Tissues from WT and *En2*−/− mice (*n* = 6 per genotype) were dissected and frozen in dry ice. Blood samples were centrifuged in an Eppendorf benchtop centrifuge for 15 min at 3,000 rpm to separate serum from cell fraction, and then frozen in dry ice. All samples were stored at −80°C until use. Total RNAs were extracted by Trizol reagent (Invitrogen), treated with DNAse, purified by the RNAeasy Kit (Qiagen), and pooled. cDNAs were synthesized from pooled RNAs (3µg) by SuperScript VILO cDNA Synthesis Kit (Invitrogen). Quantitative reverse-transcription PCR (RT-PCR) was performed in a C1000 thermal cycler (BioRad) with realtime detection of fluorescence, using the KAPA SYBR FAST master mix reagent (Resnova). Mouse mitochondrial ribosomal protein L41 (Mrpl41) was used as a standard for quantification. Primers (Sigma Genosys, UK) sequences are reported in **Table 1**. Ratios of comparative concentrations of each mRNA with respect to L41 mRNA were then calculated and plotted as the average of three to four independent reactions with technical replicates obtained from each RNA pool. Expression analyses were performed using the CFX3 Manager (BioRad) software.

#### **BIOINFORMATIC ANALYSIS**

Analysis of *En2* binding sites on GH, GHR, mGRF, SST, IGF-1, and IGF-1R gene promoters was performed using the Matinspector web-based search algorithm available from Genomatix Software2 . The algorithm calculated the similarity between the core motif and overall sequence of the *En2* consensus binding site (matrix ID: V\$EN2.01) and the gene promoter sequences of interest. The *En2* matrix was determined based on the Genomatix Matrix Library Version 9.1.

#### **IMMUNOHISTOCHEMISTRY**

Brains from adult WT and *En2*−/− mice (*n* = 3 per genotype) were used for immunohistochemical characterization of

2http://www.Genomatix.de

Abbreviations are as in the text.

hypothalamic SST. Brains were fixed by transcardial perfusion with 4% paraformaldehyde followed by 1 h post-fixation, and coronal sections (40µm thickness) were cut by a vibratome (Leica). Serial sections at level of the hypothalamus were incubated overnight with and anti-SST rabbit polyclonal (Peninsula-Bachem; 1:2000 dilution). Signals were revealed using appropriate secondary antibodies and fluorophores as described (27). Hypothalamic nuclei were identified according to the Allen Brain Atlas3 . Images were acquired at 10× objective magnification using a Zeiss AxioImager M2 microscope.

#### **IN SITU HYBRIDIZATION**

Brains from WT and *En2*−/− mice (*n* = 3 per genotype) were rapidly removed and frozen on dry ice. Coronal cryostat sections (20µm thick) were fixed in 4% paraformaldehyde. Nonradioactive *in situ* hybridization was performed as previously described (27) using digoxigenin-labeled riboprobes specific for GH [GenBank ID: X02891; (30)], mGRF (31), and IGF-1 (Gen-Bank ID: NM\_010512). The IGF-1 cDNA was cloned by PCR from hippocampal cDNA and recognizes both class 1 and class 2 IGF-1 mRNAs. Signal was detected by alkaline phosphatase-conjugated anti-digoxigenin antibody followed by alkaline phosphatase staining. The specificity of the results was confirmed by the use of sense riboprobes (not shown).

#### **ENZYME-LINKED IMMUNO-SORBENT ASSAY**

Blood samples (*n* = 5 per genotype) were collected at the time of animal sacrifice, kept in ice for 30 min, and then centrifuged in an Eppendorf benchtop centrifuge for 15 min at 3,000 rpm to separate serum. Dissected liver and hippocampal tissues (*n* = 5 per genotype) were homogenized in lysis buffer (50 mM Tris–HCl pH 7.5, 1% NP-40, 1% Triton-100, 1 mM PMSF, 10% glycerol, and protease inhibitor cocktail; Sigma-Aldrich). Homogenates were incubated in ice for 30 min, centrifuged at 12,000 rpm for 5 min at 4°C, and supernatants were recovered and stored at −80°C. Protein concentration in serum, liver, and hippocampal samples was determined by BCA method (Pierce). Samples were processed for GH (EZRMGH-45K, Millipore) and IGF-1 (ELM-IGFI; Ray-Biotech) ELISA according to manufacturers' protocol. All samples were analyzed in duplicate.

