Understanding the behavioural phenotype of the precocial spiny mouse

Behavioural Brain Research 275 (2014) 62–71

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Research report

Understanding the behavioural phenotype of the precocial spiny mouse Udani Ratnayake a,b,∗ , Tracey Quinn a , Kerman Daruwalla b , Hayley Dickinson a,1 , David W. Walker a,c,1 a

The Ritchie Centre, MIMR-PHI Institute, Monash University, Clayton, Melbourne 3168, Australia The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville 3010, Australia c Department of Obstetrics & Gynaecology, Monash University, Clayton, Melbourne 3168, Australia b

h i g h l i g h t s • • • •

The effects of sex and age on the behaviour of spiny mice (Acomys cahirinus) were examined in this study. Spiny mice were behaviourally characterised on a set of behavioural test commonly used to assess rodent models of disease. Spiny mouse demonstrate precocial development of exploratory activity, locomotor coordination and social behaviours. Fear and anxiety behaviours, learning and memory, and sensory gating can be assessed from relatively early postnatal development in the spiny mouse.

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Article history: Received 1 August 2014 Received in revised form 12 August 2014 Accepted 16 August 2014 Available online 23 August 2014 Keywords: Acomys cahirinus Exploratory activity Fear and anxiety Sensorimotor gating Motor coordination Social interaction

a b s t r a c t The use of the spiny mouse (Acomys cahirinus) in experimental research is steadily increasing, due to the precocial nature of this species and the similarities in endocrinology to the human. The characterisation of normal behavioural traits throughout development has not been comprehensively measured in the spiny mouse. Therefore the aim of this study was to behaviourally phenotype the spiny mouse, with the use of behavioural paradigms commonly used to assess behaviour in rat and mouse models of human behavioural disorders such as autism, attention-deficit disorder, and schizophrenia. Male and female spiny mice were assessed at 1–5, 10–15, 20–25, 40–45 and 80–85 days of age using the open field test, novel object recognition test, rotarod, elevated plus maze, a social interaction test, and prepulse inhibition. Exploratory activity, motor coordination, fear, anxiety and social behaviours could be accurately measured from 1 day of age. Open field exploration and motor coordination on a modified rotarod were precociously developed by 10–15 and 20–25 days of age, respectively, when they were equivalent to the performance of conventional adult mice. Learning and memory (assessed by the novel object recognition test), and sensory gating (prepulse inhibition) could be reliably determined only after 20–25 days of age, and performance on these tests differed significantly between male and female spiny mice, particularly in adulthood. This study characterises the behavioural traits of spiny mice and provides important information about critical periods of behavioural development throughout postnatal life. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The use of animal models to study human disease is essential for the better understanding of the disease process, and to develop and test potential treatments. Rats and mice are often

∗ Corresponding author at: The Florey Institute of Neuroscience and Mental Health, University of Melbourne, 30 Royal Parade, Parkville, Victoria 3010, Australia. Tel.: +61 3 9035 6624; fax: +61 3 9035 3107. E-mail address: Udani.ratnayake@florey.edu.au (U. Ratnayake). 1 Joint senior authors. http://dx.doi.org/10.1016/j.bbr.2014.08.035 0166-4328/© 2014 Elsevier B.V. All rights reserved.

used for this type of research, but from a developmental point of view these conventional laboratory rodents do not possess many of the fundamental features of human pregnancy. Many aspects of the newborn human reflect a precocial mode of development; such as relatively complete neurogenesis [1,2] (and organogenesis, in general) by birth, as well as an altricial mode of development in that the infant is highly dependent on the mother for nutrition and mobility for an extended period after birth. This pattern of development is unique to the human. The spiny mouse (Acomys cahirinus) is a small rodent-like animal which replicates many of the hormonal characteristics of human pregnancy [3] as well as the precocial mode of organ development, including

