- Email: [email protected]
Social behaviour is altered in the insulin-regulated aminopeptidase knockout mouse
Journal Pre-proof Social Behaviour is Altered in the Insulin-Regulated Aminopeptidase Knockout Mouse Peta Burns, Joshua Bowditch, James McFadyen, Richard Loiacono, Anthony L. Albiston, Vi Pham, Siew Yeen Chai
PII:
S0166-4328(19)31145-3
DOI:
https://doi.org/10.1016/j.bbr.2019.112150
Article Number:
112150
Reference:
BBR 112150
To appear in:
Behavioural Brain Research
Received Date:
22 July 2019
Revised Date:
6 August 2019
Accepted Date:
12 August 2019
Please cite this article as: Burns P, Bowditch J, McFadyen J, Loiacono R, Albiston AL, Pham V, Yeen Chai S, Social Behaviour is Altered in the Insulin-Regulated Aminopeptidase Knockout Mouse, Behavioural Brain Research (2019), doi: https://doi.org/10.1016/j.bbr.2019.112150
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
BURNS ET AL
Social Behaviour is Altered in the Insulin-Regulated Aminopeptidase Knockout Mouse Running title:
Social Behaviour and Insulin-Regulated Aminopeptidase
Peta Burnsa, Joshua Bowditchb, James McFadyena, Richard Loiaconob, Anthony L. Albistona, Vi Phamc and Siew Yeen Chaia,d, Monash Biomedicine Discovery Institute, Departments of c
Physiology and
b
Drug Discovery Biology,
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Pharmacology, Monash University, Clayton, Victoria 3800,
a
Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria
author
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dCorresponding
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3052, AUSTRALIA
Monash Biomedicine Discovery Institute,
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Department of Physiology,
Monash University, Clayton Campus,
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Victoria 3800, AUSTRALIA Ph: +61 3 9905 2515
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Fax: +61 3 990 52547
Email: [email protected]
HIGHLIGHTS
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Male mice with deletion of the gene for insulin regulated aminopeptidase (IRAP) displayed significantly more interactions with a stranger stimulus mouse than their wildtype controls. This effect was not observed in the female mice with the gene deletion.
A decrease in rearing behaviour was observed in the open field in these male IRAP knockout mice compared to wildtype.
Locomotor activity, as measured by the distance travelled, was significantly
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decreased in the open field in the male IRAP knockout compared to wildtype mice.
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ABSTRACT
Oxytocin, and the closely related neuropeptide, vasopressin, are both known to
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modulate social behaviours. The pro-social effects of oxytocin are well-documented and
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have generated much interest into its suitability as a therapeutic for disorders characterised by social dysfunction.
This study investigated the social phenotype of
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mice with a targeted deletion of the gene for insulin-regulated aminopeptidase, an enzyme involved in the degradation of oxytocin and vasopressin.
In the 3-chamber
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sociability test, a genotype effect was observed and subsequent post hoc analysis revealed that male, but not female, insulin-regulated aminopeptidase knockout mice
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made significantly more approaches to the enclosure holding a stranger mouse than did wildtype mice (p=0.0039). Male insulin-regulated aminopeptidase knockout mice also displayed decreased rearing (t=2.309, df=24, p=0.0299) and locomotor activity (t=2.134, df=24, p=0.043) in the open field test, suggestive of a reduced stress response to a novel environment. Our findings provide support for the role of insulin-regulated aminopeptidase in influencing social behaviour, possibly via modulation of oxytocin and 2
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vasopressin levels. The increase in social interaction observed in the male, but not female, insulin-regulated aminopeptidase knockout mice is in agreement with reports of sex differences in effects of oxytocin and vasopressin on social behaviours and should be explored further.
Key words: oxytocin, vasopressin, insulin-regulated aminopeptidase, social behaviour
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1. INTRODUCTION
Social behaviour, the intra-species interaction between at least two individuals, is present in many organisms. From the simple swarming demonstrated by some
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bacteria [1] to the intricate interactions in human society [2, 3], social behaviour can benefit individuals as well as entire species. Many behaviours are pro-social, increasing
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interactions between individuals and resulting in gains such as protection against
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predation, improved acquisition and/or control of resources, mating, peaceful cohabitation, transfer of information, even ease of travelling. A number of behaviours such
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as aggression and solitary lifestyle appear anti-social but are also designed to benefit the individual or species by providing protection, ensuring fitness for reproduction, and
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removing the need to share resources that may be in short supply. Aberrations in social behaviour can lead to serious consequences for the
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individual and community, and in many animal species results in the death or abandonment of the affected individual in order to protect the larger group [4]. Genetic mutations [5], defective parental care [6], environment [7], past experiences [8] and physiological dysfunction [9], can all cause abnormal social behaviour. In humans, a number of psychiatric disorders such as depression, schizophrenia, social anxiety, autism spectrum disorder [10] and post-traumatic stress disorder, present with 3
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abnormal social behaviour. Understanding the biology behind social behaviour is key to finding treatment for these conditions. Many
peptide
hormones
are
known
to
influence
social
behaviour.
Gonadotropin-releasing hormone [11], corticotropin-releasing factor and the urocortins [12-14], orexins [15, 16], oxytocin and vasopressin [17, 18] have all been shown to affect social behaviour in different species. In recent years, interest has focussed on the roles of the nonapeptides oxytocin
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(OXY) and arginine vasopressin (AVP) in social behaviours. Administration of OXY (or its species-specific homologue), has been demonstrated to exert pro-social effects when given to species ranging from fish [19] to man [20]. In humans, intranasal OXY has been
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reported to influence the development of trust [21] and empathy [22]. AVP has been demonstrated to increase risky cooperative behaviour in men, although only in a
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mutually beneficial context [23], and together with OXY, it has been shown, using
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receptor knockout mice, to be integral to social recognition [24]. Such findings have stimulated research into possible therapeutic use of these compounds for the treatment
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of disorders hallmarked by social defects, such as autism and schizophrenia. In mammals, AVP and OXY are primarily produced in the paraventricular and
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supraoptic nuclei of the hypothalamus and are then either released in the brain or transported to the posterior pituitary for release into the periphery [25]. Synthesis,
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secretion, receptor availability and degradation of these peptides all influence their overall function. Oxytocinase (OXYase) was first identified as an enzyme in the serum of pregnant women that inactivated OXY, resulting in the attenuation of the uterinecontracting action of the hormone. OXYase was cloned and sequenced [26] and shown to be homologous with insulin-regulated aminopeptidase (IRAP)[27]. This enzyme is now known to cleave both OXY and AVP [28, 29] as well as angiotensin III, somatostatin, 4
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met-enkephalin, and neurokinin A [30-32] and is proposed to be the key regulator of tissue and circulating AVP and OXY levels. A recent study in pupfish reported OXYase production reflected social status, with higher hypothalamic OXYase levels in subordinate fish compared to dominant (aggressive) fish [33], demonstrating a role for endogenous OXYase in social behaviour. IRAP knockout (KO) mice have been generated and characterised on the more recognised roles of the protein - insulin-regulated glucose handling [34] and female
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reproductive function and maternal behaviour [35]. However, the effect of IRAP gene deletion on social behaviour has not been investigated and is the focus of this study.
