these two factors [F<sub>1,33</sub> = 6.10, p < 0.05] were significant for the difference on the time of contact between sessions 4 and 3 (Figure 5C). On the whole, C and H rats spent a greater amount of time in contact with the object in the replaced object condition. In addition, H rats spent more time in contact with the objects (independently of its replacement). The post hoc analysis of the interaction indicated a higher difference in the time of contact with a replaced object in H rats than in C rats [p < 0.05] and a significant difference with animals from the non-replaced object group only in the PH condition [p < 0.001].

Results for behaviours directed towards the object were slightly similar. For the behaviour climbing onto the object, only the object replacement main effect was significant [F<sub>1,33</sub> = 11.15, p < 0.01], with more frequent rears by C and H rats onto the object in case of its replacement [p < 0.01]. The rearing condition effect was not significant but the interaction was close to significance [F<sub>1,33</sub> = 4.09, p = 0.051] and its post hoc analysis showed that H rats reacted more precisely to novelty. Indeed, the difference between session 4 and session 3 for this behaviour was significant only for the comparison between the two groups of H animals [p < 0.01]. Results for the behaviour passing the head into one object were comparable (Figure 5D). Indeed, the rearing condition factor, the
replacement of the object factor and their interaction were all significant [respectively F₁, ₃₃ = 4.56, p < 0.05; F₁, ₃₃ = 8.56, p < 0.01 and F₁, ₃₃ = 6.81, p < 0.05]. The post hoc analysis led to the same observations. First, the behaviour passing head into the object was more frequent for all rats when the object had been replaced and more frequent in the H than in the C group. As for the time of contact with the object, the post hoc analysis indicated that the interaction was due to H animals from the novel object group. Their difference between session 4 and session 3 for the behaviour passing head into the novel object was significantly higher than the difference of their counterparts from the familiar object group [p < 0.01] but also than the difference of the C rats from the replaced object group [p < 0.05]. There was no difference between the two control groups.

## Discussion

As previously reported, we found a general effect of postnatal handling on emotional reactivity in the elevated plus maze, the free exploration and the open field. However, this effect was not so straightforward in regard to some standard behavioural parameters and our data emphasise the usefulness of a precise ethological analysis.

### Elevated plus maze

In agreement with previous studies, analyses of some parameters that are classically recorded in the elevated plus maze lead to the conclusion that H rats displayed a lower emotional reactivity level than C rats in this device [38,47,63]. Indeed, H rats spent more time in the anxiogenic parts of the maze (i.e. the central part and the open arms) with a significant difference for the time spent in the open arms, a parameter that is typically associated with a low anxiety level [12,17,53]. However, the differences for the number of open arm entries or open arm entries ratio, also associated with a low anxiety level [32,46], were not significant. In addition, “locomotion” in the apparatus was more important in H rats since their total number of entries and sector crossing in the maze was significantly increased. These parameters reflect general activity in the maze but recent reviews show evidences that they are also associated to anxiety processes [65,66]. With respects to these classical parameters, PH effects are then no more so clear-cut.

Ethological measures bring important information about this issue. C rats made more SAPs in protected parts of the maze, thought not significantly, and more frequent closed arm returns. These two behaviours respectively reflect risk assessment in a potentially dangerous situation and avoidance of the situation. C animals thus showed a greater “hesitation” to leave the reassuring closed arms and it can therefore be argued that PH specifically reduces the approach / avoidance conflict and thereafter, only, increase activity in the maze. This point is particularly visible since a significant difference between C and H rats for closed arm entries was only detected when entries were corrected by the number of closed arm returns. Therefore we believe, as emphasised by several authors that closed arm and total number of arm entries should not be considered as parameters specific to locomotion [32,51,53,65,66], but also reflect emotional reactivity since they initially depend on risk assessment behaviours and approach / avoidance behaviours.

On the whole, our results from the elevated plus maze test allow concluding that PH decreases emotional reactivity by reducing the approach / avoidance conflict (i.e. fewer SAPs and closed arm returns) and that the effect on general activity is not responsible for this but rather is a consequence.

