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/lp/springer-journals/impaired-innate-and-conditioned-social-behavior-in-adult-c57bl6-j-mice-omi4tSqdZS
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- Springer Journals
- Copyright
- Copyright © 2019 by The Author(s)
- Subject
- Biomedicine; Neurosciences; Neurology; Behavioral Therapy; Psychiatry
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- 1744-9081
- DOI
- 10.1186/s12993-019-0153-3
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- See Article on Publisher Site
Abstract
Background: Signs of pervasive developmental disorder and social deficits were reported in toddlers and children whose mothers were exposed to organophosphate pesticides during pregnancy. Deficits in social preference were reported in adult male mice exposed to chlorpyrifos on gestational days 12–15. This study aimed (a) to test the hypothesis that adult female and male mice that were exposed prenatally to subtoxic doses of chlorpyrifos would be impaired in social behavior and (b) to determine if prenatal chlorpyrifos altered the expression of transcripts for oxytocin in the hypothalamus. Pregnant mice were treated by gavage with corn oil vehicle or 2.5 mg/kg or 5 mg/kg of CPF on gestational days 12–15. Social preference, social and non-social conditioned place preference tasks were tested in adults. Expression of oxytocin transcripts in hypothalamus was measured by qPCR. Results: Chlorpyrifos (5 mg/kg on GD 12–15) reduced the innate preference for a conspecific in a dose and sex dependent manner. Adult males exposed prenatally to 5 mg/kg CPF showed a reduction in social preference. Socially conditioned place preference was impaired in offspring of dams treated with either dose of CPF. Non-social appetitive place conditioning was impaired in offspring of dams exposed to 2.5 mg/kg, but not to 5 mg/kg chlorpyrifos. Prenatal chlorpyrifos treatment did not alter the expression of the oxytocin mRNA in the hypothalamus, although expression was significantly lower in females. Conclusions: Prenatal chlorpyrifos induced innate and learned social deficits and non-specific conditioning deficits in adult mice in a sex-dependent manner. Males showed specific social deficits following the higher dose whereas both males and females showed a more generalized conditioning deficit following the intermediate dose. Keywords: Chlorpyrifos, Social behavior, Prenatal exposure, Organophosphate pesticides, Oxytocin Background between ASD and residential proximity to OP applica- The role of environmental pollutants, including organo - tion during the second and third trimester of gestation in phosphate pesticides (OP) such as chlorpyrifos (CPF), as a population-based case control study in California [47]. a vulnerability factor in the etiology of autism spectrum In addition to ASD, deficits in cognition and attention disorders (ASD) has garnered attention of researchers were associated with residential proximity to OP use dur- [26] given that heritability for autism is incomplete [18]. ing gestation in the Center for the Health Assessment of In support of a link between pesticide exposure and ASD, Mothers and Children of Salinas (CHAMACOS) Study in the Childhood Autism Risks from Genetics and Envi- the Salinas Valley, California [17]. The level of OP metab - ronment (CHARGE) study found a positive association olites in maternal urine during pregnancy was associ- ated with deficits in social reciprocal behavior, which were more prominent in boys than girls in the Mount Sinai Children’s Environmental Health Center study *Correspondence: kofman@bgu.ac.il Department of Psychology, Ben-Gurion University of the Negev, POB [14]. However, a recent study on the association between 653, Beer-Sheva, Israel maternal exposure to OPs and signs indicative of ASD Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lan et al. Behav Brain Funct (2019) 15:2 Page 2 of 9 was equivocal [46]. In two major longitudinal studies in Higher order cognitive deficits were observed in chil - the USA, the CHAMACOS project [13] and the Colum- dren who had been exposed to OPs during gestation. bia project in New York [43], symptoms of pervasive Attention deficits were reported at ages 36 months and developmental disorder (PDD) in toddlers were reported 5 years [30, 43]. Lower IQ scores and impaired working in offspring of mothers who showed evidence of signifi - memory were reported in different longitudinal studies at cant exposure to organophosphate pesticides (OP) dur- 7 years [3, 12, 42]. In contrast, a more recent study found ing gestation. Prior to the publication of the fifth edition a positive association