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2. MATERIAL AND METHODS 2.1 Animals
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IRAP gene deleted mice were generated by Ozgene Pty Ltd (Perth, Australia) as
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previously described [36] and bred in-house by the Monash Animal Research Platform (MARP), Monash University. Homozygous breeding pairs were used to generate
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wildtype (WT) and KO mice. Adult male and female mice, 4-6 months of age were used in this study. C57Bl/6 mice were purchased from MARP to use as stimulus mice in the
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3-chamber sociability test. Mice were housed 2-4 per box with ad libitum mouse chow (Barastoc Ridley, Australia) and water. Lighting was maintained on a 12:12 light dark
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cycle, beginning with lights on at 0700hr. Behavioural tests were conducted between 0900-1600hr with at least 1 day
break between tests. Lighting levels in the testing room were subdued and the mice were acclimated to this lighting for at least 30 min prior to testing. Behaviours of the mice in each test were video recorded for later analysis. The testing and scoring of the behaviour were performed by an investigator who was blinded to the genotype of the 5
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mice. All animal procedures were approved by the Monash University Animal Ethics Committee and were conducted in accordance with Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (7th edition).
2.2 Three-chamber sociability test
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Both male (n=16 WT, n=21 KO) and female (n=18 WT, n=17 KO) mice were tested for social behaviour in a 3-chamber sociability test. The apparatus consisted of a rectangular, clear plexiglass box divided into 3 chambers (left, centre, and right). Each
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chamber measured 40.5cm long x 30.5cm high; the centre chamber measured 22cm wide and each of the left and right chambers measured 19cm wide). The left and right
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chambers were accessible from the central chamber via doorways that could be closed
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with guillotine doors, and each of the left and right chambers contained a cylindrical wire enclosure (9cm diameter, 12cm high) placed in the centre of the floor space.
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Each mouse tested was placed in the central chamber for a 5 minute habituation period. The guillotine doors leading to the left and right chambers were closed and the
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wire enclosures in those chambers were empty. Following habituation, a C57Bl/6 stimulus mouse (same sex and age as the test mouse and habituated to wire enclosure
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in the days prior to testing) was placed in the wire enclosure in either the left or the right chamber (alternated across tests), leaving an empty wire enclosure in the other chamber. The guillotine doors to both left and right chambers were then raised, allowing the test mouse to explore the entire 3-chamber apparatus for a further 10 minutes.
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The number of entries into, and time spent in each chamber, as well as the number of approaches to each of the wire enclosures were measured. The chambers and wire enclosures were thoroughly cleaned with disinfectant between tests.
2.3 Elevated plus maze Both male (n=11 WT, n=15 KO) and female (n=18 WT, n=17 KO) mice were tested for anxiety in the Elevated Plus Maze (EPM). The maze consisted of four arms
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(each 30cm long x 4cm wide) extending in the shape of a cross from a central, square platform (4x4cm). One set of opposing arms were “closed” with 14.0cm high walls while the other set of opposing arms were “open” with only a small edge (1.5cm high) to
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prevent falls. Mounted on a frame, the maze was set 40cm above the floor.
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Each mouse was placed on the central square platform and allowed to explore the maze for 10 minutes. Number of entries into (all four feet in an arm), and time
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spent in each arm was scored and expressed as a percentage of the total number of arm entries and percentage of the total time spent in an arm, respectively. The maze was
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2.4 Open field test
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thoroughly cleaned with 80% (v/v) ethanol between tests.
Following the difference in approach behaviour detected in the 3-chamber
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sociability test (reported in results below), male mice (n=11 WT, n=15 KO) were further assessed for exploratory activity and anxiety-like behaviour in a novel environment using the Open Field Test (OFT). The test was conducted in a plastic circular arena, 75cm in diameter, with 30cm high walls and no discerning features. A lamp was
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suspended above the centre of the arena to produce a central area (radius 10cm) more brightly lit than the surrounding perimeter. Each mouse was placed in the centre of the arena and allowed to explore for 10 minutes. Distance travelled; entries into, and time spent in the central lit area; episodes of rearing and grooming, were measured. The arena was thoroughly cleaned with 80%
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(v/v) ethanol between tests.
2.5 Measurement of Plasma Oxytocin
Following the difference in approach behaviour detected in the 3-chamber
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sociability test (reported in results below), plasma OXY levels were measured in male IRAP KO mice (n=10 WT, n=10 KO). For 2 weeks prior to blood collection, mice were
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handled every few days to accustom them to handling, restraint scruff and the light
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touch of a needle to the abdomen. On the day of blood collection, mice were anaesthetised with sodium pentobarbitone, 80mg/kg (Ilium, Troy Laboratories Pty Ltd,
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Australia) and 0.5ml of blood was drawn from the left ventricle into a 1cc syringe preloaded with 0.75mg di-sodium ethylenediaminetetraacetic acid (EDTA, Merck,
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Australia). Blood was then immediately transferred to an Eppendorf tube on ice, containing protease inhibitor cocktail (Roche, Germany). The collected blood samples
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were centrifuged at 1600g at 4°C for 15 minutes, plasma extracted and frozen at -20°C. Plasma OXY was measured using a commercially available enzyme immunoassay kit:primary antiserum=rabbit anti-peptide IgG; undisclosed secondary antibody; range 0100ng/ml (Phoenix Pharmaceuticals, California, USA).
2.6 Statistical analyses 8
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Data were analyzed using an unpaired t-test or a 2-way ANOVA, using Graph Pad Prism software (Graph Pad Software, Inc., USA). In the OFT, repeated measures ANOVA
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was used to determine locomotor activity over time.
3. RESULTS 3.1 Effect of IRAP gene deletion on social behaviour
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3.1.1 Entries into Chambers
In the 3-chamber sociability task, there was no effect of genotype on number of
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entries into either the stimulus chamber (F1,68=1.204, p=0.2763)(Fig 1a) or the empty
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chamber (F1,68=0.074, p=0.7862)(Fig. 1b). There was a significant effect of sex on the number of entries into the stimulus chamber (F1,68=5.928, p=0.0175)(Fig. 1a) and the
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empty chamber (F1,68=11.2, p=0.0013)(Fig. 1b), with females making more entries than males into both chambers.
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3.1.2 Time spent in Chambers
There was no effect of either genotype or sex on the time spent in the stimulus
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chamber (F1,68=0.207, p=0.6505 or F1,68=2.547, p=0.1151, respectively) (Fig. 1c) or the empty chamber (F1,68=0.569, p=0.4535 or F1,68=0.39, p=0.5344, respectively)(Fig. 1d). 3.1.3 Approaches to Enclosures Analysis of approaches made to the stimulus wire enclosure showed a significant effect of genotype (F1,68=10.31, p=0.0020) and no effect of sex (F1,68=3.183, p=0.0789). Post hoc Tukey’s multiple comparison test showed the male IRAP KO mice made 9
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significantly more approaches to the enclosure holding a stranger mouse than the WT males (p=0.0039) and WT females (p=0.0025)(Fig. 1e).
Analysis of approaches to the
empty enclosure showed there was a significant effect of genotype (F1,68=5.774, p=0.0190) and again no effect of sex (F1,68=0.341, p=0.5614). However, a Tukey’s multiple comparison test showed no difference between any of the individual groups (Fig. 1f).