#### **STATISTICAL ANALYSIS**

Statistical analysis was performed with SigmaPlot 11.0 and Prism 6 (GraphPad) softwares. Values were expressed as mean ± SEM and quantitative gene expression differences between WT and *En2*−/− mice were assessed by Student's *t*-test, with the level of statistical significance set at *p* < 0.05.

#### **RESULTS**

#### **EXPRESSION OF En2 AND IGF-1 SIGNALING GENES IN THE MOUSE BRAIN–PITUITARY–LIVER AXIS**

By using quantitative RT-PCR, we first investigated mRNA expression of *En2*, GH, GH receptor (GHR), mGRF, SST, IGF-1, and IGF-1 receptor (IGF-1R) in the neuroendocrine axis of WT adult male mice from our colony. mRNA expression was studied in the hypothalamus, pituitary gland, and liver (the crucial tissues involved in GH and IGF-1 synthesis), as well as hippocampus and blood cell fraction. In agreement with previous findings, we confirmed that *En2* mRNA is expressed in the hypothalamus and hippocampus (23, 25, 27). *En2* mRNA was also expressed at detectable levels in blood, pituitary gland, and liver (**Figure 1A**). As expected, GH mRNA was mainly detected in the pituitary gland, while much lower levels were present in the hippocampus, hypothalamus, liver, and blood (**Figure 1B**). Consistent with the notion that liver is the main target of GH action, we found GHR mRNA predominantly expressed in the liver, and at lower levels in the other tissues analyzed (**Figure 1C**). mRNA expression of mGRF and SST, the two hypothalamic hormones controlling GH synthesis, was mainly detected in the hypothalamus (**Figures 1D,E**). High levels of SST mRNA were also present in the hippocampus, as previously described (27). Two major different transcripts have been described for IGF-1 [class 1 and class 2; (32, 33)]. Both IGF-1 class 1 and class 2 mRNAs were predominantly expressed in the liver (**Figures 1F,G**), as expected (32). Finally, IGF-1R mRNA was mainly expressed in the pituitary gland (the target for IGF-1 negative feedback for GH production) (**Figure 1H**), but also throughout the neuroendocrine axis, consistent with the widespread action of IGF-1 on multiple tissues. These results clearly indicate that our RT-PCR protocol can detect the expression of genes belonging to the GH/IGF-1 pathway in the appropriate tissues throughout the brain–pituitary–liver axis.

#### **ALTERED GH mRNA EXPRESSION IN THE BRAIN–PITUITARY–LIVER AXIS OF En2** −**/**− **MICE**

Before investigating GH, GHR, mGRF, SST, IGF-1, and IGF-1R mRNA expression in the neuroendocrine axis of *En2*−/− mice, we first verified whether *En2* might directly regulate their transcription. Indeed, bioinformatic analysis revealed that *En2* binding sites are present in the promoters of all these genes (**Table 2**). We next studied GH and GHR mRNA expression in the neuroendocrine axis of WT and *En2*−/− adult mice. We found that GH mRNA expression was markedly deregulated throughout the neuroendocrine axis in *En2*−/− mice. A statistically significant increase of GH mRNA levels was detected in the pituitary gland (+72%, *p* < 0.001), liver (+376%, *p* < 0.001), and blood (+87%, *p* < 0.05) of *En2*−/− mice, as compared to WT littermates (**Figure 2A**). GH mRNA levels were instead significantly lower in the *En2*−/− hypothalamus (−97%, *p* < 0.001) and hippocampus (−98%, *p* < 0.001), as compared to WT (**Figure 2A**). *In situ* hybridization confirmed GH mRNA decrease in the *En2*−/− hippocampus, mainly in the CA3 subfield (**Figure 2C**). No significant differences in GHR mRNA levels were detected between genotypes in the analyzed tissues, with the exception of blood, where a marked increase was detected in mutant mice compared to controls (+312%, *p* < 0.05) (**Figure 2B**).