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the brain, where considerable neurological maturity is reached at birth [1]. The spiny mouse is a desert rodent species native to regions of Africa and the Middle East. The neonates are precocial, in that they are capable of movement from birth, and self-feeding within 4–6 days after birth [4]. Compared to other rodents, spiny mice are born with a well-developed coat, eyes and ears are open and visual and auditory functions are well developed, together with other sensory [4] and autonomic functions, including thermoregulation [5]. They are capable of locomotion and have vestibular function (i.e., negative geotaxis) from soon after birth (hours), and it is therefore possible to make meaningful quantitative and qualitative assessments of behaviour from postnatal day 1 in this species. Spiny mice have a relatively long gestation of 38–39 days and a small litter size (usually 1–3) [6]. In addition to the longer exposure to the maternal/intrauterine environment, foetal development is somewhat more comparable to the human in that, unlike rats and mice, the foetal adrenal gland secretes cortisol and dehydroepiandrosterone (DHEA) [3], and DHEA is also synthesised in the foetal and neonatal brain [3]. The presence of cortisol and not corticosterone in this species is indeed a highly significant finding, with various implications for the field of developmental endocrinology, brain–adrenal–placental interactions, and the effects of stress on the developing brain during pregnancy. The fact that the adrenal gland of this animal produces not only cortisol but also small amounts of the androgen DHEA during gestation makes it a better model of human foetal development compared to both conventional rat and mouse species. In addition, the maximum rate of brain growth occurs in the spiny mouse, as in the human infant, at near the time of birth. Neuroanatomically, cortical and limbic development at 30 days (0.75) gestation in the spiny mouse is equivalent to 24–26 weeks gestation in the human infant [1]. The advanced development of the spiny mouse at birth provides an opportunity to assess the contribution of normal and abnormal in utero development on the brain and behaviour postnatally. This is particularly relevant to the growing consensus that neuropsychiatric disorders such as autism and schizophrenia have origins in foetal life [7–9]. However, to fully understand aberrant behaviour in the spiny mouse, it is first necessary to fully characterise normal behavioural traits, and particularly for developmental studies, to describe the changes that occur from the early neonatal period into early adult life. Therefore, the aim of this study was to characterise the development of behaviour from birth in the spiny mouse, with a focus on behavioural paradigms commonly used to identify abnormal behaviour and cognition that, in conventional mice and rats, have been used as models of neuropsychiatric disease. 2. Methods 2.1. Animals Spiny mice (A. cahirinus) were obtained from the breeding colony maintained at Monash University. The mice were housed under controlled temperature (25 ± 0.5 ◦ C) and humidity (30 ± 5%) conditions, and a 12 h light–dark cycle (lights on at 0700 h), using the breeding protocols previously described [10]. All procedures received prior approval from the Monash University Animal Ethics Committee and were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

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cohorts for behavioural testings (see Fig. 1A). Cohort 1 pups (n = 8 male, n = 8 female) were assessed using, in sequence, the open field test, novel object recognition test, rotarod, and elevated plus maze at 1–5 10–15, 20–25, 40–45 and 80–85 days of age. Cohort 2 pups (n = 8 male, n = 8 female) were assessed using the social interaction test and prepulse inhibition (PPI) at similar ages, except the PPI test was not conducted at the 1–5 days of age. The pups were separated into two cohorts to allow for a 1- to 2-day rest period between tests in each age range to minimise stress effects on behaviour that would have occurred if each animal was to be subjected to all tests [11]. Testing was always conducted between 1000 and 1300 h to avoid diurnal effects [12], and all animals were habituated to the test room and apparatus for at least 30 min prior to obtaining any measurements. 2.3. Open field The open field test was conducted on the first day of each age range days 1, 10, 20, 30, 40 and 80; Fig. 1). Individual animals were placed in the centre of a square field 50 cm × 50 cm, with 40 cm high walls which are uniformly black and provide no visual cues. Lighting levels were 2.8 lux for all trials. Activity was recorded using a video camera over the next 5 min. Post-acquisition analysis of the activity using CleverSys software (CleverSys Inc., USA) was used to track the movement of the animals throughout the open field trial by identifying the nose, body and tail. This software also allowed the measurement of distance (cm) travelled and time (seconds) spent in the central zone (defined as the area of the field excluding the 10 cm outer perimeter) vs. the outer zone. 2.4. Novel object recognition test The novel object recognition test (NORT), which assesses non-spatial memory [13], was conducted immediately after the completion of the 5 min open field test which acts as a habituation trial to reduce the contribution of anxiety and stress on the outcome. Two bottles of identical shape, colour and size were placed in the open field approximately 6 cm from the walls of the enclosure to ensure the animal had an unobstructed view of the objects at all times. The animal’s behaviour and investigation of the objects was recorded for 10 mins (learning trial), after which the animal was returned to its home cage for a 1 h retention period. During this time one of the bottles was replaced with an object of a different shape and colour (the novel object); both the novel and familiar object were wiped down with 70% ethanol at this time to remove olfactory cues. The animal was then replaced in the open field (‘recall trial’) and it’s behaviour and exploration of the two objects was recorded for the next 10 min. In both the learning and recall trials behaviour was scored as positive, actual investigation when the animal’s nose was pointed at, and within 2 cm of the object, as used elsewhere [13–20]. Post-acquisition analysis with the CleverSys software was used to measure the time animals spent investigating the object. The total time spent exploring each object in the recall trial was used to calculate the ‘discrimination index’, calculated by subtracting the time spent exploring the familiar object from the time spent exploring the novel object, divided by the total time spent exploring both objects. Thus, a discrimination index of 1 indicates that the animal spent all of the time exploring the novel object, 0 indicates no preference for either object, and −1 indicates the animal spent all the time exploring the familiar object. 2.5. Rotarod