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3.2 Effect of IRAP gene deletion on anxiety in the elevated plus maze
Analysis of arm preference showed a significant effect of arm type, with both sexes spending a greater percent of time in (Males- F1,48=3642, p<0.0001; Females-
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F1,66=1191, p<0.0001), and making a greater number of entries into (Males- F1,48=1326, p<0.0001; Females- F1,66=600.5, p<0.0001) the closed, compared to the open, arms.
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Analysis of behaviour in the open arms showed there was a significant effect of sex on
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the percent of time spent in these arms (F1,57=7.891, p<0.0068), with females spending a greater percent of time out in the open arms compared to males. No genotype effect of
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percent time spent in the open arms was observed (F1,57=0.9019, p=0.3463) but there was a small, significant interaction (F1,57=4.015,
p=0.0498) and Tukey’s multiple
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comparison test showed the WT (but not IRAP KO) female mice spent significantly greater percent of their time in the open arms compared to both the WT (p=0.0094) and
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the KO (p=0.0339) males (Fig. 2a). Measurement of number of entries into the arms produced similar results, with a
sex effect (F1,57=4.52, p=0.0378) showing females made more entries into the open arms.
There was no genotype effect (F1,57=0.5204, p=0.4736) but a significant
interaction (F1,57=4.52, p=0.0378), with female WT (but not IRAP KO) mice making more entries into the open arms compared to male WT (p=0.0266) but not male IRAP 10
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KO (p=0.1579) (Fig. 2b). Females were more active overall, making significantly more arm entries (both closed and open) than males (F1,57=16.76, p=0.0001). There was no effect of genotype on total number of arm entries (F1,57=0.006, p=0.9385).
3.3 Effect of IRAP gene deletion in male mice on activity and anxiety in the open field test Male IRAP KO mice were not as active as WT mice in the OFT, measured as distance travelled (t=2.134, df=24, p=0.0432) (Fig. 3a). IRAP KO mice traversed the centre lit
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area as frequently as the WT (WT 10.5+3.16 crosses compared to KO 11.6+1.41 crosses, t=0.5451, df=33, p=0.5893). The time spent in the lit area was also not significantly different (WT 20.06+4.29s compared to IRAP KO 19.06+3.16s, t=0.1918, df=33,
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p=0.8491).
When locomotor activity was analysed in 1min intervals over the 10 min test period,
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an overall genotype difference was detected (F9,24=4.556, p=0.0432). Sidak’s multiple
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comparison test revealed that the activity level of the IRAP KO mice was significantly lower than WT mice only in the first minute (distance travelled in WT 10,556mm and
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IRAP KO 8,321mm, mean diff. 2,234, SE of diff.=633, adjusted p=0.0049) (Fig. 3b). Thereafter, both genotypes demonstrated typical habituation to the arena, with activity
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levels decreasing at the same rate over the remainder of the test period. Male IRAP KO mice made significant fewer rears in the open field test (t=2.309, df=24, p=0.0299) (Fig.
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4a) but the number of grooming episodes were not different between genotypes (t=1.506, df=24, p=0.1451) (Fig. 4b).
3.4 Effect of IRAP gene deletion in male mice on plasma oxytocin To investigate if the increase in social approach in male IRAP KO mice was due to an increase in circulating OXY levels, we measured the peptide hormone in the plasma. 11
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Plasma OXY levels of IRAP KO male mice were not significantly different from those of WT mice (t=2.082, df=18, p=0.0519) (Fig. 5).
4. DISCUSSION This study examined the social behaviour of mice predicted to have increased levels of endogenous OXY and AVP due to a gene deletion of IRAP. Male IRAP KO mice displayed increased sociability, as determined by a significant increase in the number of
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approaches to a stranger mouse in the 3-chamber sociability test. The complex nature and subtleties of social behaviour are evident in this study where standard measures of sociability (chamber entries and time spent in each chamber, [37]) indicated no
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difference between the IRAP KO and WT mice, yet further analysis of behaviour revealed there was in fact, an increase in social behaviour. The IRAP KO mice appear
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not to have been drawn to the stimulus chamber any more than the empty chamber, yet
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were inclined to approach the stranger mouse significantly more than WT mice were. That is, they were not seeking out conspecifics but in the presence of one, they were
Many
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more likely to interact with it. studies
have
demonstrated
the
positive
effects
of
exogenous
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administration of OXY on social approach behaviour. OXY has been shown to increase eye contact in men [38], increase social interaction in mice in a 3-chamber test [39], and
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the non-mammalian homologue of OXY, isotocin, is reported to stimulate social approaches in goldfish [19] . In comparison, relatively little work has been reported on the effect of AVP administration on social approach, but work in fish using the nonmammalian homologue vasotocin, suggests a negative influence [19], while work in rats suggests either a null or negative effect depending on the specific AVP receptor being investigated [40]. The increased level of social approach observed in this study is 12
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suggestive of increased activity of OXY in the male IRAP KO mice. Effects of increased AVP activity may require different tests to uncover. It has been shown to be more influential in social memory [41], empathy and territorial aggression [42] rather than social approach or interaction. While the observed behaviours can be interpreted as indicating activity of one or the other peptide, in fact, both may be influencing the behaviour together or either one separately. IRAP is an enzyme involved in the breakdown of both OXY and AVP [29, 30]
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therefore, IRAP KO mice are predicted to have elevated levels of both these peptides. Wallis [43] reported IRAP KO mice to have higher levels of AVP in plasma and lower brain levels, but did not measure OXY. Our study showed no difference in plasma OXY
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levels of male IRAP KO mice compared to WT mice. This may indicate that peripheral OXY levels are not involved in the behaviours observed in this study, however, potential
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actions of IRAP on OXY and AVP in local tissue, which would not be reflected in
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circulating levels, cannot be discounted. We can only speculate as to the involvement of central levels of OXY in these animals as there is currently very little consensus on
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whether peripheral levels of OXY reflect central levels [44-46]. Nevertheless, where behaviour is being modified it would appear reasonable to assume some central effect is
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involved and studies in fish support this [33]. An additional consideration is that the production and processing of both OXY and AVP would be altered in response to
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behavioural testing [47]. Investigation of plasma and cerebrospinal fluid levels of both nonapeptides in IRAP KO mice following exposure to the novel environments, apparatus and stimulus mice of the various tests will be required before drawing any definitive conclusions. In contrast to males, female IRAP KO mice showed no change in sociability. Sex differences have been well documented in regard to OXY and AVP synthesis, receptors 13
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in the brain, and effects when administered exogenously [48]. Often, administration of the peptides has opposing effects on social behaviour in males and females. OXY has been reported to increase social behaviour in naïve male, but not female, mice [49]. Intranasal administration of OXY or AVP has been shown to increase brain activity in areas important to reward, arousal and social bonding in men but not in women [50]. In men but not in women, AVP has been reported to reciprocate cooperation from human partners, suggesting a role in inter-male cooperation [50] and yet it has also been
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reported to elicit agonistic responses in males and affiliative responses in females [51]. The differences in social behaviour observed between males and females may reflect the different brain regions activated, the specific task presented, or different social
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strategies adopted by the sexes [50-53].