between maternal DMP during of the Diagnostics and Statistical Manual in Mental Dis- pregnancy and maternal-reported measures of executive orders (DSM V) [1], PDD was the term used for sociali- function in children aged 6–9 [15]. zation and communication deficits that did not meet full Studies focusing on sex differences in the effects of criteria for autism. gestational OP exposure have led to inconclusive results The possibility of OP exposure as a risk factor for ASD [7]. On one hand boys showed greater deficits than girls is not limited to agricultural environments, as OPs are on social [14] and working memory tests [21] in corre- ubiquitous in food, dust, and air. It was estimated that lation with gestational exposure to chlorpyrifos (CPF); children under age 5 potentially inhale 2 ng/kg/day and however in a sample of children genetically at high-risk potentially ingest close to 5 ng/kg/day in their home and for ASD, OP exposure was more likely to be associated daycare environments [20, 29, 33, 55]. Moreover, expo- with ASD in the girls than the boys [38]. Since the effects sure to pesticides from environmental contact in infants of prenatal OPs on behavioral outcomes have not been and toddlers exceeds that of adults because the former adequately explored in the pre-clinical literature, in the have a higher metabolic rate and more frequent hand-to- current study, both male and female mice were assessed mouth contact [27]. for the effects of gestational CPF on social behavior. Eec ff ts of exposure to OPs during gestation on infant Long‑term effects of prenatal exposure to CPF on rodent and child development behavior In North America [13, 30, 55, 56] and Asia [24, 53], an Prenatal exposure to CPF resulted in delayed motor association was found between the level of organophos- development, impaired innate social preference and con- phate dialkylphosphate (DAP) metabolites in pregnant ditioned social preference, and restricted interest in adult mothers’ urine or in neonate cord blood and lower birth male mice [25], behaviors used to model symptoms of weight. Reviews of the studies on the long-term effects ASD in preclinical studies [34, 44]. While delayed reflexes of maternal exposure to pesticides during gestation are not specifically related to the autism spectrum, delays have yielded conflicting conclusions, supporting [19] or in reaching motor milestones, abnormal reflexes, and opposing [31] the view that CPF exposure has deleteri- postural asymmetries were reported to be prevalent in ous effects on infant development. The former review infants and toddlers who were later diagnosed with ASD was based on a meta-analysis of longitudinal studies after [2]. stratification of the birth and development scores by eth - Exposure to CPF during late gestation enhanced offen - nic origin [19]. There was a negative relation between sive posture, but not attacks, in adult male mice [45] and DAP metabolites and birth weight in blacks and between increased social discrimination in adult female mice [9]. DAP and gestational length in whites. In addition to Dietary CPF exposure during gestation extending to weight, delayed development of neonatal reflexes [11, postnatal day 14 led to enhanced social investigation of 56] and lower scores on the Bayley Mental Development familiar conspecifics in female mice and both familiar Index (MDI) at 12 and 24 months and on the Psychomo- and novel mice in males [52]. In contrast, gestational CPF tor Development Index at 36 months [43] were associated had no effect on investigation of a conspecific in adult with higher maternal exposure to OPs. OPs are rapidly female BTBR mice, which have been used as a model for metabolized such that maternal urinary diethylphosphate autism, possibly because of their low baseline level of (DEP) or dimethylphosphate (DMP) metabolites are not social preference [8]. Exposure of wild type Reelin posi- a direct reflection of foetal exposure; however, these lev - tive mice to CPF oxon from embryonic day 13.5 through els were associated with residential proximity to pesti- weaning also did not affect social preference in male or cide use in the CHAMACOS cohort [4]. In the Columbia female adult offspring at postnatal day 30 [35]. University longitudinal studies, CPF exposure was also Notably, many of the assays that have been used to derived from monitoring maternal air exposure, maternal assess the long-term effects of gestational CPF exposure blood and neonate cord blood. As described above, these on social behavior involve an element of learning, raising measures were related to changes in psychomotor and the question of whether the behavioral