# The positive effects of postnatal handling on defensive burying are more obvious in a situation that enlarges the potential coping responses

Vincent Roy, Pierre Chapillon

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Vincent Roy, Pierre Chapillon. The positive effects of postnatal handling on defensive burying are more obvious in a situation that enlarges the potential coping responses. Behavioural Brain Research, 2002, 136, pp.67 - 73. 10.1016/S0166-4328(02)00095-5. hal-01576707

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Roy V, Chapillon P. The positive effects of postnatal handling on defensive burying are more obvious in a situation that enlarges the potential coping responses. Behav Brain Res. 2002 Oct 17;136(1):67-73. doi: [10.1016/s0166-4328(02)00095-5](https://doi.org/10.1016/s0166-4328(02)00095-5).

The positive effects of postnatal handling on defensive burying are more obvious in
a situation that enlarges the potential coping responses

Vincent ROY *, Pierre CHAPILLON

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

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

Abstract

A recent study reported few effects of postnatal handling in the defensive burying test. Since the importance of testing conditions has been emphasized in this paradigm, the lack of handling effects in this test could be attributed to the testing situation. Our experiment was carried out to test whether postnatal handling in DA/HAN strain rats have positive effects in two situations of the defensive burying test: a no-hide situation, in which avoidance of the probe was not possible, and a hide situation, in which animals were allowed to avoid the probe by sheltering in a hiding compartment. Our results showed no difference between control and handled rats defensive reactions in the no-show situation. However, the general coping style of handled rats was different, mostly by a higher level of exploration of the apparatus and of the probe. In the hide situation, the time spent burying the probe was significantly lower in handled rats and they also spent less time in the hiding compartment. Those results, along with approach / avoidance behaviours directed towards the probe demonstrate that postnatal handling do have some positive effects in the defensive burying test. In addition, our results also point out that testing situations in which complex coping strategies are available are appropriate to test the effects of early stimulation such as handling.

**Keywords:** Postnatal handling, DA/HAN rats, Defensive behaviours, Probe burying, Coping.
# 1 Introduction

Postnatal handling, consisting of a short daily maternal separation between birth and weaning, has been found to considerably reduce emotional reactivity of rats in a large amount of behavioural tests including conditioned and unconditioned conflict tests [1, 13, 22, 21].

A recent study by Merloo et al. (1999) reported beneficial effects of neonatal handling in Wistar rats. Clear results were obtained from the elevated plus maze and were strengthened by physiological data from conditioned fear and novelty reaction tests. Surprisingly, this study did not prove substantial effects of postnatal handling in the defensive burying test, a paradigm that was yet validated as an appropriate model of animal anxiety [25,27]. It seems that at least two explanations could be advanced.

First, burying was also described as a predominant defensive behaviour of rodents in response to various stimuli [3, 11, 18, 20]. Recently, it was proposed that behavioural responses observed in classical and more ethoexperimental tests are measuring different components of anxiety [2] and are sustained by different neurobiological substrates [1]. Therefore, it is possible that handling does not influence defensive reactions as it does in the reactions elicited in classical tests of anxiety.

The second explanation could also emerge from testing conditions. Indeed, situations that are highly anxiogenic, or oppositely not enough anxiogenic, may not be appropriate for the detection of handling effects. A series of studies emphasized the importance of testing conditions in the defensive burying paradigm. In particular the opportunity to avoid the threatening source (i.e. by using a double compartment apparatus or a larger apparatus) introduces variations in the defensive behaviour and enlarges the potential responses of rats and gerbils [5,13,18,26]. According to their anxiety state, rats may adopt an active avoidance strategy, by exploring and burying the probe, or a passive avoidance strategy [26]. This passive avoidance strategy, consisting in a hiding or sheltering response, has already been reported during expositions to predators or to their odours and takes part of the rodent's defensive repertory [3,6,31]. Because of a possible competitive occurrence with burying [26], this hiding response may be helpful to separate animals with different profiles in the defensive burying test.

Our experiment was designed to show that testing conditions can influence the effects of postnatal handling in DA/HAN strain rats subjected to the defensive burying test. Given the above considerations, we performed a detailed behavioural description of C and H rats submitted to a no-hide situation of test, with no escape available, or to a hide situation in which a hiding compartment was accessible. Since a preliminary study in
our laboratory showed beneficial effects of handling in the elevated plus maze and the free exploration test (unpublished data) we predicted that H rats would be less affected than C rats in the no-hide situation, especially on behavioural parameters related to classical test of anxiety (i.e. locomotion and exploration). In the hide situation, due to the larger diversity of potential responses, we expected to find more pronounced differences between C and H rats, and possibly on defensive parameters.