2.2. Behavioural testing procedures One male and one female spiny mouse were selected from the litters of at least 16 dams and randomly assigned to one of two

A rotarod trial was conducted 1–2 days after the open field and NORT test. A standard rat accelerating rotarod apparatus has an inner axle with a diameter of 2.5 cm with 12 cm walls. This

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Fig. 1. The test battery to investigate the behavioural phenotype of spiny mice (A). Spiny mice were allocated to cohort 1 or 2 at birth, and the sequence of tests applied at each of the developmental period as shown. The modified rotarod with rungs is shown in panel B.

apparatus was adapted by adding 12 plastic rungs, placed 1.5 cm beyond the surface of the inner axle (see Fig. 4B). The addition of the rungs made it a requirement that the animal place all 4 paws correctly on the rungs in order to stay on the rotarod as it rotated. The addition of rungs was done to make the rotarod test more challenging for spiny mice which are more agile, particularly as neonates, compared to rats and mice. The animal is placed on the rungs when the apparatus is at rest, and accelerating rotation is immediately commenced with velocity increasing by 7.2 revolutions/min/min. As habituation, all animals were allowed two learning trials on the rotarod, and then the latency to fall was determined over 5 subsequent trials (T1–T5). An interval of 10 min was allowed for recovery between T2 and T3 and a maximum latency of 5 min was allowed for each trial. 2.6. Elevated plus maze 1–2 days after the rotarod test, the elevated plus maze (EPM), consisting of 40 cm (length; L) and 10 cm (width; W) arms raised 50 cm above the floor, was used to assess anxiety and fear behaviours [21]. Two opposing arms were without sidewalls, and at right angles to these were two arms with 20 cm high walls. All four arms were connected to a central 10 cm × 10 cm platform, on which the animal was placed at the start of the test, facing one of the open arms. Exploratory behaviour was recorded for 5 min by a video camera placed above the apparatus and CleverSys software was used post acquisition to track the movement of the animal and determine the time spent in each arm throughout the trial. 2.7. Social interaction test Social interaction test was first conducted at 1–5 days of age, and again at 11–13 days of age (1–2 days after the PPI test). The social interaction apparatus consists of an open top rectangular box 60 cm × 30 cm × 45 cm (L × W × height; H) divided into three chambers 20 cm × 30 cm (L × W) with 8 cm × 8 cm opening cut into them to allow the test animal to move between chambers. Chambers were numbered 1–3 from left to right for reference. Chambers 1 and 3 contained large cylinders measuring 30 cm × 8 cm (H × diameter; D) drilled with numerous 0.6 cm (D) holes to confine the stranger spiny mouse during the test, but to allow adequate airflow and olfactory stimulation for the test spiny mouse. The stranger spiny mouse was a spiny mouse age- and sex-matched to the test spiny mouse. The social interaction test consisted of the test spiny mouse being habituated to the social interaction apparatus for a 5 min period by placing it alone in the middle chamber, after which it was taken out and the stranger spiny mouse was placed in the cylindrical enclosure in chamber 1 or 3. (The placement of the stranger