change is an indi- mental development [42, 54]. rect consequence of a more general deficit or advantage Lan et al. Behav Brain Funct (2019) 15:2 Page 3 of 9 induced by CPF. For example, increased discrimination had been treated with vehicle (p < .001) or 2.5 mg/kg CPF or investigation of familiar mice by female mice exposed (p = .007). There were no group differences among female to CPF suggests that the CPF-treated female mice had an mice exposed prenatally to CPF or vehicle. The female advantage in social learning assays [9, 52]. Thus, although offspring of dams treated with 2.5 mg/kg CPF had lower our main aim in the present study was to explore both SP than the male offspring treated with 2.5 mg/kg CPF innate (social preference) and conditioned (conditioned (p = .007). In conclusion, the male, but not the female social place preference) social behaviors, we addressed mice exposed to CPF showed significantly less SP than the possibility that a more general change in learning the offspring of the vehicle-treated dams, replicating our would be found. previous result [25] and suggesting that CPF exposure during gestation elicits a deficit in social behavior (Fig. 1). Oxytocin involvement in the long‑term effects of prenatal Social conditioned place preference (SCPP) CPF The change in preference for the initially non-preferred Although the lethal effect of CPF is attributed to inhi - bedding prior to and following social conditioning was bition of acetylcholinesterase (AChE), sub-toxic doses measured. A change in the positive direction indicated equal to or greater than those used in our studies pro- that the conditioned stimulus, the bedding, acquired duced transient or no significant inhibition of brain a positive value after being associated with social living AChE in pups [45, 52] with brain AChE being less inhib- conditions (the unconditioned stimulus). The Levene test ited than serum AChE. Fetuses treated with 5 mg/kg for homogeneity of variance was significant for the SCPP CPF/day during mid or late gestation did not show sig- test, F = 2.62, p = .04, leading to the decision to analyze nificant changes in forebrain or brainstem markers for the data with non-parametric statistics. The Kruskal– development at gestational days 17 or 21 [39]; however, Wallis test for the treatment effect indicated that there long-term changes in forebrain integrity and choliner- was a significant effect of prenatal CPF exposure, gic markers were observed postnatally [40]. In addition H = 10.29, p = .006, with the control group, who were off - to long-term effects on cholinergic markers, other neu - spring of dams treated with the oil vehicle, ranking 32.18, rotransmitter systems and expression of a multitude of compared to the offspring of mice treated with 2.5 mg/kg genes have been reported following gestational CPF [5, CPF (16.36) or 5.0 mg/kg CPF (21.39). The effect of sex 22, 32, 48]. was also statistically significant, H = 5.97, p = .015. These In the current study, we tested whether the expression analyses showed that the offspring of mice treated with of oxytocin (OT) transcripts was altered in the hypo- thalamus as reported previously [49]. OT is a peptide that mediates social behavior. OT antagonists attenu- ated social learning [10, 23], and social recognition was impaired in rodents lacking OT receptors [50]. Con- versely, OT agonists facilitated social behavior, and potentiated prosocial behaviors induced by 3,4-methyl- enedioxy-N-methylamphetamine (MDMA) [10, 41]. The current study examined innate and learned social behavior in male and female adult mice exposed during gestation to CPF. A non-social control experiment was used to examine the specificity of the deficit, and the expression of mRNA for OT in the hypothalamus was assayed as a putative mechanism for the reported deficits. Results Social preference (SP) Fig. 1 Social preference test. Mean preference for a same sex conspecific. Individual mice are shown as symbols (Δ male O female). In the SP task, significant main effects for treatment, F All mice are offspring of dams age 90 days, which were given CPF (2, 44) = 4.32, p = .02, and sex were found, F(1,44) = 4.61, (2.5 or 5 mg/kg) or peanut oil vehicle by gavage on GD 12–15. Social p = .04. The Levene’s test for homogeneity of variance was preference is calculated as the ratio of the time spent in the chamber not significant (F = .75, p = .59). There was also a signifi - containing the mouse to the total time spent in the apparatus, cant sex × treatment interaction, F(2,44) = 7.33, p = .002. measured in seconds. Group differences: Solid line male p < .001; dashed line p = .007. Male vehicle: n = 11, male 2.5 mg/kg: n = 10, The Tukey post hoc test revealed that male offspring of male 5 mg/kg: n = 9, female vehicle: n = 8; female 2.5 mg/kg: n = 7; dams that had been treated with 5 mg/kg CPF had sig- female 5 mg/kg: n = 5 nificantly lower SP than the male offspring whose dams Lan et al. Behav Brain Funct (2019) 15:2 Page 4 of 9 CPF did not acquire the socially conditioned bedding preference, in contrast to the offspring of vehicle-treated mice, and that female mice did not learn this condition- ing as well as males. Food conditioned place preference (FCPP) The change in preference for the initially non-preferred bedding following food conditioning was measured. Because the experiment was designed as a biased place preference, two mice were removed from the analysis because they did not show a preference for either bed- ding (one female offspring of a dam treated with vehicle and one female offspring of a dam treated with 5 mg/ kg CPF). A positive change in value indicated that the mice learned to associate the initially non-preferred bed- ding with a positive appetitive reward and to increase their preference for that bedding. The Levene’s test for homogeneity of variance was not significant, F = .39, p = .84. A significant main effect for treatment was found, F(2,45) = 9.24, p = .0004, and a significant sex × treat- ment interaction F(2,45) = 3.55, p = .04. The male mice that were offspring of dams that had been treated with 2.5 mg/kg CPF on GD 12–15 dose showed less robust conditioning on the FCPP (Tukey’s test p < .001), com- pared to the male offspring of the vehicle treated group, whereas there was no significant appetitive conditioning deficit in the male offspring of dams treated with 5 mg/ kg CPF. The female offspring of dams that had been Fig. 2 Top panel: social conditioned place preference test. Mean treated with 2.5 mg/kg CPF on GD 12–15 dose showed preference for the bedding that was conditioned with another an FCPP deficit compared to the female offspring of the conspecific. Individual mice are shown as symbols (Δ male O female). dams treated with 5 mg/kg CPF p = .046, but they did All mice are offspring, aged 90 days, of dams that were given CPF not differ from the offspring of dams treated with vehicle (2.5 or 5 mg/kg) or peanut oil vehicle by gavage on GD 12–15. (Fig. 2). These findings indicate that prenatal exposure to The main effect of treatment is depicted by the thin triple bracket, indicating that the vehicle exposed group showed a larger change CPF reduced the ability of mice to learn to associate a cue in preference than each of the CPF-exposed groups (p = .006). The with a non-social reward. thick bracket indicates the main effect of sex (p = .015). Male: vehicle: n = 16, 2.5 mg/kg: n = 8, 5 mg/kg: n = 15, female: n = 3 in each OT transcripts group. Bottom panel: food conditioned place preference test. Mean preference for the bedding that was conditioned with an appetitive CPF administration did not change the expression of stimulus reward. Individual mice are shown as symbols (Δ male the OT mRNA in the hypothalamus. After removing O female). All mice are offspring aged 90 days of dams, that were two outliers, whose values were more than three stand- given CPF (2.5 or 5 mg/kg) or peanut oil vehicle by gavage on GD ard deviations above mean, an ANOVA was conducted 12–15. The significant comparisons are depicted by dashed brackets for the effect of treatment and sex. There was no sig - (p = .001) or double bracket (p < .05). Male: vehicle: n = 11, 2.5 mg/kg: n = 8, 5 mg/kg: n = 12, female: vehicle: n = 8, 2.5 mg/kg: n = 7. 5 mg/ nificant main effect for treatment, F (2,32) = .92, p = .41, kg: n = 5 nor a sex × treatment interaction, F(2,32) = .02, p = .98. However, a main effect for sex was found, F (1,32) = 4.51, p = .04. Females expressed less OT in the hypothalamus compared to males (Fig. 3). mice after prenatal exposure to CPF, commensurate with the suggestion that exposure to pesticides during mid to Discussion late gestation can reveal sex-specific effects [7]. Adminis - In addition to replicating our previous findings that pre - tration of CPF on GD 12–15, at doses below the thresh- natal CPF induced deficits in the SP and SCPP task in old for observable signs of toxicity, elicited sex-selective males [25], the current study revealed both social and deficits in social behavior and caused a reduced ability non-social conditioning deficits in both male and female to establish conditioning in the FCPP task. The main Lan et al. Behav Brain Funct (2019) 15:2 Page 5 of 9 oxon, showed more social investigation than vehicle- treated mice. u Th s, a range of effects