Anxiety is considered to be closely linked with social behaviour [54]. The EPM is
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a well validated test of anxiety in mice [55, 56] that measures unconditioned behaviour
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when exploring an apparatus that offers safety in the closed arms versus exposure in the open arms. A higher exploration of the open arms correlates with lower levels of
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anxiety. Assessment of anxiety on the EPM showed male IRAP KO mice to be no different to WT mice, suggesting that the genotype effect observed in males in the
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sociability test was not due to differences in trait anxiety levels. Female mice displayed lower levels of anxiety than males in the EPM, yet there was no effect of sex on social
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approach behaviour. It would appear that the general anxiety levels indicated by behaviour in the EPM are not reflective or predictive of social behaviour. However, this interpretation may be complicated by the finding that while anxiety is the primary factor driving open-arm entries of male rats and mice, activity is the primary factor for females. That is, the behaviour of females in the EPM is a less sensitive measure of anxiety than in males [57, 58]. This makes comparison of anxiety between the sexes 14
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difficult, limiting the conclusions that can be drawn when relating the behaviour of the female mice in the sociability test to anxiety levels. Anxiety in the OFT is assessed by observing behaviour in the brightly lit centre region. No genotype difference was observed in the number of crossings male mice made into this centre area, further supporting the finding from the EPM tests. However, unexpectedly, activity in the open field, as measured by both distance travelled and rearing episodes, was decreased in male IRAP KO mice. Locomotor activity in an open
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field is affected by a number of factors including motor function, pre-test stress levels [59], stress response to the novel environment of the open field [60], and inherent curiosity or motivation to explore a novel environment [61]. We have previously
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reported that the locomotor activity of IRAP KO mice, as measured in a locomotor cell, is not different from WT mice [36]. This is supported by the present study, where activity
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in both the EPM and 3-chamber sociability test, measured as number of entries into the
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arms and chambers respectively, was not different between genotypes. Taken together, impaired motor function does not appear to be the cause of the altered activity in the
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OFT. Mice were not stressed prior to testing in the OFT but the large open arena would be expected to cause some level of stress once the test commenced.
One possible
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interpretation of the results then, is that the decreased locomotor activity indicates lower levels of stress in response to the novel environment of the open field and/or
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attenuated curiosity/motivation to explore it. A similar behavior is observed in mice that voluntarily exercise. They show decreased anxiety, yet a decreased explorative behaviour in the open field, interpreted as improved stress coping mechanisms [62]. Rearing, an indicator of exploratory behaviour [63] and often closely associated with locomotor activity [64] was also reduced in IRAP KO mice in the OFT.
A similar
reduction in rearing in a stressful environment has been observed in OXY-treated, but 15
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not AVP-treated, rats [65], supporting the proposal that altered OXY levels may be affecting the behaviour of the IRAP KO male mice. An alternative explanation may be that AVP is increased in IRAP KO mice, thus heightening their response to stress [66] and decreasing all exploratory behaviour. Both OXY and AVP are able to regulate the stress response via modulation of the hypothalamo-pituitary-adrenal axis, but often have opposing effects. AVP potentiates the activity of corticotropin releasing factor, strengthening the stress response,
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particularly during prolonged stress [67, 68]. OXY is generally considered to be an anxiolytic, reducing both endocrine and behavioural responses to stressors [65, 69, 70]. Temporal analysis of the OFT revealed that the distance travelled by the IRAP KO
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mice was decreased only in the first minute of the test, thereafter, they travelled similar distances and acclimated to the arena at the same rate as WT mice. That the difference
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in activity in the OFT is restricted to the first minute of the trial argues against the idea
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that increased AVP levels are driving a stress response in the IRAP KO mice, as a response strong enough to significantly decrease activity might reasonably be assumed
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to have a more sustained effect.
There are a number of different theories as to how OXY and AVP may influence
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social behaviour. They may promote or reduce sociability per se; they may alter stress, levels, thereby allowing for increased/decreased social interaction; or they may alter
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the salience of social cues, thus altering the social response of the participant. Underlying all of these options is an understanding that the effects of both OXY and AVP are altered by internal states such as history, context and personality. For example, OXY can increase nurturing and bonding in a female towards it’s offspring and at the same time, promote aggression towards out-group or threatening subjects in defence of that offspring [71].
Whether increased social approach behaviour in the 3-chamber 16
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sociability test was due to an increased motivation to socialise or a decreased stress response to a novel social situation, or both, is unclear. Results from the OFT suggest that the environment in the sociability test may have been less stressful for the IRAP KO mice but what influence this had on their motivation to make social approaches is not known. In this study, male IRAP KO mice displayed two behaviours that may be beneficial to those suffering social behaviour disorders: improved social interaction
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and reduced novelty-induced stress response. Further work into the benefits of manipulating endogenous levels of OXY and/or AVP levels, substrates of IRAP, is supported. In addition, the absence of an effect of IRAP gene deletion on the social
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approach behaviour of female mice highlights sex differences that should be both
protein.
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DECLARATION OF INTEREST
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further explored, and taken into consideration in interpreting future work with this
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None.
ACKNOWLEDGEMENTS
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This work was supported by the National Health and Medical Research Council project grant (APP 1007907) and S.Y. Chai was funded by a National Health and Medical
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Research Council Senior Research Fellowship.
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FIGURE LEGENDS Figure 1 Behaviour of insulin-regulated aminopeptidase knockout (open bars) and wildtype (closed bars) mice in a 3-chamber sociability test. a) Female mice made more entries into the stimulus chamber and b) the empty chamber, compared to males. c) & d) There was no difference between sexes or genotypes in the amount of time spent in the stimulus chamber or the empty chamber.
e) Male knockout mice made more
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approaches to the stimulus mouse enclosure than wildtype males. This effect of genotype was not observed in the female mice. f) There was a small overall effect of genotype and not of sex in the number of approaches to the empty enclosure. Values are
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mean + SEM, *=p<0.05, **=p<0.01. Columns sharing the same letter are not significantly
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different (panels a, b & e).
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Figure 2
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Activity of insulin-regulated aminopeptidase knockout (open bars) and wildtype (closed bars) mice in an Elevated Plus Maze. a) Female mice spent more time in, and b) made more entries into, the open arms of the maze than male mice. Values are mean + SEM,
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*=p<0.05, **=p<0.01. Columns sharing the same letter are not significantly different.
Figure 3
Activity of male insulin-regulated aminopeptidase knockout (IRAP KO) and wildtype (WT) mice in an Open Field Test. a) Total distance travelled by IRAP KO mice was less
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than WT. b) Distance travelled was significantly different between IRAP KO and WT
Figure 4
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mice only in the first minute of the trial. Values are mean + SEM, *=p<0.05, **=p<0.01.
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Behaviour of male insulin-regulated aminopeptidase knockout (IRAP KO) and wildtype (WT) mice in an Open Field Test. a) IRAP KO mice displayed a reduced number of rearing events compared to WT mice. b) There was no genotype difference in the number of grooming events. Values are mean + SEM, *=p<0.05.