spiny mouse into chamber 1 or 3 was alternated between trials to account for any preference for a particular chamber of the apparatus.) The test spiny mouse was placed back in the middle of chamber 2 and video recording was taken as the spiny mouse explored the apparatus. Post-acquisition analysis consisted of determining the number of crossings into the chamber containing the stranger spiny mouse, and time spent interacting with the stranger spiny mouse, as measured using the CleverSys software. Interaction with the stranger spiny mouse was deemed to occur when the animal’s nose was pointed at, and within 2 cm of the stranger spiny mouse’s enclosure. 2.8. Prepulse inhibition Prepulse inhibition (PPI) test was first conducted at 10 days of age, as this test gave unreliable results when used with younger animals. Acoustic startle PPI was measured using the SR-LAB startle apparatus (San Diego Instruments, San Diego, USA). The soundproof startle chamber contained a clear Plexiglas cylinder resting on a piezoelectric transducer that detected the vibration caused by the movement of the animal. A computer connected to the apparatus recorded maximum startle responses and controlled the timing and presentation of the acoustic stimuli. Each test began with 2 min of apparatus acclimatisation, followed by 5 consecutive pulse-alone trials presented to habituate and stabilise the animal’s startle response. Each animal was then presented with a total of 35 pseudo-random trials of the 10 pulse-alone trials, 5 trials where no acoustic stimulation occurred (i.e., background noise), and 20 trials where a prepulse sound of 2, 4, 8 or 16 dB above background (70 dB) and 20 ms duration was presented 100 ms before the startle pulse. The session was concluded with 5 consecutive pulse alone trials. The interval between successive trials was variable with a mean of 20 s, but ranging from 10 to 30 s. The acoustic startle pulse itself was white noise at 115 dB and of 40 ms duration. The result was expressed as relative inhibition of the startle response due to the prepulse as follows: %PPI = (amplitude of startle pulse alone) − (amplitude of prepulse + startle pulse)]/(amplitude of startle pulse) × 100%). %PPI was calculated for all of the prepulse intensities. A higher %PPI score indicates a greater reduction in startle magnitude due to the prepulse, relative to the ‘pulse alone’ trials. 2.9. Statistics All data are presented as means ± SEM. All body weight and behavioural data were analysed using a two-way repeated measures analysis of variance (ANOVA) for the effect of age (PAGE ). Sex (PSEX ) and the interaction between age and sex (PINT ) using the

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Fig. 2. Locomotor activity in the open field and in the central zone of the open field, with age, in male and female spiny mice. Total distance travelled (A) and average velocity (B) in the open field test with age in male and female spiny mice. Distance and time spent in the central zone as a percentage is shown in (C) and (D), respectively. The data are shown as means ± SEM.

statistical package SPSS. However a three-way repeated measures analysis of variance (ANOVA) was used to analysed the effect of arm (PARM ) and chamber/enclosure (PCHAMBER/ENCLOSURE ), for the elevated plus maze and social interaction, respectively, in addition to PAGE , PSEX and PINT . The p-values for the main effects and interactions are listed on figures. A Bonferroni post hoc test was used as required and any asterisks on figures refer to these values. p < 0.05 was accepted as statistically significant unless otherwise stated, however a p value of <0.1 was considered noteworthy.

3. Results An important aim of this study was to determine how behaviour changed after birth, and at which postnatal age reliable results could be first obtained from the different tests outlined above in Sections 2.2–2.8. Body weight and size are important parameters that determine neonatal performance for specific tasks. The increase of body weight with age is shown in Table 1. As expected, body weight increased with age (ANOVA effect age: F4,120 = 798.4, p < 0.0001), but with a significant difference between male and female spiny mice (ANOVA effect sex: F1,30 = 5.75, p < 0.05). Post hoc analysis (Table 1) revealed that males weigh significantly more than female spiny mice from 40 to 45 days of age (ANOVA effect age × sex: F4,120 = 3.11, p < 0.05).