### Free exploration

A significantly higher proportion of H animals entered the new compartment of the free exploration paradigm and, as a result, the classically used latency to enter the new compartment was lower in H rats. This result is in accordance with previous reports from several authors that tested the effects of PH in emergence tests [16,25,26,44]. However, as in the elevated plus maze, it seems difficult to dissociate this effect from a more general effect of PH on general activity, especially since H animals were prompter to enter the corridor. Again, analysis of risk assessment behaviours brings information about this issue. The free exploration paradigm represents a situation in which the
conflict between familiarity and novelty is maximal [41] and this gives a great value to risk
assessment behaviours. Our results show that H animals entered the new compartment promptly but
this was not associated with a total suppression of risk assessment behaviours before the first
entrance. Indeed, H animals made approximately as much SAPs and head scans as C animals before
first entrance. However, the behaviour "return into the familiar compartment", which presents
similarities with closed arm returns in the elevated plus maze, was significantly less frequent in H
animals. Therefore, the contribution of ethological parameters again lies in the possibility to
improve the discussion of the results. Actually, if H animals enter more rapidly the new
compartment, this observation seems to be attributable to an approach / avoidance conflict reduction
and can not be confounded with a general activity enhancement.

Open field

In the open field, our results first confirmed the importance of a several session procedure [14]. Indeed, no difference between C and H rats was found during session 1 on standard parameters such as locomotion, motionless time or rearing. This result is consistent with previous works that used a single open field session [58,63] or with the first session of work that used multiple sessions [31,38]. Analyses of sessions 1 to 3 showed some evolutions for these parameters and differences between groups for locomotion and motionless time. Locomotion in H rats was relatively stable and their motionless time increased very slightly. On the contrary, motionless time in C rats increased inversely to locomotion. Consequently, differences between C and H rats on session 3 were significant. This is in agreement with the literature both for the session and PH effects. Indeed, a high locomotion on session 1 could represent a high emotional reactivity level and then, on consecutive expositions, a low emotional reactivity level [14]. This explanation was also proposed by Levine et al. [31] who described an inversion in locomotion between C and H rats among sessions.

Risk assessment behaviours in our open field study were limited to the latency to leave the starting
place and to the number of SAPs. We found no differences between C and H rats for these two
parameters that were yet reported to discriminate control and enriched BALB/c mice in the open
field [54]. In addition, Choleris et al. (2001) recently reported the ability of SAPs (but also "return"
behaviours and thigmotaxis) to emphasise the anxiogenic properties of diazepam and
chlordiazepoxide in mice that were tested in the open field. The absence of a difference for risk
assessment behaviours in our results thus seems questionable and two valuable (non-exclusive)
explanations could emerge from our testing conditions. First, a ceiling effect due to insufficiently
stressful conditions could be responsible for our results since SAPs scores and latencies to leave the
starting place were relatively low. Another possibility would be that these parameters are not as
sensitive in rats as in mice. Secondly, the approach / avoidance conflict in a circular open field is
certainly reduced in comparison to a square open field in which corners represent special locations
to initiate risk assessment behaviours. A study by Vallée et al. (1997) in which the time spent in the
corners of the field was used to differentiate C and H rats accounts for this explanation.

As an alternative to risk assessment, the behaviours displayed towards the objects were very
informative. First, exploration of the objects by H rats was greater and more diversified. This effect
was detectable as early as session 1, when locomotion and rearing were still equivalent between C
and H rats, and it was also pronounced during sessions 2 and 3. Therefore, this reinforces the idea
that locomotion on session 1 represents, to a larger extent in C rats, a motivation to escape the
apparatus while it rather represents a motivation to explore the apparatus in H rats [31]. In addition,
the object replacement procedure on session 4 provided additional information. Rats from the two
rearing conditions readily detected the novel object but while H rats significantly increased their
exploration behaviours towards the object that was replaced (i.e. locomotion, time of contact with
the object, behaviours directed towards the object), C rats avoided the novelty by increasing their
motionless time. One could therefore make the proposition that the emotional reactivity decrease
observed in H rats in this test is based on a prompter habituation to novelty than in C rats.

Furthermore, this result emphasise that non-exploration of a novel object in a spatial task should not
be interpreted exclusively in terms of memory processes impairment (e.g.[60]) but should also be
discussed in terms of emotional processes [39,41].