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Figure 5
Plasma oxytocin levels of male insulin-regulated aminopeptidase knockout (IRAP KO)
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mice were not different from those of wildtype (WT) mice. Values are mean + SEM.
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PII:
S0166-4328(19)31145-3
DOI:
https://doi.org/10.1016/j.bbr.2019.112150
Article Number:
112150
Reference:
BBR 112150
To appear in:
Behavioural Brain Research
Received Date:
22 July 2019
Revised Date:
6 August 2019
Accepted Date:
12 August 2019
Please cite this article as: Burns P, Bowditch J, McFadyen J, Loiacono R, Albiston AL, Pham V, Yeen Chai S, Social Behaviour is Altered in the Insulin-Regulated Aminopeptidase Knockout Mouse, Behavioural Brain Research (2019), doi: https://doi.org/10.1016/j.bbr.2019.112150
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
BURNS ET AL
Social Behaviour is Altered in the Insulin-Regulated Aminopeptidase Knockout Mouse Running title:
Social Behaviour and Insulin-Regulated Aminopeptidase
Peta Burnsa, Joshua Bowditchb, James McFadyena, Richard Loiaconob, Anthony L. Albistona, Vi Phamc and Siew Yeen Chaia,d, Monash Biomedicine Discovery Institute, Departments of c
Physiology and
b
Drug Discovery Biology,
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Pharmacology, Monash University, Clayton, Victoria 3800,
a
Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria
author
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dCorresponding
re
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3052, AUSTRALIA
Monash Biomedicine Discovery Institute,
na
Department of Physiology,
Monash University, Clayton Campus,
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Victoria 3800, AUSTRALIA Ph: +61 3 9905 2515
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Fax: +61 3 990 52547
Email: [email protected]
HIGHLIGHTS
1
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Male mice with deletion of the gene for insulin regulated aminopeptidase (IRAP) displayed significantly more interactions with a stranger stimulus mouse than their wildtype controls. This effect was not observed in the female mice with the gene deletion.
A decrease in rearing behaviour was observed in the open field in these male IRAP knockout mice compared to wildtype.
Locomotor activity, as measured by the distance travelled, was significantly
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decreased in the open field in the male IRAP knockout compared to wildtype mice.
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ABSTRACT
Oxytocin, and the closely related neuropeptide, vasopressin, are both known to
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modulate social behaviours. The pro-social effects of oxytocin are well-documented and
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have generated much interest into its suitability as a therapeutic for disorders characterised by social dysfunction.
This study investigated the social phenotype of
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mice with a targeted deletion of the gene for insulin-regulated aminopeptidase, an enzyme involved in the degradation of oxytocin and vasopressin.
In the 3-chamber
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sociability test, a genotype effect was observed and subsequent post hoc analysis revealed that male, but not female, insulin-regulated aminopeptidase knockout mice
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made significantly more approaches to the enclosure holding a stranger mouse than did wildtype mice (p=0.0039). Male insulin-regulated aminopeptidase knockout mice also displayed decreased rearing (t=2.309, df=24, p=0.0299) and locomotor activity (t=2.134, df=24, p=0.043) in the open field test, suggestive of a reduced stress response to a novel environment. Our findings provide support for the role of insulin-regulated aminopeptidase in influencing social behaviour, possibly via modulation of oxytocin and 2
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vasopressin levels. The increase in social interaction observed in the male, but not female, insulin-regulated aminopeptidase knockout mice is in agreement with reports of sex differences in effects of oxytocin and vasopressin on social behaviours and should be explored further.
Key words: oxytocin, vasopressin, insulin-regulated aminopeptidase, social behaviour
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1. INTRODUCTION
Social behaviour, the intra-species interaction between at least two individuals, is present in many organisms. From the simple swarming demonstrated by some
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bacteria [1] to the intricate interactions in human society [2, 3], social behaviour can benefit individuals as well as entire species. Many behaviours are pro-social, increasing
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interactions between individuals and resulting in gains such as protection against
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predation, improved acquisition and/or control of resources, mating, peaceful cohabitation, transfer of information, even ease of travelling. A number of behaviours such
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as aggression and solitary lifestyle appear anti-social but are also designed to benefit the individual or species by providing protection, ensuring fitness for reproduction, and
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removing the need to share resources that may be in short supply. Aberrations in social behaviour can lead to serious consequences for the
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individual and community, and in many animal species results in the death or abandonment of the affected individual in order to protect the larger group [4]. Genetic mutations [5], defective parental care [6], environment [7], past experiences [8] and physiological dysfunction [9], can all cause abnormal social behaviour. In humans, a number of psychiatric disorders such as depression, schizophrenia, social anxiety, autism spectrum disorder [10] and post-traumatic stress disorder, present with 3
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abnormal social behaviour. Understanding the biology behind social behaviour is key to finding treatment for these conditions. Many
peptide
hormones
are
known
to
influence
social
behaviour.
Gonadotropin-releasing hormone [11], corticotropin-releasing factor and the urocortins [12-14], orexins [15, 16], oxytocin and vasopressin [17, 18] have all been shown to affect social behaviour in different species. In recent years, interest has focussed on the roles of the nonapeptides oxytocin
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(OXY) and arginine vasopressin (AVP) in social behaviours. Administration of OXY (or its species-specific homologue), has been demonstrated to exert pro-social effects when given to species ranging from fish [19] to man [20]. In humans, intranasal OXY has been
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reported to influence the development of trust [21] and empathy [22]. AVP has been demonstrated to increase risky cooperative behaviour in men, although only in a
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mutually beneficial context [23], and together with OXY, it has been shown, using
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receptor knockout mice, to be integral to social recognition [24]. Such findings have stimulated research into possible therapeutic use of these compounds for the treatment
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of disorders hallmarked by social defects, such as autism and schizophrenia. In mammals, AVP and OXY are primarily produced in the paraventricular and
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supraoptic nuclei of the hypothalamus and are then either released in the brain or transported to the posterior pituitary for release into the periphery [25]. Synthesis,
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secretion, receptor availability and degradation of these peptides all influence their overall function. Oxytocinase (OXYase) was first identified as an enzyme in the serum of pregnant women that inactivated OXY, resulting in the attenuation of the uterinecontracting action of the hormone. OXYase was cloned and sequenced [26] and shown to be homologous with insulin-regulated aminopeptidase (IRAP)[27]. This enzyme is now known to cleave both OXY and AVP [28, 29] as well as angiotensin III, somatostatin, 4
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met-enkephalin, and neurokinin A [30-32] and is proposed to be the key regulator of tissue and circulating AVP and OXY levels. A recent study in pupfish reported OXYase production reflected social status, with higher hypothalamic OXYase levels in subordinate fish compared to dominant (aggressive) fish [33], demonstrating a role for endogenous OXYase in social behaviour. IRAP knockout (KO) mice have been generated and characterised on the more recognised roles of the protein - insulin-regulated glucose handling [34] and female
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reproductive function and maternal behaviour [35]. However, the effect of IRAP gene deletion on social behaviour has not been investigated and is the focus of this study.