3.1. Open field Up to 5 days of age, spiny mouse neonates showed little mobility in the open field (Fig. 2A); moreover, after crawling to the side they preferred to remain against the wall, as shown by the very small amount of time (<5%) spent in the central zone (Fig. 2C). However, from 10 days of age they were very active, travelling on average 1700 cm over 5 min at a speed of ∼60 mm/s (Fig. 2B and D). Thus, the main age effect identified by ANOVA for total distance travelled (ANOVA main effect age: F4,60 = 66.35, p < 0.0001, Fig. 2A) and average velocity (ANOVA main effect age: F4,60 = 66.50, p < 0.0001, Fig. 2B) occurred because of the significant increases between 1–5 and 10–15 days of age, with no change thereafter. There was no effect of sex on the distance travelled or average velocity in the open field at any age. Throughout the 5 min trial in the open field, there was a progressive decrease in the distance travelled when measured at 1 min intervals (ANOVA interval effect: F1,60 = 102.83, p < 0.0001), which was similar for males and females and at all ages measured. Distance travelled and time spent in the central zone, expressed as a percentage of total distance and time in the open field, respectively, increased significantly up to 20–25 days of age (ANOVA main effect age: F4,60 = 29.27, p < 0.0001, Fig. 2C and D), with no difference between males and females on either of these measures.

3.2. Novel object recognition test (NORT) Table 1 Body weight (g) of male and female spiny mice with age. All data are shown as means ± SEM. Data were analysed by 2-way ANOVA. Post hoc test revealed **p < 0.01 compared to males. D, days; g, grams. Postnatal age (days)

Males (g)

1–5 10–15 20–25 40–45 80–85

6.43 10.87 17.76 30.25 36.20

± ± ± ± ±

0.28 0.39 0.86 0.79 0.78

Females (g) 7.23 10.94 17.17 27.36 34.27

± ± ± ± ±

0.34 0.35 0.65 0.44** 0.68

At 1–5 days of age there was little evidence that spiny mouse neonates spent any significant time ‘exploring’ objects in the learning trial, and therefore the discrimination index could not be determined in the recall trial. Time spent investigating the objects in the learning trial increased with age (ANOVA main effect age: F4,60 = 14.92, p < 0.0001, Fig. 3A), and after 40 days of age males were more active in this respect than females (ANOVA main effect sex: F4,60 = 5.81, p < 0.05, Fig. 3A). While males spend more time exploring objects during the learning trial, the discrimination index

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in the closed arms increased with age (ANOVA effect Arms × Age: F3,45 = 6.12, p = 0.01, Fig. 5A), although post hoc analysis revealed this was significant only after 40 days of age. The time spiny mice spent in the closed arms was significantly greater than the open arm of the EPM (ANOVA effect Arms: F1,45 = 164.23, p = 0.0001, Fig. 5B), and the time spent in the closed arms continued to increase with age (ANOVA effect Arms × Age: F3,45 = 3.82, p = 0.05, Fig. 5A). There was no effect of sex on time spent or distance travelled in either of the arms (Fig. 5A). 3.5. Social interaction Spiny mice spent a significantly greater amount of time in the chamber that contained the stranger mouse enclosure compared to the empty chamber, at all ages (ANOVA effect Chamber: F1,60 = 6.11, p < 0.05, Fig. 6A). However, post hoc analysis revealed that at 1–5 days of age male and female spiny mice spend significantly more time than the overall average in the stranger chamber than the empty chamber (Fig. 6A). Furthermore, at 80–85 days it is only the female spiny mice that show this preference (Fig. 6A). Overall spiny mice also spent significantly more time actually interacting with the enclosed stranger mouse compared to the empty enclosure (ANOVA effect Enclosure: F1,60 = 4.14, p < 0.05, Fig. 6B). There was no difference between male and female spiny mice in the spent in either chamber or the time spend exploring each enclosure (Fig. 6A and B). Fig. 3. Time spent exploring objects in the learning trial of the novel object recognition test with age in male and female spiny mice (A). The discrimination index calculated in recall trail of the novel object recognition test, with age in male and female spiny mice (B). The data are shown as means ± SEM. Post hoc test revealed *p < 0.01 and **p < 0.01.

calculated in the recall was significantly less for males, than for females (ANOVA main effect sex: F1,48 = 6.89, p < 0.05, Fig. 3B). A significantly greater discrimination index was obtained for the spiny mouse neonate from 20 to 25 days of age and older (ANOVA main effect age: F3,48 = 9.68, p < 0.0001, Fig. 3B), although there was a significant interaction effect of sex and age (ANOVA age × sex effect: F3,48 = 4.75, p < 0.01, Fig. 3B). Post hoc analysis revealed that male spiny mice explored the novel object significantly less at 10–15 (p < 0.01) and 80–85 (p < 0.05) days of age.