In conclusion, our results extend the usefulness of risk assessment and object exploration behaviours to better dissociate emotional processes from general activity in “exploration” tests. Therefore, these behaviours should be used more systematically to avoid false negative when a treatment (e.g. pharmacological, lesional or early stimulation) does not provide significant results with classically recorded parameters. The research for brain systems that mediates these behaviours should also be continued as to provide important information in regard to emotional processes and decision making in stressful situations.

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## Tables and Figures

<figure><img src="image_2.png" /><figcaption>Figure 1: Elevated plus maze</figcaption></figure>

Open arm Entries, Closed arm Entries and Closed arm Returns in the elevated plus maze for C (n = 14) and H rats (n = 16). Data are given as mean ± SEM. Statistic: Student t test, ** p < 0.01.
<table><caption>Table 1: Elevated plus maze</caption><thead><tr><th></th><th colspan="2">Controls</th><th colspan="2">Handled</th><th></th></tr></thead><tbody><tr><td>Time in the centre</td><td>196.21</td><td>±23.65</td><td>235.94</td><td>±16.47</td><td></td></tr><tr><td>Time in open arms</td><td>16.43</td><td>±4.57</td><td>37.88</td><td>±8.06</td><td>*</td></tr><tr><td>Time in open arm's extremities</td><td>2.29</td><td>±1.68</td><td>6.75</td><td>±2.73</td><td></td></tr><tr><td>Time in closed arms</td><td>387.36</td><td>±26.01</td><td>326.19</td><td>±19.14</td><td>0.06</td></tr><tr><td>Time in closed arm's extremities</td><td>185.86</td><td>±16.61</td><td>176.25</td><td>±14.73</td><td></td></tr><tr><td>Open arms entries</td><td>1.86</td><td>±0.36</td><td>3.19</td><td>±0.71</td><td></td></tr><tr><td>Entries into open arm's extremities</td><td>0.36</td><td>±0.25</td><td>1.00</td><td>±0.39</td><td></td></tr><tr><td>Closed arms entries</td><td>11.07</td><td>±0.70</td><td>12.81</td><td>±0.62</td><td>0.07</td></tr><tr><td>Total number of arms entries</td><td>12.93</td><td>±0.89</td><td>16.00</td><td>±1.06</td><td>*</td></tr><tr><td>Total number of sector crossing</td><td>42.29</td><td>±2.86</td><td>55.00</td><td>±3.45</td><td>**</td></tr><tr><td>Number of rearing</td><td>19.00</td><td>±1.78</td><td>24.88</td><td>±1.88</td><td>*</td></tr><tr><td>Closed arms returns</td><td>2.71</td><td>±0.50</td><td>1.19</td><td>±0.26</td><td>**</td></tr><tr><td>Closed arms entries (correction)</td><td>8.36</td><td>±0.87</td><td>11.63</td><td>±0.57</td><td>**</td></tr><tr><td>Head scans</td><td>21.29</td><td>±1.42</td><td>25.81</td><td>±1.44</td><td>*</td></tr><tr><td>Total Head dips</td><td>9.00</td><td>±1.46</td><td>15.06</td><td>±0.97</td><td>**</td></tr><tr><td>Unprotected head dips</td><td>1.07</td><td>±0.25</td><td>4.88</td><td>±1.31</td><td>*</td></tr><tr><td>Protected SAPs</td><td>4.36</td><td>±0.72</td><td>2.93</td><td>±0.73</td><td></td></tr><tr><td>Unprotected SAPs</td><td>1.00</td><td>±0.30</td><td>1.48</td><td>±0.41</td><td></td></tr><tr><td>Number of open arms entries (%)</td><td>13.36</td><td>±2.27</td><td>18.10</td><td>±3.13</td><td></td></tr></tbody></table>

Behaviours of C (n = 14) and H (n = 16) rats in the elevated plus maze. Data are given as mean ± SEM. Statistic: Student's t Tests, * p < 0.05, ** p < 0.01.
<figure><img src="image_3.png" /><figcaption>Figure 2: Free exploration</figcaption></figure>