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2. MATERIAL AND METHODS 2.1 Animals
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IRAP gene deleted mice were generated by Ozgene Pty Ltd (Perth, Australia) as
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previously described [36] and bred in-house by the Monash Animal Research Platform (MARP), Monash University. Homozygous breeding pairs were used to generate
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wildtype (WT) and KO mice. Adult male and female mice, 4-6 months of age were used in this study. C57Bl/6 mice were purchased from MARP to use as stimulus mice in the
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3-chamber sociability test. Mice were housed 2-4 per box with ad libitum mouse chow (Barastoc Ridley, Australia) and water. Lighting was maintained on a 12:12 light dark
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cycle, beginning with lights on at 0700hr. Behavioural tests were conducted between 0900-1600hr with at least 1 day
break between tests. Lighting levels in the testing room were subdued and the mice were acclimated to this lighting for at least 30 min prior to testing. Behaviours of the mice in each test were video recorded for later analysis. The testing and scoring of the behaviour were performed by an investigator who was blinded to the genotype of the 5
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mice. All animal procedures were approved by the Monash University Animal Ethics Committee and were conducted in accordance with Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (7th edition).
2.2 Three-chamber sociability test
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Both male (n=16 WT, n=21 KO) and female (n=18 WT, n=17 KO) mice were tested for social behaviour in a 3-chamber sociability test. The apparatus consisted of a rectangular, clear plexiglass box divided into 3 chambers (left, centre, and right). Each
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chamber measured 40.5cm long x 30.5cm high; the centre chamber measured 22cm wide and each of the left and right chambers measured 19cm wide). The left and right
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chambers were accessible from the central chamber via doorways that could be closed
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with guillotine doors, and each of the left and right chambers contained a cylindrical wire enclosure (9cm diameter, 12cm high) placed in the centre of the floor space.
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Each mouse tested was placed in the central chamber for a 5 minute habituation period. The guillotine doors leading to the left and right chambers were closed and the
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wire enclosures in those chambers were empty. Following habituation, a C57Bl/6 stimulus mouse (same sex and age as the test mouse and habituated to wire enclosure
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in the days prior to testing) was placed in the wire enclosure in either the left or the right chamber (alternated across tests), leaving an empty wire enclosure in the other chamber. The guillotine doors to both left and right chambers were then raised, allowing the test mouse to explore the entire 3-chamber apparatus for a further 10 minutes.
6
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The number of entries into, and time spent in each chamber, as well as the number of approaches to each of the wire enclosures were measured. The chambers and wire enclosures were thoroughly cleaned with disinfectant between tests.
2.3 Elevated plus maze Both male (n=11 WT, n=15 KO) and female (n=18 WT, n=17 KO) mice were tested for anxiety in the Elevated Plus Maze (EPM). The maze consisted of four arms
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(each 30cm long x 4cm wide) extending in the shape of a cross from a central, square platform (4x4cm). One set of opposing arms were “closed” with 14.0cm high walls while the other set of opposing arms were “open” with only a small edge (1.5cm high) to
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prevent falls. Mounted on a frame, the maze was set 40cm above the floor.
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Each mouse was placed on the central square platform and allowed to explore the maze for 10 minutes. Number of entries into (all four feet in an arm), and time
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spent in each arm was scored and expressed as a percentage of the total number of arm entries and percentage of the total time spent in an arm, respectively. The maze was
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2.4 Open field test
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thoroughly cleaned with 80% (v/v) ethanol between tests.
Following the difference in approach behaviour detected in the 3-chamber
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sociability test (reported in results below), male mice (n=11 WT, n=15 KO) were further assessed for exploratory activity and anxiety-like behaviour in a novel environment using the Open Field Test (OFT). The test was conducted in a plastic circular arena, 75cm in diameter, with 30cm high walls and no discerning features. A lamp was
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suspended above the centre of the arena to produce a central area (radius 10cm) more brightly lit than the surrounding perimeter. Each mouse was placed in the centre of the arena and allowed to explore for 10 minutes. Distance travelled; entries into, and time spent in the central lit area; episodes of rearing and grooming, were measured. The arena was thoroughly cleaned with 80%
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(v/v) ethanol between tests.
2.5 Measurement of Plasma Oxytocin
Following the difference in approach behaviour detected in the 3-chamber
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sociability test (reported in results below), plasma OXY levels were measured in male IRAP KO mice (n=10 WT, n=10 KO). For 2 weeks prior to blood collection, mice were
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handled every few days to accustom them to handling, restraint scruff and the light
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touch of a needle to the abdomen. On the day of blood collection, mice were anaesthetised with sodium pentobarbitone, 80mg/kg (Ilium, Troy Laboratories Pty Ltd,
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Australia) and 0.5ml of blood was drawn from the left ventricle into a 1cc syringe preloaded with 0.75mg di-sodium ethylenediaminetetraacetic acid (EDTA, Merck,
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Australia). Blood was then immediately transferred to an Eppendorf tube on ice, containing protease inhibitor cocktail (Roche, Germany). The collected blood samples
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were centrifuged at 1600g at 4°C for 15 minutes, plasma extracted and frozen at -20°C. Plasma OXY was measured using a commercially available enzyme immunoassay kit:primary antiserum=rabbit anti-peptide IgG; undisclosed secondary antibody; range 0100ng/ml (Phoenix Pharmaceuticals, California, USA).
2.6 Statistical analyses 8
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Data were analyzed using an unpaired t-test or a 2-way ANOVA, using Graph Pad Prism software (Graph Pad Software, Inc., USA). In the OFT, repeated measures ANOVA
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was used to determine locomotor activity over time.
3. RESULTS 3.1 Effect of IRAP gene deletion on social behaviour
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3.1.1 Entries into Chambers
In the 3-chamber sociability task, there was no effect of genotype on number of
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entries into either the stimulus chamber (F1,68=1.204, p=0.2763)(Fig 1a) or the empty
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chamber (F1,68=0.074, p=0.7862)(Fig. 1b). There was a significant effect of sex on the number of entries into the stimulus chamber (F1,68=5.928, p=0.0175)(Fig. 1a) and the
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empty chamber (F1,68=11.2, p=0.0013)(Fig. 1b), with females making more entries than males into both chambers.
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3.1.2 Time spent in Chambers
There was no effect of either genotype or sex on the time spent in the stimulus
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chamber (F1,68=0.207, p=0.6505 or F1,68=2.547, p=0.1151, respectively) (Fig. 1c) or the empty chamber (F1,68=0.569, p=0.4535 or F1,68=0.39, p=0.5344, respectively)(Fig. 1d). 3.1.3 Approaches to Enclosures Analysis of approaches made to the stimulus wire enclosure showed a significant effect of genotype (F1,68=10.31, p=0.0020) and no effect of sex (F1,68=3.183, p=0.0789). Post hoc Tukey’s multiple comparison test showed the male IRAP KO mice made 9
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significantly more approaches to the enclosure holding a stranger mouse than the WT males (p=0.0039) and WT females (p=0.0025)(Fig. 1e).
Analysis of approaches to the
empty enclosure showed there was a significant effect of genotype (F1,68=5.774, p=0.0190) and again no effect of sex (F1,68=0.341, p=0.5614). However, a Tukey’s multiple comparison test showed no difference between any of the individual groups (Fig. 1f).