#### **ALTERED EXPRESSION OF mGRF AND SST IN THE HYPOTHALAMUS OF En2** −**/**− **MICE**

We then investigated the expression of mGRF (also known as growth hormone releasing hormone, GHRH) and SST, the two hypothalamic hormones regulating GH synthesis, in the

3http://www.brain-map.org/

**FIGURE 1 | mRNA expression of En2 and genes involved in the GH/IGF-1 pathway in the neuroendocrine axis of WT mice**. En2 **(A)**, GH **(B)**, GHR **(C)**, mGRF **(D)**, SST **(E)**, IGF-1 **(F,G)**, and IGF-1R **(H)** mRNA expression levels in the hippocampus, hypothalamus, pituitary gland, liver, and blood, obtained by quantitative RT-PCR. For each mRNA, relative expression levels

(normalized to L41) are reported on a log scale. Two different transcripts (class 1 and class 2) were analyzed for IGF-1. Values are plotted as mean ± SEM of three independent experiments. Abbreviations: hi, hippocampus; hy, hypothalamus; pit, pituitary gland; liv, liver; bld, blood (cell fraction). Other abbreviations are as in the text.

The promoter regions of the six genes belonging to the GH/IGF-1 pathway were analyzed for the presence of En2 binding site using the Matinspector program. Core similarity (Core) indicates the match against the nucleotides representing the core motif of the En2 binding site (AATT), where 1 is 100% identity. Matrix similarity (Matrix) is the overall similarity to the En2 binding motif, where 1 is 100% identity. For each sequence, the start/end position respect to ATG is indicated, together with the strand on which they are present. For each promoter, several regions containing the En2 binding site were found; the table reports only those with highest values for core and matrix similarity. Genomatix Promoter database (GXP) accession numbers are given for each gene. Abbreviations are as in the text.

brain–pituitary–liver axis of WT and *En2*−/− adult mice. As compared to WT controls, a marked increase of mGRF mRNA (+53%, *p* < 0.05; **Figure 3A**) and a significant decrease of SST mRNA (−13%, *p* < 0.01; **Figure 3B**) was found in the *En2*−/− hypothalamus. Significantly higher mRNA levels for the two hormones were also found in blood (mGRF: +125%, *p* < 0.05) and liver (mGRF: +129%, *p* < 0.01; SST: +147%, *p* < 0.001) from *En2*−/− mice (**Figures 3A,B**). According to our previous study (27), lower levels of SST mRNA were found in the *En2*−/− hippocampus, as compared to WT (−10%, *p* < 0.05; **Figure 3B**). *In situ* hybridization

and immunohistochemistry experiments, respectively, confirmed the increased expression of mGRF mRNA and decreased levels of SST protein in the dorsomedial/ventromedial paraventricular nuclei of the *En2*−/− hypothalamus, as compared to WT (**Figure 3C**).

#### **ALTERED IGF-1 mRNA EXPRESSION IN THE BRAIN–PITUITARY–LIVER AXIS OF En2** −**/**− **MICE**

We next analyzed mRNA expression of IGF-1 and its receptor in the brain–pituitary–liver axis of WT and *En2*−/− adult mice.

**FIGURE 2 | Expression of GH and GH receptor mRNAs in the neuroendocrine axis of WT and En2** −**/**− **mice**. **(A)** GH mRNA quantitative RT-PCR. **(B)** GHR quantitative RT-PCR. Values are plotted as each gene/L41 comparative quantitation ratios normalized on the expression of WT (mean ± SEM of three replicates from pools of six animals per genotype; \*p < 0.05, \*\*\*p < 0.001; Student's t-test, En2−/− vs. WT). **(C)** Representative pictures of GH mRNA in situ hybridization on the dorsal hippocampus from WT and En2−/− mice. Insets show the CA3 subfield. Scale bar: 200µm (whole hippocampi) and 125µm (insets). Abbreviations are as in **Figure 1**.