3.6. Prepulse inhibition Prepulse inhibition (PPI) increased significantly with age between 10–15 and 40–45 days of age, similarly for males and females (ANOVA effect Age: F3,57 = 2.89, p < 0.05, Fig. 7A). PPI increased in spiny mice as the intensity of the prepulse increased, however this was not evident until 20–25 days of age (ANOVA effect Intensity: F1,47 = 38.42, p < 0.0001, Fig. 7B–D). At 80–85 days of age, female spiny mice have significantly lower PPI compared to males, except when the intensity of the pre-pulse was 16 dB (ANOVA effect Sex: F1,11 = 10.59, p < 0.01, Fig. 7D). 4. Discussion

Spiny mouse neonates up to 5 days of age had little ability to walk on the rotarod, and fell off after 20–30 s, when the rotational speed was approximately 3 RPM. Performance on the rotarod did not noticeably improve with repetition, and therefore the average latency over the 5 trials was calculated. The average latency to fall increased significantly with age (ANOVA main effect age: F4,70 = 322.2, p < 0.0001), with most animals reaching the maximum time for the test (i.e., latency of 300 s) by 40–45 days of age (Fig. 4). The increase in latency for males appears to be greater than for female spiny mice, although this did not quite reach significance (ANOVA main effect sex: F1,70 = 3.33, p = 0.07, Fig. 4).

The aim of this study was to assess the behavioural phenotype of spiny mice from birth to adulthood on a battery of behavioural tests commonly used in rats and mice. This study demonstrates that these behavioural tests can also be used with spiny mice, but with some alterations in equipment and considerations regarding the timing of development of spiny mice, relative to other laboratory rodents, because the spiny mouse is clearly more precocial, specifically with respect to the development of the brain and other organs [1,5,6,22]. This study shows that comprehensive assessment of behaviour can occur from soon after birth, which is not possible in conventional rodents, and thus provides the opportunity to characterise the early onset of behavioural abnormalities that may arise because of perturbations that have occurred in utero; for example, those arising from maternal immune activation [23,24], foetal growth retardation, and birth asphyxia [25].

3.4. EPM

4.1. Exploratory behaviours and motor coordination

At 1–5 days of age, nearly all of the spiny mouse neonates fell from the EPM. However, from 10 to 15 days of age the spiny mice were able to move freely between the closed and open arms, and showed a significant preference for the closed arms (Fig. 5). Spiny mice travelled a significantly greater distance in the closed arms compared to the open arms of the EPM (ANOVA effect Arms: F1,45 = 67.91, p = 0.0001, Fig. 5A) and the distance travelled

The open field test is one of the oldest and most extensively used tests, which assesses normal spontaneous exploratory activity in rats and mice [26,27]. Spiny mice possess an inherent curiosity of a novel environment from an early age [28], and due to their precocial development [1,5,6,22] it is possible to assess exploratory activity and motor ability from as early as postnatal day 1, unlike commonly used laboratory rodents where locomotor immaturity

3.3. Rotarod test

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Fig. 4. Latency to fall (average of 5 trials, where the maximum latency for each trial is 300 s) during the rotarod test with age in female and male spiny mice. The data are shown as means ± SEM.

prevents meaningful testing until 7–8 days of age [29]. The relatively complete neurogenesis of essential brain regions from birth underlies the greater motor function of spiny mice compared to mice and rats [30,31]. The more advanced development of the axial muscles also determines the greater motor ability from birth. Exploration of the open field increased in spiny mice between 1–5 and 10–15 days of age, suggesting a greater interest in a novel environment with age. In a previous study [32], 70- to 80-day-old C57BL/6J male mice travelled approximately the same distance in a similar size open field as do the 10- to 15-day-old juvenile spiny mice. Spiny mice are very agile, and the height of walls of the open field, and the elevated plus maze had to be increased to prevent them escaping [unpublished observations]. The performance of spiny mice in the accelerating rotarod test is also consistent with more advanced motor capacity and coordination from soon after birth. Although the latency for spiny mice to fall

from the rotarod test increased with age, 1- to 5-day-old neonates were able to spend a significant amount of time on the modified apparatus, which required them to coordinate their fore- and hindlegs by grasping the rungs. By 40 days of age this test is no longer challenging and this finding needs to be taken into consideration for future studies when assessing spiny mice older than 40 days of age. In contrast, the rotarod test is regularly used and remains a challenge for mice and rats well into adulthood [29,32]. Adult mice are able to remain on the rotarod for an average of 120 s [32], whereas spiny mice of a similar age consistently remain aboard for the full 300 s of the test. The enhanced ability of spiny mice at earlier ages on these tests is likely to be underpinned by more advanced patterns of brain development in spiny mice during prenatal and postnatal life [30,31]. The rotarod was modified by adding rungs above the base of the central axle to increase the motor demands for the spiny mouse, as the central axle of the accelerating rotarod