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3.2 Effect of IRAP gene deletion on anxiety in the elevated plus maze
Analysis of arm preference showed a significant effect of arm type, with both sexes spending a greater percent of time in (Males- F1,48=3642, p<0.0001; Females-
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F1,66=1191, p<0.0001), and making a greater number of entries into (Males- F1,48=1326, p<0.0001; Females- F1,66=600.5, p<0.0001) the closed, compared to the open, arms.
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Analysis of behaviour in the open arms showed there was a significant effect of sex on
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the percent of time spent in these arms (F1,57=7.891, p<0.0068), with females spending a greater percent of time out in the open arms compared to males. No genotype effect of
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percent time spent in the open arms was observed (F1,57=0.9019, p=0.3463) but there was a small, significant interaction (F1,57=4.015,
p=0.0498) and Tukey’s multiple
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comparison test showed the WT (but not IRAP KO) female mice spent significantly greater percent of their time in the open arms compared to both the WT (p=0.0094) and
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the KO (p=0.0339) males (Fig. 2a). Measurement of number of entries into the arms produced similar results, with a
sex effect (F1,57=4.52, p=0.0378) showing females made more entries into the open arms.
There was no genotype effect (F1,57=0.5204, p=0.4736) but a significant
interaction (F1,57=4.52, p=0.0378), with female WT (but not IRAP KO) mice making more entries into the open arms compared to male WT (p=0.0266) but not male IRAP 10
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KO (p=0.1579) (Fig. 2b). Females were more active overall, making significantly more arm entries (both closed and open) than males (F1,57=16.76, p=0.0001). There was no effect of genotype on total number of arm entries (F1,57=0.006, p=0.9385).
3.3 Effect of IRAP gene deletion in male mice on activity and anxiety in the open field test Male IRAP KO mice were not as active as WT mice in the OFT, measured as distance travelled (t=2.134, df=24, p=0.0432) (Fig. 3a). IRAP KO mice traversed the centre lit
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area as frequently as the WT (WT 10.5+3.16 crosses compared to KO 11.6+1.41 crosses, t=0.5451, df=33, p=0.5893). The time spent in the lit area was also not significantly different (WT 20.06+4.29s compared to IRAP KO 19.06+3.16s, t=0.1918, df=33,
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p=0.8491).
When locomotor activity was analysed in 1min intervals over the 10 min test period,
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an overall genotype difference was detected (F9,24=4.556, p=0.0432). Sidak’s multiple
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comparison test revealed that the activity level of the IRAP KO mice was significantly lower than WT mice only in the first minute (distance travelled in WT 10,556mm and
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IRAP KO 8,321mm, mean diff. 2,234, SE of diff.=633, adjusted p=0.0049) (Fig. 3b). Thereafter, both genotypes demonstrated typical habituation to the arena, with activity
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levels decreasing at the same rate over the remainder of the test period. Male IRAP KO mice made significant fewer rears in the open field test (t=2.309, df=24, p=0.0299) (Fig.
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4a) but the number of grooming episodes were not different between genotypes (t=1.506, df=24, p=0.1451) (Fig. 4b).
3.4 Effect of IRAP gene deletion in male mice on plasma oxytocin To investigate if the increase in social approach in male IRAP KO mice was due to an increase in circulating OXY levels, we measured the peptide hormone in the plasma. 11
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Plasma OXY levels of IRAP KO male mice were not significantly different from those of WT mice (t=2.082, df=18, p=0.0519) (Fig. 5).
4. DISCUSSION This study examined the social behaviour of mice predicted to have increased levels of endogenous OXY and AVP due to a gene deletion of IRAP. Male IRAP KO mice displayed increased sociability, as determined by a significant increase in the number of
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approaches to a stranger mouse in the 3-chamber sociability test. The complex nature and subtleties of social behaviour are evident in this study where standard measures of sociability (chamber entries and time spent in each chamber, [37]) indicated no
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difference between the IRAP KO and WT mice, yet further analysis of behaviour revealed there was in fact, an increase in social behaviour. The IRAP KO mice appear
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not to have been drawn to the stimulus chamber any more than the empty chamber, yet
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were inclined to approach the stranger mouse significantly more than WT mice were. That is, they were not seeking out conspecifics but in the presence of one, they were
Many
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more likely to interact with it. studies
have
demonstrated
the
positive
effects
of
exogenous
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administration of OXY on social approach behaviour. OXY has been shown to increase eye contact in men [38], increase social interaction in mice in a 3-chamber test [39], and
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the non-mammalian homologue of OXY, isotocin, is reported to stimulate social approaches in goldfish [19] . In comparison, relatively little work has been reported on the effect of AVP administration on social approach, but work in fish using the nonmammalian homologue vasotocin, suggests a negative influence [19], while work in rats suggests either a null or negative effect depending on the specific AVP receptor being investigated [40]. The increased level of social approach observed in this study is 12
BURNS ET AL
suggestive of increased activity of OXY in the male IRAP KO mice. Effects of increased AVP activity may require different tests to uncover. It has been shown to be more influential in social memory [41], empathy and territorial aggression [42] rather than social approach or interaction. While the observed behaviours can be interpreted as indicating activity of one or the other peptide, in fact, both may be influencing the behaviour together or either one separately. IRAP is an enzyme involved in the breakdown of both OXY and AVP [29, 30]
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therefore, IRAP KO mice are predicted to have elevated levels of both these peptides. Wallis [43] reported IRAP KO mice to have higher levels of AVP in plasma and lower brain levels, but did not measure OXY. Our study showed no difference in plasma OXY
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levels of male IRAP KO mice compared to WT mice. This may indicate that peripheral OXY levels are not involved in the behaviours observed in this study, however, potential
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actions of IRAP on OXY and AVP in local tissue, which would not be reflected in
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circulating levels, cannot be discounted. We can only speculate as to the involvement of central levels of OXY in these animals as there is currently very little consensus on
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whether peripheral levels of OXY reflect central levels [44-46]. Nevertheless, where behaviour is being modified it would appear reasonable to assume some central effect is
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involved and studies in fish support this [33]. An additional consideration is that the production and processing of both OXY and AVP would be altered in response to
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behavioural testing [47]. Investigation of plasma and cerebrospinal fluid levels of both nonapeptides in IRAP KO mice following exposure to the novel environments, apparatus and stimulus mice of the various tests will be required before drawing any definitive conclusions. In contrast to males, female IRAP KO mice showed no change in sociability. Sex differences have been well documented in regard to OXY and AVP synthesis, receptors 13
BURNS ET AL
in the brain, and effects when administered exogenously [48]. Often, administration of the peptides has opposing effects on social behaviour in males and females. OXY has been reported to increase social behaviour in naïve male, but not female, mice [49]. Intranasal administration of OXY or AVP has been shown to increase brain activity in areas important to reward, arousal and social bonding in men but not in women [50]. In men but not in women, AVP has been reported to reciprocate cooperation from human partners, suggesting a role in inter-male cooperation [50] and yet it has also been
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reported to elicit agonistic responses in males and affiliative responses in females [51]. The differences in social behaviour observed between males and females may reflect the different brain regions activated, the specific task presented, or different social
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strategies adopted by the sexes [50-53].