IGF-1 mRNA exists in two major forms (class 1 and class 2), class 2 mRNA transcription being directly regulated by GH (33, 34). We found that levels of IGF-1 class 1 mRNA were significantly reduced in the hippocampus but not other tissues of *En2*−/− mice, as compared to WT (−18%, *p* < 0.05 for all comparisons) (**Figure 4A**). In keeping with GH mRNA expression data (**Figure 2**), IGF-1 class 2 mRNA was significantly up-regulated in the liver (+43%, *p* < 0.001) and down-regulated in the hypothalamus (−19%, *p* < 0.001) of *En2*−/− mice, as compared to WT littermates; increased levels of IGF-1 class 2 mRNA were also detected in *En2*−/− pituitary gland (+16%, *p* < 0.01) and blood (+312%, *p* < 0.001) (**Figure 4B**). *In situ* hybridization with a riboprobe specific for both class 1 and class 2 IGF-1 mRNAs confirmed that IGF-1 mRNA levels are decreased in the *En2*−/− hippocampus (**Figure 4D**). Finally, IGF-1R mRNA levels did not differ between genotypes in hypothalamus, hippocampus, and pituitary gland, while a significant increase was found in blood (+84%, *p* < 0.01) and liver (+52%, *p* < 0.05) from *En2*−/− mice compared to WT controls (**Figure 4C**). **Table 3** summarizes mRNA expression data for all the analyzed genes in the brain–pituitary–liver axis of WT and *En2*−/− adult mice.

#### **REDUCED LEVELS OF GH PROTEIN IN THE En2** −**/**− **HIPPOCAMPUS**

The altered levels of GH and IGF-1 mRNA expression detected in the *En2*−/− neuroendocrine axis prompted us to investigate the levels of these hormones in the hippocampus, serum, and liver of both genotypes. ELISA assays revealed a significant reduction (−54%, *p* < 0.05) of GH protein levels in the hippocampus of

*En2*−/− mice, as compared to WT littermates, while no difference was detected in serum samples (**Figure 5A**). Hippocampal, liver, and serum IGF-1 protein levels did not significantly differ between the two genotypes (**Figure 5B**).

Scale bar: 200µm. Abbreviations are as in **Figure 1**.

#### **DISCUSSION**

#### **BRIEF SUMMARY OF RESULTS**

In this study, we analyzed the expression of GH, IGF-1, their receptors, and regulatory hormones in the brain–pituitary–liver axis of adult *En2*−/− mice, a mouse model for ASD. We found that in mutant mice, GH and IGF-1 mRNA levels were significantly higher in the pituitary gland and liver, respectively,but this increase was not paralleled by higher levels of circulating hormones. In *En2* mutants, GH and IGF-1 mRNA levels were instead significantly down-regulated in the hippocampus, and this reduction was accompanied by a significant decrease of GH but not IGF-1 protein levels.

#### **En2 INTERACTS WITH IGF-1 SIGNALING IN THE MOUSE NEUROENDOCRINE AXIS**

Our results, schematically summarized in **Figure 6**, indicate that regulatory mechanisms controlling GH and IGF-1 mRNA

expression in the neuroendocrine system are altered in *En2*−/− mice. The presence of *En2* mRNA in the hypothalamus–pituitary– liver axis (**Figure 1**) and that of an *En2* binding site in the promoters of GH, IGF-1, and other genes of the pathway (**Table 2**) suggest that *En2* might directly contribute to their transcriptional control. Indeed, recent studies revealed an unprecedented interaction between *En2* and IGF-1 signaling. In the absence of *En2*, IGF-1 has a stronger mitogenic effect on cerebellar GNPs, due the increased activity of downstream effectors of IGF-1 signaling, such as S6 kinase (29). Thus, *En2* appears to negatively regulate IGF-1 signaling during postnatal cerebellar development. Our results strengthen this link between *En2* and IGF-1 signaling, and suggest that a direct transcriptional control of *En2* onto genes belonging to the IGF-1 pathway takes place also in the brain–pituitary–liver axis.

Summary of quantitative RT-PCR data. Data are presented as the mean percentage of the decrease/increase of each mRNA in En2−/− mice, as compared to WT littermates.

Statistical significance: \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001 (En2−/− vs. WT). Abbreviations are as in the text.