have been found in female mice, but there is no conclusive evidence of a sociability deficit following exposure to CPF or its oxon metabolite. Furthermore, this study revealed a more general con- ditioning deficit in both sexes which was evident in the offspring of mice exposed to the 2.5 mg/kg dose but not 5.0 mg/kg CPF. Although the source of this non-linear dose response is not clear, a similar phenomenon was reported by Levin et al. [28] who showed that a dose of 1 mg/kg of CPF on GD 17–20 induced more deleterious effects, compared to a dose of 5 mg/kg in females when working and reference memory in the radial-arm maze Fig. 3 Quantitative PCR for oxytocin mRNA levels in the were tested in adult offspring in rats. A tentative hypoth - hypothalamus of adult offspring of mothers exposed to CPF on GD esis could involve the possible effect of CPF on increas - 12–15 (normalized to GAPDH mRNA levels). Male: vehicle: n = 10, ing stimulus salience and discriminability, as pointed out 2.5 mg/kg: n = 8, 5 mg/kg: n = 4, female: vehicle: n = 7, 2.5 mg/kg: n = 6. 5 mg/kg: n = 3. The bracket indicates the main effect of sex, by De Felice et al. [9] and in a test of fear conditioning p < .05 in mice treated with CPF on post-natal days 4–10 [37]. In that study, the mice treated with CPF showed better discrimination between the conditioned stimulus and the difference between the sexes was the fact that the female interstimulus interval than the control mice. Thus, we mice were not impaired in the SP test, an assay of inher- speculate that the low dose of CPF may impair condition- ent sociability that is commonly used to investigate social ing, but that this effect is countered by the enhanced cue deficits in genetic models of ASD [34]. Male offspring salience in mice that were exposed to the higher dose of of the dams that had been treated with 5 mg/kg CPF CPF. However, further research addressing the effects of showed a deficit in both innate and learned social pref - perinatal CPF on cue salience and conditioning would be erence tests. These findings are in accord with findings required. that boys who had been exposed to OPs during gestation The current study is in accord with some of the stud - had more social deficits than girls [14]. In addition to the ies on humans that reported more severe cognitive and social deficits, offspring of dams that had been treated social deficits in boys who had been exposed to gesta - with 2.5 mg/kg CPF showed a deficit in the non-social tional OPs [14, 21]; however, there is no evidence for a appetitive conditioning task, the FCPP. specific social deficit in female offspring. While the sociability deficit in males was consistent, Although sex differences were not reported in the SP the female offspring of dams that had been gavaged with test, per se, estrogen alpha and beta mRNA expression CPF did not show a deficit in the social preference test were correlated with social interaction in regions asso- even thought they were impaired in the SCPP. It is dif- ciated with social recognition, such as amygdala, dorsal ficult to draw conclusions regarding social behavior of septum and medial preoptic area [6]. Estrogen replace- female offspring in this study for several reasons: (a) in ment has both prosocial or asocial effects, depending on general female mice showed a weak social preference context [16]. Further investigation of the effects of gesta - whether innate or learned and (b) the number of females tional exposure to CPF should more closely control for in the studies was very low in some groups (3–5), reduc- hormonal influences by monitoring the hormonal state of ing the power of the analysis. De Felice et al. [9] pointed the female. out that female mice exposed to gestational CPF showed Prenatal exposure to CPF did not affect the OT gene more social discrimination, while gestational exposure to expression in the hypothalamus. This contrasted with CPF had no effect in a strain with inherently low social increased OT peptide expression in the hypothalamus in behavior, the BTBR mice [8]. Venerosi et al. [51] found males reported in adult mice that had been exposed to that 6 mg/kg exposure to CPF on GD 15–18 in outbred 6 mg/kg of CPF on GD 15–18 [49]. Another study by the CD-1 mice led to more frequent social vocalization and same group showed that CPF administration on GD 15 to investigation, an effect that was eliminated if the mice lactation day 14 did not change the expression of OT in continued to be exposed to CPF postnatally. Mullen et al. the hypothalamus, but decreased the expression of OT in [35] also found that adult female, but not male mice, the amygdala in males [52]. Together these data suggest which had been exposed during late gestation to CPF that exposure on different periods of development may Lan et al. Behav Brain Funct (2019) 15:2 Page 6 of 9 result in different alternations of brain function, an issue did not induce overt cholinergic symptoms in dams, such which requires further research. as tremor or lacrimation. Exposure to CPF during the second trimester of gesta- All the tests were performed blindly by having a tion led to impaired social preference and conditioned research assistant or investigator remove the identities social preference in male, but not female adult offspring. of the treatment groups and number the cages consecu- However, conditioning impairments in the FCPP sug- tively. The code was broken only when the data were ana - gest that the CPF exposure impairs both social and non- lyzed. On each testing day, mice from each group was social learning for positive reinforcement. There was no tested. The order of testing was counterbalanced. The effect of CPF on the expression of OT transcripts in the protocols were approved by the Institutional Committee hypothalamus; however further studies are required to for the Ethical Care and Use of the Animals in Experi- examine the effects on OT and its receptor in brain areas mentation of Ben-Gurion University of the Negev. relevant to reward conditioning. Social preference Conclusions At 90 days of age innate social preference was tested Exposure to low dose pesticides is a global health prob- on one female and one male per litter [25]. The test - lem which can impair brain development in utero. In the ing box (24 × 16 cm) was divided into 3 chambers present study, prenatal CPF induced innate and learned (8 × 16 cm each). The middle chamber (8 × 16 cm) that social deficits and non-specific conditioning deficits in had a small opening on either side to allow the mouse adult mice in a sex-dependent manner. Male offspring of to pass freely between chambers. Each side chamber dams treated with 5 mg/kg CPF showed specific social contained an overturned perforated oval plastic cup deficits, whereas both males and female offspring of (10.4 × 9.4 cm × 12.2 cm high), one of which enclosed dams treated with 2.5 mg/kg CPF showed a more gener- an unfamiliar, untreated conspecific of the same age and alized conditioning deficit. The mechanism for this defi - sex. The perforations allowed nose to nose contact but cit requires further investigation, as in this study, we did prevented other direct contact. This apparatus is similar not find a relation between the behavioral findings and to that used in the classical studies of social preference hypothalamic oxytocin. and social novelty to test genetic mouse models of autism [34, 36]. Mice serving as social stimuli were habituated to Materials and methods the apparatus the day before testing by confining them Mice breeding and prenatal treatment to the container for 10 min. The experimental mouse All experiments were conducted on C57BL/6 (B6) mice, was placed in the middle chamber and allowed to freely using one naive male and one naïve female mouse per lit- explore the 3 chambers for 10 min without the object ter for each of the behavioral assessments and another or mouse and then tested the following day for 10 min male and female from the same litter for the OT gene and in the presence of the empty container or the container receptor expression evaluation. Dams and sires for breed- under which the stimulus mouse was confined. The sides ing were purchased from Harlan, Israel. Animals were containing the social stimulus vs empty container were housed in polycarbonate breeding cages in a temperature counterbalanced among mice. The time spent and num - controlled environment (22 ± 1 °C) under a 12-h reversed ber of entries into the chamber containing the stimulus light–dark cycle (21:00–9:00 lights on) and ad libitum mouse and that with the empty container were measured food (SSNIFF V1154-703 10 mm gamma irradiated offline by an experimenter blind to the treatment condi - 25 kGy) and water. After mating two females to one male, tion using Ethovision software. Social preference was females were examined daily for the presence of vaginal calculated as the ratio of the time spent in the chamber plug, designating gestational day (GD) 0. On GD 12, the containing the mouse to the total time spent in the appa- dams were randomly assigned to one of three prenatal ratus (measured in seconds). treatments: high (5 mg/kg) or low dose of CPF (2.5 mg/ kg), or corn oil (vehicle). Dams were housed individually Social conditioned place preference (SCPP) after the plug was