Anxiety is considered to be closely linked with social behaviour [54]. The EPM is
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a well validated test of anxiety in mice [55, 56] that measures unconditioned behaviour
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when exploring an apparatus that offers safety in the closed arms versus exposure in the open arms. A higher exploration of the open arms correlates with lower levels of
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anxiety. Assessment of anxiety on the EPM showed male IRAP KO mice to be no different to WT mice, suggesting that the genotype effect observed in males in the
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sociability test was not due to differences in trait anxiety levels. Female mice displayed lower levels of anxiety than males in the EPM, yet there was no effect of sex on social
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approach behaviour. It would appear that the general anxiety levels indicated by behaviour in the EPM are not reflective or predictive of social behaviour. However, this interpretation may be complicated by the finding that while anxiety is the primary factor driving open-arm entries of male rats and mice, activity is the primary factor for females. That is, the behaviour of females in the EPM is a less sensitive measure of anxiety than in males [57, 58]. This makes comparison of anxiety between the sexes 14
BURNS ET AL
difficult, limiting the conclusions that can be drawn when relating the behaviour of the female mice in the sociability test to anxiety levels. Anxiety in the OFT is assessed by observing behaviour in the brightly lit centre region. No genotype difference was observed in the number of crossings male mice made into this centre area, further supporting the finding from the EPM tests. However, unexpectedly, activity in the open field, as measured by both distance travelled and rearing episodes, was decreased in male IRAP KO mice. Locomotor activity in an open
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field is affected by a number of factors including motor function, pre-test stress levels [59], stress response to the novel environment of the open field [60], and inherent curiosity or motivation to explore a novel environment [61]. We have previously
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reported that the locomotor activity of IRAP KO mice, as measured in a locomotor cell, is not different from WT mice [36]. This is supported by the present study, where activity
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in both the EPM and 3-chamber sociability test, measured as number of entries into the
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arms and chambers respectively, was not different between genotypes. Taken together, impaired motor function does not appear to be the cause of the altered activity in the
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OFT. Mice were not stressed prior to testing in the OFT but the large open arena would be expected to cause some level of stress once the test commenced.
One possible
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interpretation of the results then, is that the decreased locomotor activity indicates lower levels of stress in response to the novel environment of the open field and/or
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attenuated curiosity/motivation to explore it. A similar behavior is observed in mice that voluntarily exercise. They show decreased anxiety, yet a decreased explorative behaviour in the open field, interpreted as improved stress coping mechanisms [62]. Rearing, an indicator of exploratory behaviour [63] and often closely associated with locomotor activity [64] was also reduced in IRAP KO mice in the OFT.
A similar
reduction in rearing in a stressful environment has been observed in OXY-treated, but 15
BURNS ET AL
not AVP-treated, rats [65], supporting the proposal that altered OXY levels may be affecting the behaviour of the IRAP KO male mice. An alternative explanation may be that AVP is increased in IRAP KO mice, thus heightening their response to stress [66] and decreasing all exploratory behaviour. Both OXY and AVP are able to regulate the stress response via modulation of the hypothalamo-pituitary-adrenal axis, but often have opposing effects. AVP potentiates the activity of corticotropin releasing factor, strengthening the stress response,
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particularly during prolonged stress [67, 68]. OXY is generally considered to be an anxiolytic, reducing both endocrine and behavioural responses to stressors [65, 69, 70]. Temporal analysis of the OFT revealed that the distance travelled by the IRAP KO
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mice was decreased only in the first minute of the test, thereafter, they travelled similar distances and acclimated to the arena at the same rate as WT mice. That the difference
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in activity in the OFT is restricted to the first minute of the trial argues against the idea
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that increased AVP levels are driving a stress response in the IRAP KO mice, as a response strong enough to significantly decrease activity might reasonably be assumed
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to have a more sustained effect.
There are a number of different theories as to how OXY and AVP may influence
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social behaviour. They may promote or reduce sociability per se; they may alter stress, levels, thereby allowing for increased/decreased social interaction; or they may alter
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the salience of social cues, thus altering the social response of the participant. Underlying all of these options is an understanding that the effects of both OXY and AVP are altered by internal states such as history, context and personality. For example, OXY can increase nurturing and bonding in a female towards it’s offspring and at the same time, promote aggression towards out-group or threatening subjects in defence of that offspring [71].
Whether increased social approach behaviour in the 3-chamber 16
BURNS ET AL
sociability test was due to an increased motivation to socialise or a decreased stress response to a novel social situation, or both, is unclear. Results from the OFT suggest that the environment in the sociability test may have been less stressful for the IRAP KO mice but what influence this had on their motivation to make social approaches is not known. In this study, male IRAP KO mice displayed two behaviours that may be beneficial to those suffering social behaviour disorders: improved social interaction
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and reduced novelty-induced stress response. Further work into the benefits of manipulating endogenous levels of OXY and/or AVP levels, substrates of IRAP, is supported. In addition, the absence of an effect of IRAP gene deletion on the social
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approach behaviour of female mice highlights sex differences that should be both
protein.
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DECLARATION OF INTEREST
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further explored, and taken into consideration in interpreting future work with this
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None.
ACKNOWLEDGEMENTS
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This work was supported by the National Health and Medical Research Council project grant (APP 1007907) and S.Y. Chai was funded by a National Health and Medical
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Research Council Senior Research Fellowship.
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FIGURE LEGENDS Figure 1 Behaviour of insulin-regulated aminopeptidase knockout (open bars) and wildtype (closed bars) mice in a 3-chamber sociability test. a) Female mice made more entries into the stimulus chamber and b) the empty chamber, compared to males. c) & d) There was no difference between sexes or genotypes in the amount of time spent in the stimulus chamber or the empty chamber.
e) Male knockout mice made more
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approaches to the stimulus mouse enclosure than wildtype males. This effect of genotype was not observed in the female mice. f) There was a small overall effect of genotype and not of sex in the number of approaches to the empty enclosure. Values are
-p
mean + SEM, *=p<0.05, **=p<0.01. Columns sharing the same letter are not significantly
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different (panels a, b & e).
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Figure 2
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Activity of insulin-regulated aminopeptidase knockout (open bars) and wildtype (closed bars) mice in an Elevated Plus Maze. a) Female mice spent more time in, and b) made more entries into, the open arms of the maze than male mice. Values are mean + SEM,
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*=p<0.05, **=p<0.01. Columns sharing the same letter are not significantly different.
Figure 3
Activity of male insulin-regulated aminopeptidase knockout (IRAP KO) and wildtype (WT) mice in an Open Field Test. a) Total distance travelled by IRAP KO mice was less
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than WT. b) Distance travelled was significantly different between IRAP KO and WT
Figure 4
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mice only in the first minute of the trial. Values are mean + SEM, *=p<0.05, **=p<0.01.
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Behaviour of male insulin-regulated aminopeptidase knockout (IRAP KO) and wildtype (WT) mice in an Open Field Test. a) IRAP KO mice displayed a reduced number of rearing events compared to WT mice. b) There was no genotype difference in the number of grooming events. Values are mean + SEM, *=p<0.05.
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Figure 5
Plasma oxytocin levels of male insulin-regulated aminopeptidase knockout (IRAP KO)
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mice were not different from those of wildtype (WT) mice. Values are mean + SEM.
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