**FIGURE 6 | Schematic summary of SST, mGRF, GH, and IGF-1 expression in the En2** −**/**− **neuroendocrine axis**. Red and blue arrows, respectively, indicate up- and down-regulations observed in En2−/− mice as compared to WT controls. Double-arrowed gray lines indicate comparable levels between WT and En2−/− mice. Increased levels of GH and IGF-1 mRNA, respectively observed in En2−/− pituitary and liver, are not paralleled by higher levels of circulating hormones, suggesting that a complex post-translational control of GH and IGF-1 synthesis takes place in mutant mice. GH mRNA and protein levels are instead significantly down-regulated in En2−/− hippocampus. Arrowed red lines connecting different organs indicate the action of circulating hormones onto their target tissues. The mouse brain sagittal section is a Nissl stain taken from the Allen Mouse Brain Atlas (see text footnote 2). Abbreviations are as in the text.

#### **DIFFERENTIAL EXPRESSION OF GH AND IGF-1 mRNAs IN THE BRAIN AND PERIPHERAL TISSUES OF En2** −**/**− **MICE**

IGF-1 mRNA exists in two main isoforms, derived from alternative splicing of the two first exons contained in the 50 untranslated region (50 UTR) of the mRNA precursor. Class 1 transcripts contain exon 1, whereas class 2 transcripts contain exon 2 (33). Inclusion of exon 1 or exon 2 is mutually exclusive, resulting in different 50 UTRs of the IGF-1 mRNA containing different leader sequences of the pre-pro-IGF-I peptide. In addition, transcription of class 1 and class 2 mRNAs is differently controlled; class 2 mRNA being directly regulated by GH. It is, however, important to note that both class 1 and class 2 transcripts code for the same IGF-1 mature peptide (33).

In *En2* mutants, we described a differential expression of IGF-1 mRNAs in different tissues of the brain–pituitary–liver axis. A significant increase of IGF-1 class 2 mRNA was detected in liver and blood, whereas IGF-1 class 1 mRNA was significantly decreased in the *En2*−/− hippocampus (**Figure 4**). The negative regulation exerted by *En2* onto IGF-1 signaling in the cerebellum (29), therefore, appears to occur also at a transcriptional level in the hippocampus.

Interestingly,*En2*−/− mice showed increased hypothalamic levels of mGRF and decreased levels of somatostatin (SST), the two hormones controlling GH synthesis. Previous studies from our laboratory already showed that loss of *En2* results in lower levels of SST mRNA and protein in the hippocampus and cerebral cortex. Here, we extended this observation, demonstrating that lower levels of SST are also present in the *En2*−/− hypothalamus; hypothalamic levels of mGRF mRNA were instead increased in mutant mice (**Figure 3**). Deregulation of hypothalamic hormones controlling GH synthesis might result in increased levels of circulating GH and IGF-1 in mutant mice. However, we showed that serum levels of both hormones, as well as IGF-1 levels in liver did not differ between WT and *En2*−/− mice (**Figure 5**). Indeed, behavioral studies did not reveal gross weight and growth abnormalities in *En2* mutants (23), suggesting that differences in GH and IGF-1 mRNA levels might be blunted at the protein level, via multiple post-transcriptional control mechanisms. Indeed, a tight dependence of IGF-1 translation via several miRNA binding site in the 30 UTR of the transcripts has been described (33, 35).

#### **FUNCTIONAL CONSEQUENCES OF REDUCED GH LEVELS IN THE En2** −**/**− **HIPPOCAMPUS**

The decreased expression of GH mRNA and protein observed in the *En2*−/− hippocampus might contribute to learning deficits observed in *En2* mutants. Indeed, *En2*−/− mice show impaired learning in hippocampal-dependent tasks, such as the Morris water maze and contextual fear conditioning (22, 23). Profound effects on cognitive function have been demonstrated for GH. GH-deficient spontaneous dwarf rats display marked deficits in hippocampus-dependent spatial learning and memory, accompanied by an imbalance in hippocampal glutamatergic/GABAergic synapses and neurogenesis (36). Chronic stress, which is known to affect hippocampal function, also reduces hippocampal GH levels (37). Restoration of normal GH levels in the hippocampus (obtained via viral-mediated gene transfer) is able to reverse stress-dependent behavioral impairment, as tested by hippocampus-dependent tasks (contextual fear conditioning) (37). Taken together, these results indicate that reduced levels of hippocampal GH detected in *En2*−/− mice might contribute to hippocampal dysfunction observed in these mutants.

#### **CONCLUSION**

To our knowledge, this is the first demonstration that brain GH levels are reduced in a mouse model of ASD. Considering the important role of GH on cognitive functions, our data strengthen the notion that reduced expression of GH in the hippocampus may be implicated in learning disabilities associated to ASD.