Numbers of SAPs directed towards the new compartment (left) and numbers of Returns to the familiar compartment (right) before first entry for C and H rats (n = 12 for each group). Data are given as mean + SEM. Statistics: Mann-Whitney U test, * p < 0.05.
<table><caption>Table 2: Free exploration</caption><thead><tr><th></th><th colspan="2">Controls</th><th colspan="2">Handled</th><th></th></tr></thead><tbody><tr><td>Latency to enter the corridor</td><td>95.08</td><td>±38.81</td><td>15.08</td><td>±7.46</td><td>*</td></tr><tr><td>Latency to enter the new compartment</td><td>433.92</td><td>±66.81</td><td>112.92</td><td>±37.70</td><td>**</td></tr><tr><td>Number of head scans</td><td>5.92</td><td>±1.33</td><td>4.75</td><td>±1.42</td><td></td></tr><tr><td>SAPs towards the new compartment</td><td>2.42</td><td>±0.45</td><td>1.92</td><td>±0.63</td><td></td></tr><tr><td>Returns to the familiar compartment</td><td>3.83</td><td>±0.74</td><td>1.75</td><td>±0.88</td><td>*</td></tr></tbody></table>

Behaviours of C (n = 12) and H (n = 12) rats in the free exploration paradigm. Data are given as mean ± SEM. Statistic: Mann-Whitney U test, * p < 0.05, ** p < 0.01.
<table><caption>Table 3: Open field, sessions 1 to 3</caption><thead><tr><th rowspan="2"></th><th colspan="2">Session 1</th><th colspan="2">Session 2</th><th colspan="2">Session 3</th><th rowspan="2"></th></tr><tr><th>Control</th><th>Handled</th><th>Control</th><th>Handled</th><th>Control</th><th>Handled</th></tr></thead><tbody><tr><td>Locomotion in peripheral parts</td><td>26.84 ±2.09</td><td>25.83 ±2.07</td><td>21.11 ±1.98</td><td>25.83 ±2.86</td><td>17.84 ±2.65</td><td>24.56 ±2.21*</td><td>S, I</td></tr><tr><td>Locomotion in the central part</td><td>1.37 ±0.24</td><td>1.28 ±0.29</td><td>1.05 ±0.26</td><td>0.67 ±0.29</td><td>0.79 ±0.30</td><td>1.06 ±0.32</td><td></td></tr><tr><td>Total locomotion</td><td>28.21 ±2.16</td><td>27.11 ±2.24</td><td>22.16 ±2.13</td><td>26.50 ±3.03</td><td>18.63 ±2.87</td><td>25.61 ±2.41</td><td>S</td></tr><tr><td>Number of Rears</td><td>13.11 ±1.39</td><td>10.78 ±1.05</td><td>9.00 ±1.24</td><td>9.44 ±1.08</td><td>8.84 ±1.59</td><td>9.61 ±1.09</td><td>S</td></tr><tr><td>Motionless time</td><td>18.21 ±4.38</td><td>16.56 ±4.78</td><td>56.42 ±12.19</td><td>31.06 ±5.43</td><td>91.05 ±15.03</td><td>31.50 ±4.14***</td><td>H, S, I</td></tr><tr><td>Number of SAPs</td><td>1.21 ±0.40</td><td>0.50 ±0.19</td><td>1.00 ±0.29</td><td>1.00 ±0.37</td><td>0.47 ±0.19</td><td>0.67 ±0.28</td><td></td></tr><tr><td>Latency to leave starting place (sec)</td><td>11.58 ±1.19</td><td>14.94 ±1.94</td><td>10.63 ±2.24</td><td>18.61 ±5.92</td><td>11.11 ±4.05</td><td>6.78 ±1.53</td><td></td></tr><tr><td>Time of contact with objects</td><td>51.47 ±4.33</td><td>69.28 ±6.32</td><td>46.32 ±4.63</td><td>63.22 ±3.34</td><td>35.26 ±4.44</td><td>46.50 ±3.00</td><td>H, S</td></tr><tr><td>Number of “object climbing”</td><td>6.00 ±0.76</td><td>8.39 ±1.00</td><td>6.32 ±1.01</td><td>8.89 ±0.99</td><td>5.89 ±0.88</td><td>7.94 ±0.94</td><td>H</td></tr><tr><td>Number of “head in one object”</td><td>0.32 ±0.13</td><td>1.94 ±0.53</td><td>1.00 ±0.36</td><td>2.72 ±0.45</td><td>1.74 ±0.49</td><td>2.50 ±0.56</td><td>H, S</td></tr></tbody></table>