Fig. 5. Distance (A) and time (B) in closed and open arms of the elevated plus maze with age in male and female spiny mice. The data are shown as means ± SEM. *p < 0.05 compared to open arm.

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Fig. 6. Time spent in each chamber and time spent interacting enclosed stranger mouse (as a percentage of the total trial) with age during the social interaction test. The data are shown as means ± SEM.

was not sufficiently challenging for these animals, who often voluntarily jump off the apparatus before it has accelerated by any significant amount [unpublished observations].

4.2. Anxiety behaviours Anxiety-related behaviours have conventionally been assessed by measuring exploration in the central zone of the open field, and selection between the open and closed arms of the elevated plus maze [21]. The validity of these tests depends on the ability of the animal to distinguish between ‘safe’ and ‘less safe’ situations. The open field test suggests that spiny mice do not make this distinction until about 10 days of age; while 1- to 5-day-old neonates preferred to remain near a wall, they nevertheless did not show ‘wall hugging’ behaviour (i.e., thigmotaxis). The distance travelled and time spent in the central zone increased progressively between 10 and 40 days of age, although the greater part of the time (∼90%) was always spent in the outer zone, suggesting that from 10 days spiny mice show the thigmotaxis expected of small rodents. The results of the EPM test are consistent with these findings, in that 1- to 5-day-old neonates were apparently unaware of the two predominating aspects (closed/safe vs. open/unsafe) of this apparatus, but after 10 days they spent significantly more time in the closed, safe arms. To an extent these results must depend on changes of the visual acuity with postnatal age. Structurally, the visual cortex continues to develop in early postnatal life in the spiny mouse [33], so that poor vision may account for the lack of exploration (open field) and lack of appreciation of the difference between open and closed arm of the EPM. However, spiny mice appear to display inherently lower levels of anxiety and fear compared to conventional rodents, as our wild-type adult spiny mice demonstrate comparable patterns of open field, central zone activity as do adult rats selectively bred from a lowanxiety behaviour line [34]. Spiny mice also show greater activity in open (dangerous/fearful) arms of the EPM compared to adult mice [35].

4.3. Memory and learning behaviours The novel object recognition test involves making alterations to previously stored information, and is thus an assessment of memory and learning [13,14,36]. An essential requirement of this test is that an animal has an innate preference for novel objects [13,14,36], and unlike the Morris water maze, this test requires little pre-training and does not involve a high fear and anxiety component. (Of note, the Morris water maze cannot be used with spiny mice, a desert dwelling species, as they express excessive fear by displaying distressed vocal calls when in contact with water and subsequent inactivity and stress behaviours in the home cage [unpublished observations].) In the spiny mouse, novel object recognition was shown to be an age-dependent and sex-dependent behaviour. At <10 days of age, spiny mouse neonates do not appear to identify a new object as ‘novel’, which again, may be the consequence of poor visual acuity, particularly since they spent <5% of the trial time exploring any object. After 10 days of age they spend up to 25% of the time exploring objects, suggesting that by this age they do possess a capacity to identify novel objects, an essential requirement of this test, and after 40 days of age this preference is greater for male compared to female spiny mice. However, at 20 days of age, the males showed a complete lack of interest for the novel object in the recall trial, which may be associated with the adrenal and gonadal hormone changes that occur at about this time [3]. There was also a decline in the discrimination index in adult spiny mice at 80–85 days, which was considerably greater in males. 4.4. Sensorimotor gating Prepulse inhibition (PPI) is used extensively in humans, rats and mice as a measure of sensorimotor gating. Sensorimotor gating is assumed to involve activation of inhibitory networks in the brain, although the exact location of these is still a matter of some debate [37], and may differ from species to species. Sensorimotor gating deficits are often shown in patients with behavioural and psychiatric disorders such as schizophrenia and attention