#### **AUTHOR CONTRIBUTIONS**

Giovanni Provenzano and Elena Clementi equally contributed to this study. Giovanni Provenzano designed and performed experiments, analyzed data and wrote the paper. Elena Clementi designed and performed experiments, analyzed data. Sacha Genovesi, Manuela Scali, Prem Prakash Tripathi, and Paola Sgadò performed experiments. Yuri Bozzi provided funding, conceived the study, analyzed data and wrote the paper.

#### **ACKNOWLEDGMENTS**

We are grateful to the technical/administrative staff of the Centre for Integrative Biology (University of Trento). We thank Prof. Emiliana Borrelli (University of California, Irvine, CA, USA) for the kind gift of GH and mGRF cDNAs. This work was funded by grants from the Italian Ministry of University and Research (PRIN 2008 grant 200894SYW2\_002 and PRIN 2010-11 grant 2010N8PBAA\_002) and the University of Trento (CIBIO start-up) to Yuri Bozzi. Paola Sgadò is supported by Provincia Autonoma di Trento (Italy) under the Marie Curie-People cofunding action of the European Community.

#### **REFERENCES**

**Conflict of Interest Statement:** The Reviewer, Dr Alessandro Sale, declares that despite being affiliated with the same institution as author Dr Manuela Scali and having collaborated with her in the past, the review process was handled objectively and no conflict of interest exists. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 27 May 2014; accepted: 18 August 2014; published online: 01 September 2014. Citation: Provenzano G, Clementi E, Genovesi S, Scali M, Tripathi PP, Sgadò P and Bozzi Y (2014) GH dysfunction in Engrailed-2 knockout mice, a model for autism spectrum disorders. Front. Pediatr. 2:92. doi: 10.3389/fped.2014.00092*

*This article was submitted to Child and Neurodevelopmental Psychiatry, a section of the journal Frontiers in Pediatrics.*

*Copyright © 2014 Provenzano, Clementi, Genovesi, Scali, Tripathi, Sgadò and Bozzi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Region specific up-regulation of oxytocin receptors in the opioid Oprm1−/− mouse model of autism

#### **Valentina Gigliucci 1 , Marianna Leonzino1,2, Marta Busnelli 1,2, Alessandra Luchetti 3 , Viola Stella Palladino3 , Francesca R. D'Amato3,4 and Bice Chini 1,2\***

1 Institute of Neuroscience, National Research Council, Milan, Italy

2 Dipartimento di Biotecnologie e Medicina Traslazionale, Università degli Studi di Milano, Milan, Italy

3 Institute of Cellular Biology and Neurobiology, National Research Council, Rome, Italy

4 IRCCS Santa Lucia Foundation, Rome, Italy

#### **Edited by:**

Yuri Bozzi, University of Trento, Italy

#### **Reviewed by:**

Valery Grinevich, German Cancer Research Center (DKFZ) and University of Heidelberg, Germany Valentina Colonnello, Albert Ludwigs University of Freiburg, Germany

#### **\*Correspondence:**

Bice Chini, Institute of Neuroscience, CNR, via Vanvitelli 32, Milano 20129, Italy e-mail<EMAIL_ADDRESS>
impairments, and restricted and repetitive behaviors and interests. Recently, altered motivation and reward processes have been suggested to participate in the physiopathology of ASDs, and µ-opioid receptors (MORs) have been investigated in relation to social reward due to their involvement in the neural circuitry of reward. Mice lacking a functional MOR gene (Oprm1−/− mice) display abnormal social behavior and major autistic-like core symptoms, making them an animal model of autism.The oxytocin (OXT) system is a key regulator of social behavior and co-operates with the opioidergic system in the modulation of social behavior. To better understand the opioid-OXT interplay in the central nervous system, we first determined the expression of the oxytocin receptor (OXTR) in the brain of WT C57BL6/J mice by quantitative autoradiography; we then evaluated OXTR regional alterations in Oprm1−/− mice. Moreover, we tested these mice in a paradigm of social behavior, the male–female social interaction test, and analyzed the effects of acute intranasal OXT treatment on their performance. In autoradiography, Oprm1−/− mice selectively displayed increased OXTR expression in the Medial Anterior Olfactory Nucleus, the Central and Medial Amygdaloid nuclei, and the Nucleus Accumbens. Our behavioral results confirmed that Oprm1−/− male mice displayed social impairments, as indicated by reduced ultrasonic calls, and that these were rescued by a single intranasal administration of OXT. Taken together, our results provide evidence of an interaction between OXT and opioids in socially relevant brain areas and in the modulation of social behavior. Moreover, they suggest that the oxytocinergic system may act as a compensative mechanism to bypass and/or restore alterations in circuits linked to impaired social behavior.