# 2 Material and methods

## 2.1 Animals

Forty-two DA/HAN male rats from our own breeding colony were used in this study. 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 guide for care and use of laboratory animals established by the National Institute of Health of the United States of America and with applicable guidelines from the Ministère de l'Agriculture, France.

## 2.2 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. Handling 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, non-handled animals remained in their home cages and were only manipulated once a week for cage cleaning. This procedure based on previous works [8,10,23] was already used in our laboratory and showed significant effects in DA/HAN rats subjected to classical tests of anxiety [22].

At day 22, the pups were sexed and weaned. Males from the same condition were housed by group of 4 (± 1) and left undisturbed until behavioural testing.
## 2.3 Defensive burying test

Animals were tested in one of two different situations of the defensive burying test between 9 and 10 weeks of age. The test was performed under red light condition (approximately 30 lux) during the first part of the dark phase (between 14h00 and 18h00). Animals were habituated to the testing room for a period of at least 30 min before the test.

Animals were tested in a wooden apparatus (90 cm long, 15 cm large and 30 cm high) divided into two compartments (Fig. 1). The first compartment, named the shock compartment, was 60 cm long and the second one, named the hide compartment was 30 cm long. The two compartments were separated by a wooden wall and interconnected by a small removable door. To allow measurement of locomotion and avoidance of the probe in the shock compartment, this latter was virtually divided into two equal parts: part 1, where the probe was inserted, and part 2 which gives access to the hide compartment when the door was open. The ceiling and one side of the shock compartment were made of transparent Plexiglas to allow behavioural observations whereas the sides and the ceiling of the hide compartment were made of wood to provide a maximum darkness inside. The floor of the two compartments was covered by 4 cm of clean sawdust. The probe was composed of two electric wires wrapped around a wooden cylinder (6 cm long and 1 cm in diameter) and connected to a power generator that delivered 1 mA average shocks.

### 2.3.1 No-hide situation

In this situation, the rats (C group, n = 11 and H group, n = 10) were tested in the shock compartment only, with no access to the hide compartment (i.e. the removable door was in place). At the beginning of the test, a rat was introduced in the part 2 of the shock compartment, with its head facing the part 1. The probe was not in place and the behaviour of the rat was recorded for a 5 minutes period of habituation. Behavioural parameters such as locomotion betweens part 1 and 2, time spent in each part, total number of rears and time spent digging sawdust were recorded. At the end of the habituation period, the probe was mounted on the end wall of the part 1, 2 cm above the sawdust and the rat's behaviour was video recorded for a 15 minutes period after the first shock. As during the habituation period, locomotion, numbers of rears and the time spent digging were recorded. In addition, behaviours directed towards the probe were measured: the time spent burying the probe, the number of sniffing (closer than 5 millimetres), stretched attended postures (SAPs) and additional shock. The times spent
in part 1 and 2 of the shock compartment were also recorded as well as the latency to
come back in part 1 after the first probe shock.

2.3.2 Hide situation

In this situation, the animals were given free access to the hide compartment, by
removing the small door between the shock and the hide compartments. As for the no-
hide situation, the test began with the introduction of a rat, in part 2 of the shock
compartment, for a 5 minutes period of habituation. After introduction of the probe and
first shock, the behaviours of rats were also recorded for 15 minutes. The same
behavioural parameters as those measured in the no-hide situation were recorded, in
addition to the time spent in the hide compartment.

The mean height of bedding material that is usually measured in the classical version of defensive burying was not recorded because this measure does not seem reliable when the testing apparatus is large [18] According to the description of Pinel et al., we observed that some rats engaged defensive burying from the opposite location of the probe, accumulating bedding material in front of the probe instead of on the probe.

**2.4 Data collection and statistics**

Data were collected from video analysis by using Etholog software version 2.25 [15]. All data are given as mean values ± SEM and were analysed with Statistica (version 5.1). Non-normal distribution of several variables made necessary the use of non-parametric tests. Fisher's exact probability was used to compare the proportions of rats that displayed defensive burying in the two groups for each situation. Mann-Whitney U-tests were performed to compare the rats from different rearing condition in the two different situations of test.