discovered and then housed with their First the preferred bedding of each mouse was tested in a litter until weaning. CPF (99.5% purity, Chem Service, preference test similar to the SP described in the previous Inc.) or corn oil (Willi Food, Yavneh, Israel) was admin- section, except that instead of choosing between a cup istered by gavage to pregnant females daily from GD 12 concealing a conspecific and an empty cup, in this test to 15 in a volume of 0.1 ml/10 g body weight using a 22 the mouse had to choose between two novel types of bed- gauge stainless steel feeding tube (Solomon Instech, Inc.). ding, either aspen sawdust or shredded paper. Pilot data Pups were weaned at 28 days and housed in same sex on mice that did not participate in this study indicated littermate cages of 2–5 pups. The doses that were selected that there was no overall preference for either type of Lan et al. Behav Brain Funct (2019) 15:2 Page 7 of 9 bedding, but that individual mice tended to show a pref- hypothalamus was removed when the ventral surface was erence for one or the other bedding. A negatively biased exposed. In order to isolate OT RNA we homogenized paradigm was used in order to reverse the initial prefer- brain tissues using Precellys Lysing Kit (Ref. KT03961-1- ence by associating the non-preferred bedding to social 993.2). Then the tissues were run for 30 s twice in a homog - reward. Hence, the non-preferred bedding served as the enizer (maximum speed) (Minilys , Bertin Instruments) conditioned stimulus to be associated with the social liv- and RNA was isolated using Biological Industries EZ-RNA ing condition, whereas the initially preferred bedding was II kit, following its protocol. For reverse transcription, we associated with the isolation condition. Conditioning was used QIAGEN QuantiTect Reverse Transcription Kit (Ref. accomplished by housing the experimental mouse with 205311), using the recommended kit protocol. We reverse- its non-preferred bedding and a cage mate for 24 h fol- transcribed 200 ng RNA to cDNA. Finally, for quantita- lowed by 24 h housing alone with the initially preferred tive PCR (qPCR) we used BioRad SsoAdvanced Universal bedding. The housing conditions were alternated for a SYBR Green Supermix (Cat. No. 172-5271) kit and BioRad total of 10 days with food and water available ad lib. The CFX384 machine, following the kit protocol: conditioned preference was assessed for each mouse as described for social preference. Each side contained 1. 95 °C, 5 min a cupful of one of the two beddings (social and isola- 2. 95 °C, 15 s tion) spread on the floor of the chamber. The day before 3. 60 °C, 30 s the test the experimental mouse was allowed to freely 4. Repeat steps 2–3 for 39 more times explore the chamber for 10 min in the absence of sawdust 5. Melting curve: 65–95 °C, .5 °C increments, 5 s/step. and on the test day they were place in the middle cham- ber and allowed to explore the 3 chambered apparatus in Each qPCR reaction final volume was 12 μl, with cDNA the presence of the two bedding stimuli. The sides of the equivalent to 10 ng RNA, and primer concentrations of positively or negatively conditioned beddings were coun- 300 nM each. Each combination of a sample and a primer terbalanced among the mice. Time spent in each cham- set ran in triplicates. Primer sequences for OT were: For- ber, measured in seconds, was scored via the Ethovision ward-GAC CTG GAT ATG CGC AAG TGT and reverse- software by an observer blind to the treatment condition. GAA GCA GCC CAG CTC GTC. The sequences of the ‘housekeeping’ gene glyceraldehyde 3-phosphate dehy- drogenase (GAPDH) were: Forward-AGA GAC AGC Food conditioned place preference (FCPP) CGC ATC TTC TTG and reverse-GTA ACC AGG CGT A deficit in SCPP could indicate either a social deficit or CCG ATA CG. a more general impairment in contextual conditioning. In order to test the specificity of the deficit, we tested the mouse’s ability to learn contextual conditioning to a Statistical analysis specific bedding that was associated with a non-social Before analyzing the data using analysis of variance appetitive cue (soup croutons, Osem Ltd.). During the (ANOVA) the Levene’s test for homogeneity of variance conditioning phase, each mouse was housed individually was performed. If the assumption of homogeneity of vari- in a cage with either the novel aspen or shredded paper ance was rejected, the data were analyzed by the non-par- bedding. Regular chow was restricted to the equivalent of ametric Kruskal–Wallis test. This was done in the case of 9% of body weight in grams, such that a mouse weigh- Social Conditioned Place Preference. If the Levene’s test ing 