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Fig. 7. Prepulse inhibition (PPI) with age (A), and PPI to varying prepulse intensities at 10–15 (B), 20–25 (C), 40–45 (D), 60–65 (E) and 80–85 days of age. The data are shown as means ± SEM.

deficit/hyperactivity disorder [38]. PPI refers to the inhibition of the response to a startle stimulus, such as an acoustic pulse, when preceded by a low amplitude stimulus by a short (ms) time interval [39]. In humans, auditory PPI is thought to involve projections from multiple brain regions including the anterior and superior colliculi, pedunculopontine tegmental nucleus, and caudal pontine reticular nucleus [40,41]. In rats, the limbic cortex, striatum and pallidum have also been implicated in mediating PPI [42,43]. Neurochemical substrates involved in PPI may include muscarinic, GABAergic and dopaminergic circuits [40,42]. However, the neural circuitry involved in PPI in the spiny mouse is currently not known.

In this study we show that the relative decrease of the acoustic startle response by a low amplitude prepulse (%PPI) increases with postnatal age, although consistent results could not be obtained until after 20 days of age. Although infants begin to demonstrate measurable PPI [44], other studies have shown that reliable measures of PPI cannot be obtained in early postnatal life; indeed, in humans this may not be until 8 years of age [45], and in rats until 70 days of age [46]. The early development of PPI in the spiny mouse may allow the emergence of this important neurological function to be correlated with the development of specific brain stem networks and neurotransmitter systems. In adult spiny mice, at 80–85 days of

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age, males exhibited significantly greater PPI compared to females, a finding that also applies to healthy humans [47].

4.5. Social interaction Abnormalities in social behaviours are a common symptom of many neuropsychiatric disorders, such as schizophrenia [48,49] and autism [50,51]. The assessment of social interaction in this study involved the exploration of an apparatus with a stranger spiny mouse contained in an enclosure that allowed olfactory and somatosensory contact by the ‘test’ spiny mouse. It was found that spiny mice spent more time in the chamber that contained the stranger spiny mouse enclosure and more time interacting with the stranger spiny mouse, although this behaviour was more dramatic at 1–5 days of age, consistent with the idea that spiny mice are socially curious from a young age. We have previously reported that 25-day-old spiny mice show significant preference for a stranger enclosure and chamber [24], but these animals were tested only once at this age. In the present study, we show that social preference is a profoundly inherent behaviour that can be measured from soon after birth. In this study, animals were tested on each behavioural paradigm several times, and therefore training or habituation to the investigators and handling may be a confounder, particularly for those tests involving an element of motor coordination or novelty such as the social interaction tests. In other rodent studies, these tests have been shown to be particularly vulnerable to effects of training [32], and future studies may need to be designed to minimise any such training effects.

5. Conclusion This study has identified key behavioural milestones in the ontogeny of behaviour of spiny mice, which we suggest can be divided into the following three developmental stages: neonatal (1–10 days of age), pre-pubertal (20–45 days of age), and mature adulthood (>80 days of age). The neonatal period is characterised by unusual patterns of behaviour including limited exploratory activity (Fig. 2), the inability to assess memory and learning (Fig. 3B), and an undeveloped capacity for sensorimotor gating (Fig. 7) compared to older spiny mice. The pre-pubertal period is defined by an increased ability of most behaviours including exploration, learning and memory, motor coordination and prepulse inhibition. At adulthood, the performance of spiny mice on most behavioural tests does not change further, or, in some cases, even appears to decline, particularly in males. A unique pre-pubescent period has been identified in the spiny mouse, when a significant increase in adrenal androgen (DHEA) production occurs at around 20 days of age, concurrent with the emergence of a functional zona reticularis, as shown by an increase in the p450c17: 3BHSD enzyme ratio in this zone of the adrenal cortex at this time [3]. This increase in androgen production may be analogous with adrenarche in humans, which is followed by decreased androgen production following puberty [52,53] Puberty in the spiny mouse occurs after 40 days of age, when sexual and mating behaviours emerge [10]. Thus, the period between 10 and 40 days of age in the spiny mouse may prove to be fruitful in delineating neuronal networks that determine the development of specific behaviours, and in determining how these might be affected by complications of pregnancy and maternal care [54–56], in addition to genetic and environmental factors, that are now widely thought to cause abnormal behaviour in adult life; i.e., the developmental origins of mental illness.

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