**3 Results**

Only rats that detected the probe in less than 5 minutes and that received at least one shock from the probe (measured by a startle response) were kept for statistical analysis.

**3.1 Habituation sessions**

In the no-hide situation, H animals displayed a higher locomotion during the habituation
period [U (10, 11) = 16, p < 0.01] but did not differ from C rats on other parameters. In
particular, no difference was found between the two groups for the time spent in
proximal and part 2 of the shock compartment.
During the habituation period of the hide situation test, locomotion was higher in H animals [U (11, 10) = 20.5, p < 0.05]. Moreover, H animals spent more time in the part 1 of the shock compartment [U (11, 10) = 16, p < 0.01] and less time in the hide compartment [U (11, 10) = 17, p < 0.01] than C rats.

## 3.2 Testing sessions

Fig. 2 shows the proportion of rats that displayed defensive burying in each group for the two test situations.

In the no-hide situation, 9 out of 10 C rats and 9 out of 11 H rats buried the probe. Those proportions were not statistically different [Fisher's exact probability, p = 1]. In the hide situation, the proportion of C rats that buried the probe was 8 out of 11 and quite the same than in the no-hide situation [Fisher's exact probability, p = 0.587]. In the H group, this proportion dropped to 3 out of 10 and the difference with the no-hide situation was significant [Fisher's exact probability, p = 0.030]. Therefore, the proportion of C animals that displayed defensive burying in the hide situation was higher than in the H group (8 out of 11 vs. 3 out of 10), although this result didn't reach significance [Fisher's exact probability, p = 0.086].

### 3.2.1 No-hide situation

Significant behavioural differences were observed between C and H rats during the 15 min of test (see Table 1). The latency to come back in part 1 was higher in C animals but this result did not reach significance [U (10, 11) = 28, p = 0.057]. Locomotion was higher in H rats [U (10, 11) = 9.5, p < 0.01] as well as total numbers of rears and sniffing [respectively U (10, 11) = 6, p < 0.001 and U (10, 11) = 18, p < 0.01]. H animals spent more time in part 1 [U (10, 11) = 11, p < 0.01] than C rats and therefore less time in the part 2 [U (10, 11) = 11, p < 0.01]. Importantly, the time spent burying the probe (see Fig. 2) was higher in C animals but the difference was not significant [U (10, 11) = 41.5, p = 0.34]. The total number of SAPs did not differ between the two groups.

### 3.2.2 Hide situation

The 15 minutes of test in the hide situation also led to several differences between C and H animals (see Table 2). As in the first situation, the latency to come back in part 1 was longer in controls [U (11, 10) = 13, p < 0.01] (see Fig. 3). H animals had a higher locomotion [U (11, 10) = 3, p < 0.001] and spent more time in parts 1 and 2 of the shock compartment [respectively U (11, 10) = 21.5, p < 0.05 and U (11, 10) = 4, p < 0.001]. Consequently, C animals spent more time in the hide compartment [U (11, 10) = 8, p < 0.05].
0.001]. Nevertheless, C animals spent more time burying the probe [U (11, 10) = 23, p < 0.05] than their H counterparts (see Fig. 2). In contrary to the no-hide situation, the number of sniffing was not different between the two groups and C rats made more SAPs towards the probe [U (11, 10) = 24, p < 0.05]. On the other hand, time spent digging sawdust was longer for H than for C animals in this situation [U (11, 10) = 20, p < 0.05].

### 3.2.3 Comparisons of the two situations

As shown in Fig. 3, the time spent burying the probe was not different for C rats in the two situations. On the contrary, H rats spent significantly more time burying the probe in the no-hide situation [U (11, 10) = 22.5, p < 0.05]. H rats were also faster to reenter part 1 of the shock compartment when hiding was possible thought not significantly. In C rats, the tendency was inversed since their latency to reenter part 1 was slightly higher in the hide situation.

Comparisons of sniffing, additional contacts, SAPs, and latency to re-enter part 1 of the shock compartment were not significant. Other comparisons between the two situations were not performed since some behaviours not related to the probe may have occurred in the hiding compartment.