30 g, received 2.7 g chow each day. Water was given was not significant, data were analyzed by two-way analy - ad lib. For the positive conditioned stimulus, 10 croutons sis of variance (ANOVA) for the effect of prenatal treat - were hidden in the bedding each day, whereas nothing ment (vehicle, 2.5 or 5 mg/kg) × sex (male, female) for was hidden in the bedding that was not associated with each of the dependent variables. If there was a significant reward (the stimulus that predicted no reward). The effect of treatment or a significant interaction involving habituation and preference testing occurred as described treatment, a post hoc Tukey test was performed. for the SCPP. A change in the bedding baseline prefer- Authors’ contributions ence was measured as an indication for conditioning the OK, AL and DT designed the study. AL, DS and MP conducted the experi- bedding stimulus to the food treat. Data were analyzed ments. All authors participated in data analysis and writing. All authors read by an observer blind to the treatment condition using the and approved the final manuscript. Ethovision software. Author details Department of Psychology, Ben-Gurion University of the Negev, POB 653, Oxytocin transcript analysis Beer-Sheva, Israel. Department of Life Sciences, Ben-Gurion University of the Negev, POB 653, Beer-Sheva, Israel. Zlotowski Center for Neuroscience, On PND 90, naïve mice that were littermates of those Ben-Gurion University of the Negev, POB 653, Beer-Sheva, Israel. tested in the behavioral studies were sacrificed and the Lan et al. Behav Brain Funct (2019) 15:2 Page 8 of 9 Acknowledgements 12. Engel SM, Wetmur J, Chen J, Zhu C, Barr DB, Canfield RL, et al. Prenatal The authors gratefully acknowledge the Israel Science Foundation for grant exposure to organophosphates, paraoxonase 1, and cognitive develop- 1530/14. ment in childhood. Environ Health Perspect. 2011;119(8):1182–8. 13. Eskenazi B, Marks AR, Bradman A, Harley K, Barr DB, Johnson C, et al. Competing interests Organophosphate pesticide exposure and neurodevelopment in young The authors declare that they have no competing interests. Mexican-American children. Environ Health Perspect. 2007;115(5):792–8. 14. Furlong MA, Engel SM, Barr DB, Wolff MS. Prenatal exposure to organo - Availability of data and materials phosphate pesticides and reciprocal social behavior in childhood. Raw data will be made available in an open access file at request to the cor - Environ Int. 2014;70:125–31. responding author. 15. Furlong MA, Herring A, Buckley JP, Goldman BD, Daniels JL, Engel LS, et al. Prenatal exposure to organophosphorus pesticides and childhood Consent for publication neurodevelopmental phenotypes. Environ Res. 2017;158:737–47. Not applicable. 16. Garcia AN, Bezner K, Depena C, Yin W, Gore AC. The effects of long-term estradiol treatment on social behavior and gene expression in adult Ethics approval and consent to participate female rats. Horm Behav. 2017;87:145–54. Protocol BGU-IL-66-13, Israel Ministry of Health, Ben-Gurion University of 17. Gunier RB, Raanan R, Castorina R, Holland NT, Harley KG, Balmes JR, et al. the Negev Committee for Care and Use of Animals. Committee has AALAC Residential proximity to agricultural fumigant use and respiratory health approval. in 7-year old children. Environ Res. 2018;164:93–9. 18. Hallmayer J, Cleveland S, Torres A, Phillips J, Cohen B, Torigoe T, et al. Funding Genetic heritability and shared environmental factors among twin pairs Funded by grant 1530/14 by the Israel Science Foundation to OK. This is a with autism. Arch Gen Psychiatry. 2011;68(11):1095–102. non-commercial government funded agency. 19. Harley KG, Engel SM, Vedar MG, Eskenazi B, Whyatt RM, Lanphear BP, et al. Prenatal exposure to organophosphorous pesticides and fetal growth: pooled results from four longitudinal birth cohort studies. Environ Health Publisher’s Note Perspect. 2016;124(7):1084–92. Springer Nature remains neutral with regard to jurisdictional claims in pub- 20. Heudorf U, Angerer J, Drexler H. Current internal exposure to pesticides lished maps and institutional affiliations. in children and adolescents in Germany: urinary levels of metabolites of pyrethroid and organophosphorus insecticides. Int Arch Occup Environ Received: 10 September 2018 Accepted: 19 February 2019 Health. 2004;77(1):67–72. 21. Horton MK, Kahn LG, Perera F, Barr DB, Rauh V. Does the home environ- ment and the sex of the child modify the adverse effects of prenatal exposure to chlorpyrifos on child working memory? 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Behavioral and Brain Functions – Springer Journals
Published: Mar 1, 2019