Behaviours of C (n = 19) and H (n = 18) rats in the open field during sessions 1 to 3. Data are given as mean ± SEM. Statistics: H, postnatal handling significant main effect; S, session significant main effect; I, significant interaction between these two factors; Post-hoc comparisons: * p < 0.05, *** p < 0.001, Comparison of C and H rats in one session.
<table><caption>Table 4: Reaction to novel object in the open field</caption><thead><tr><th rowspan="2">Difference (session 4 – session 3)</th><th colspan="2">Control</th><th colspan="2">Handled</th><th rowspan="2"></th></tr><tr><th>Familiar object</th><th>Novel object</th><th>Familiar object</th><th>Novel object</th></tr></thead><tbody><tr><td>Locomotion in peripheral parts</td><td>5.50 ±2.26</td><td>-2.11 ±2.99</td><td>0.89 ±1.95</td><td>8.44 ±1.43 *</td><td>I</td></tr><tr><td>Locomotion in central part</td><td>0.50 ±0.73</td><td>0.67 ±0.37</td><td>1.22 ±0.72</td><td>1.00 ±0.47</td><td></td></tr><tr><td>Total locomotion</td><td>6.00 ±2.46</td><td>-1.44 ±3.31</td><td>2.11 ±2.42</td><td>9.44 ±1.43 *</td><td>I</td></tr><tr><td>Number of rears</td><td>2.90 ±2.09</td><td>-0.67 ±2.12</td><td>2.33 ±1.96</td><td>4.22 ±1.22</td><td></td></tr><tr><td>Motionless time</td><td>-17.90 ±15.05</td><td>30.55 ±15.00 #</td><td>1.00 ±4.60</td><td>-15.89 ±5.61 *</td><td>I</td></tr><tr><td>Number of SAPs</td><td>-0.40 ±0.31</td><td>0.22 ±0.28</td><td>-0.33 ±0.29</td><td>-0.11 ±0.39</td><td></td></tr><tr><td>Time of contact with objects</td><td>-1.60 ±2.28</td><td>3.11 ±1.69</td><td>-2.44 ±2.70</td><td>13.78 ±2.52 * ###</td><td>H, O, I</td></tr><tr><td>Number of “object climbing”</td><td>-0.60 ±0.54</td><td>0.00 ±0.24</td><td>-1.11 ±0.59</td><td>1.33 ±0.33</td><td>O</td></tr><tr><td>Number of “head into one object”</td><td>-0.20 ±0.13</td><td>-0.11 ±0.26</td><td>-0.33 ±0.29</td><td>1.22 ±0.40 * ##</td><td>H, O, I</td></tr></tbody></table>

Differences Session 4 – Session 3 for C (familiar object, n = 10; novel object, n = 9) and H (familiar object, n = 9; novel object, n = 9) rats in the open field. Data are given as mean ± SEM. Statistics: H, postnatal handling significant main effect; O, Object replacement significant main effect; I, significant interaction between these two factors; Post-hoc comparisons: * p < 0.05, Comparison of C and H rats within the same object situation; # p < 0.05, ## p < 0.01, ### p < 0.001, Comparison of rats from the same rearing condition with respect to object replacement.
<figure><img src="image_4.png" /><figcaption>Figure 2: Motionless time in the open field.</figcaption></figure>

Motionless time (seconds) in the open field during session 1 to 3. Data are given as mean + SEM. Statistics: significant main effects of PH [p < 0.01] and Session [p < 0.001] and significant effect of interaction between these two factors [p < 0.01]. Post hoc comparisons: *** p < 0.001, Comparison of C and H rats within session 3.
<figure><img src="image_5.png" /><figcaption>Figure 3: Time of contact with the objects.</figcaption></figure>

Time of contact with the object in the open field (seconds). Data are given as mean ± SEM.
Statistics: significant main effects of PH [p < 0.01] and Session [p < 0.001]. Post hoc
comparisons: *** p < 0.001, Comparison of sessions 1 and 3.
<figure><img src="image_6.png" /><figcaption>Figure 4: Open field, Object replacement</figcaption></figure>

Difference (session 4 – session 3) in total locomotion (A), motionless time (B), time of contact with the familiar / new object (C) and frequency of “head into an object” (D) in the open field. Data are given as mean ± SEM. Post hoc comparison: * p < 0.05, Comparison of C and H rats in the novel object situation; **, p < 0.01, ***, p < 0.001, Comparison of the two groups of H rats with respect to object replacement.