## 4 Discussion

The main goal of our study was to test the effects of handling in DA/HAN rats submitted to the defensive burying test. As a previous study found few effects of handling in this paradigm [12], we performed a detailed analysis of rat's behaviour in two different situations of defensive burying: one in which sheltering from the probe was very limited and one in which a hiding compartment was accessible.

In the no-hide situation, which corresponds to the classical situation of the defensive burying test, our results agree with those of Merloo et al. (1999). As observed by the authors, we found that a high proportion of rats buried the probe irrespectively of being handled or not in infancy. Moreover, the time spent burying the probe was higher in C rats but the difference with H rats was not significant.

Nevertheless, some differences were observed between C and H rats. In particular, H rats were faster to come back in part 1 of the shock compartment, thought not significantly, they spent more time in the proximity of the probe and showed more sniffing towards it. H rats also had a higher locomotion during the test and their total number of rears was more important. This lesser aversion towards the probe, along with more frequent exploration related behaviours certainly reflect a lower level of anxiety in
H animals. The higher locomotion level in H rats during the habituation period strengthens this observation. Indeed, a higher level of exploration in a mildly stressful novel apparatus has already been reported to reflect a lower level of anxiety in H rats [8,14].

Therefore, an opposition seems to appear between behavioural parameters related to locomotion and exploration, generally used in classical test of anxiety, and the behavioural parameters related to defensive reactions. A possible explanation for those results is that proximity of the probe during the test, and no way to escape, may have rendered the situation too anxiogenic to separate C and H rats on parameters related to the burying response. Besides, a recent study in our laboratory showed that BALB/c mice reared in an enriched environment buried cat feces as frequently and as long as control mice, though, enriched mice were found to be less reactive in other tests of anxiety and presented a lower corticosterone response after exposition to the cat feces [21].

The hide situation test confirmed this analysis. It was previously showed that passive avoidance of the threatening source was adopted by rats when burying was not possible and / or escape was possible [18,26]. The hide situation that we used allowed both passive avoidance (i.e. hiding response) and active avoidance (i.e. burying response and exploration of the probe). This diversity in potential responses might have rendered the situation less threatening for rats, at least for H rats. The results that we obtained confirm this latter hypothesis. The proportion of H rats that buried the probe and the time they spent burying were significantly decreased in this situation whereas it did not vary in C animals. Moreover, in H rats, the latency to come back in the part 1 was shorter than in the no-hide situation. In addition, the time spent in the hide compartment was not excessive and the possibility to hide or shelter from the threatening source did not suppress exploration activities in H animals. Oppositely, C rats spent most of their time in the hide compartment when allowed and exploration of the apparatus was highly decreased. Though, the burying response towards the probe remained unmodified and oppositely to H animals their latency to come back in part 1 was increased. Besides, this latency was significantly higher in C animals whereas the difference did not reach significance in the hide situation.

Taken together, these results indicate that control and handled rats did not use similarly the possible coping strategies offered by the second situation of test. It seems that fear of the probe, and the anxiety resulting, remained unchanged in C animals that used the hide compartment to develop an important hiding response. Besides, this hiding response was so important in some C rats that they did not buried the probe because
they spend most of their time in the hide compartment, and showed a very high latency
to come back in part 1 after being shocked. H animals also used the hide compartment
as a rapid possible way to escape the probe after the first shock but only for a very short
time. Therefore, oppositely to C rats, exploration of the shock compartment was higher
along with a decreased burying response. This last observation, along with our previous
results, traduces a decreased anxiety in H animals, both in their defensive response and
in their general coping style in front of the testing situation.

Finally, the effects of postnatal handling on coping reactions lead to an apparent non-
avoidance of the danger represented by the probe. This could appear as a non-adaptive
behaviour but, in fact, we think that the effects of handling must be positive. Indeed,
whatever the situation, H animals did not make more additional contacts to the probe
than C ones and their exploration of the probe generally began with SAPs, reflecting a
cautious level of risk assessment [17,29]. In that way, the behaviour of H rats is as
adaptive as the behaviour of C rats. In addition, it may be even more adaptive once the
first threat of the shock has gone, when the H rats explore the probe instead of
sheltering. Actually, exploration of a threatening source in order to recover information
on it might be adaptive. For example, this argument was advanced in case of ground
squirrels confrontation with rattlesnakes [16]. Exploration of the apparatus in order to
find possible ways of escape would also be more adaptive. This latter observation
agrees with previous reports in which H rats were faster to learn an avoidance task
[7,24], certainly by adopting a more active strategy than C rats.

**5 Conclusion**

In addition to the well-known effects in classical tests of anxiety, our study
demonstrated that postnatal handling in rats is efficient in decreasing anxiety as
measured by a defensive task, the probe burying test. Therefore, if the reactions
observed in defensive tests and in classical tests of anxiety have different
neurobiological substrates [1,2], it appears that postnatal handling affect both of them.
Moreover, in accordance with previous reports, our study emphasized the importance of
testing conditions in the defensive burying paradigm. In particular, the possibility to
escape from the probe influences the occurrence of defensive burying and a detailed
behavioural analysis appears necessary to distinguish animals from different anxiety
profiles when escape is not available.
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**Fig. 1:** Representation of the different parts of the apparatus showing the location of the wire probe and the removable door separating the shock and the hiding compartments.

**Table 1: Behaviours of C and H rats in the No-hide and in the Hide situation**

Data are given as mean values ± SEM. Statistic: Mann-Whitney U test (\*\* p < 0.01; \*\*\* p < 0.001 comparisons between C and H rats within same situation; ° p < 0.05 comparison between H rats from the two situation of test). Comparisons for the parameters in the upper part of the table were not realized between the two situations.

**Fig. 2: Proportion and time of Burying**

A: Proportion of C and H rats that displayed a burying response in each situation of test. Fisher's exact probability (° p < 0.05 H Hide compared to H No-hide).

B: Time in seconds, spent burying the probe for C and H animals in each situation of test. Mann Whitney U tests (* p < 0.05 C rats compared to H rats, ° p < 0.05 H Hide compared to H No-hide).
<figure><img src="image_3.png" /><figcaption>Fig. 1: Apparatus</figcaption></figure>
<table><caption>Table 1: Behaviours of C and H rats in the No-hide and in the Hide situation</caption><thead><tr><th rowspan="2"></th><th colspan="2">No-hide situation</th><th colspan="2">Hide situation</th></tr><tr><th>C rats (n=10)</th><th>H rats (n=11)</th><th>C rats (n=11)</th><th>H rats (n=10)</th></tr></thead><tbody><tr><td>Time in part 1</td><td>94.0 ±32.0</td><td>273.9 ±22.5 **</td><td>83.7 ±28.5</td><td>197.1 ±17.2 *</td></tr><tr><td>Time in part 2</td><td>806.0 ±32.0</td><td>626.1 ±22.5 **</td><td>221.6 ±41.8</td><td>425.7 ±18.2 ***</td></tr><tr><td>Time in the hiding compartment</td><td></td><td></td><td>594.7 ±61.5</td><td>277.2 ±23.0 ***</td></tr><tr><td>Locomotion</td><td>10.5 ±3.3</td><td>28.8 ±3.0 **</td><td>21.82 ±4.3</td><td>50.9 ±2.4 ***</td></tr><tr><td>Number of rears</td><td>21.9 ±4.0</td><td>53.6 ±4.1 ***</td><td>13.82 ±3.0</td><td>45.6 ±3.6 ***</td></tr><tr><td>Latency to come back in part 1</td><td>347.0 ±114.4</td><td>88.1 ±31.5</td><td>449.0 ±112.7</td><td>29.9 ±5.9 **</td></tr><tr><td>Time burying the probe</td><td>28.5 ±9.9</td><td>9.8 ±2.7</td><td>27.9 ±10.1</td><td>2.6 ±2.3 * °</td></tr><tr><td>Time digging sawdust</td><td>22.2 ±6.4</td><td>30.3 ±5.9</td><td>4.6 ±1.6</td><td>20.3 ±4.5 *</td></tr><tr><td>SAP towards the probe</td><td>3.3 ±1.1</td><td>4.2 ±1.7</td><td>6.0 ±1.4</td><td>1.9 ±0.7 *</td></tr><tr><td>Sniffing</td><td>5.6 ±1.6</td><td>14.6 ±2.4 **</td><td>7.0 ±2.2</td><td>11.9 ±1.9</td></tr><tr><td>Additional shock</td><td>0.2 ±0.1</td><td>1.0 ±0.3</td><td>0.6 ±0.3</td><td>0.8 ±0.2</td></tr></tbody></table>
<figure><img src="image_4.png" /><figcaption>Fig. 2: Proportion and time of burying</figcaption></figure>