Autism spectrum disorders (ASDs) are characterized by impaired communication, social

**Keywords: oxytocin receptor,** µ**-opioid receptor, brain autoradiography, social behavior, autism, Oprm1**−**/**− **mice, ultrasonic vocalizations**

#### **INTRODUCTION**

Autism spectrum disorders (ASDs) are characterized by a triad of symptoms that includes impaired communication, social impairments, and restricted and repetitive behaviors and interests (1). The prevailing hypothesis regarding the physiopathology of ASDs identifies the area of social cognition as a primary deficit. In particular, it focuses on the impaired capabilities of affected people to attribute mental states to others (and oneself) in order to explain and predict behavior (i.e., the theory of mind hypothesis proposed by Baron-Cohen and colleagues (2, 3). However, altered affectivity is evident in autistic children (4) and, as recently proposed by Chevallier and others (5), motivation and reward processes might also participate to the physiopathology of ASDs. Reward circuitry dysfunctions might lead to deficits in social seeking and maintenance, resulting in reduced social capabilities and interests, and if appearing early in life, in social learning. Deficit in social cognition will thus be a consequence, rather than a cause, of impaired social behavior. Excessive brain opiate activity has been proposed in the past as a neurochemical feature in autism (6) and due to their involvement in the neural circuitry of reward, µ-opioid receptors (MORs) have also been investigated in relation to social reward, emotion, and social behavior (7–9), and represent a key target to understand the neurobiological basis of social reward dysfunction in humans and animals.

In humans,MOR activation in specific brain regions such as the amygdala, the periaqueductal gray, and the subgenual cingulated cortex is believed to be protective or adaptive in relation to social rejection. In fact, positron emission tomography (PET) scanning showed that social rejection, exemplified by a paradigm in which the test subject shows interest toward another individual who does not return it, could increase the binding of endogenous opioids to MOR in these areas,where greater binding also seemed to correlate to better resiliency. In a contest of social acceptance, MOR activation in the ventral striatum was shown to correlate with desire for social interaction (10). In a similar study, the G variant of the A118G polymorphism of the MOR-encoding gene (OPRM1) correlated with a greater sensitivity to social rejection, reflected in a higher activation of specific brain regions, such as the dorsal anterior cingulate cortex and the anterior insula. Genetic variations in the OPRM1 gene can also influence individuals' ability to engage in social interactions. People carrying the G allele of the A118G polymorphism of the OPRM1 gene seem to experience a greater pleasure during social situations and they tend to engage in affectionate relationships more easily in comparison with people carrying the more common A variant of the gene (11). A crucial role of MOR in partner preference has also been established by studies in prairie and mountain voles. In the monogamous prairie vole, dorsal striatal MOR has been proved fundamental for the development of partner preference, which leads to the establishment of pair bonding, although pharmacological disruption of MOR signaling did not consistently alter the pattern of mating behaviors (9, 12).

In species where individuals develop selective affective bonds during their life, such as sheep or primates, MOR signaling has been implicated in the modulation of the mother–infant attachment: in infant rhesus macaques the genetic variant C77G of the MOR gene, which increases its affinity for β-endorphin (13), has been associated with increased attachment to the mother and stronger protest response and distress during separation (14). At the same time, the maternal attachment toward the offspring seems to be subjected to MOR effects. Higham and colleagues (15) evidenced that free-ranging macaque females carrying at least one copy of the minor allele (G) of the OPRM1 gene were more possessive toward their infants than mothers homozygous for the C allele. Mouse pups where the MOR gene (*Oprm1)* has been permanently disrupted (*Oprm1*−/− mice) produce fewer ultrasound vocalizations (USVs) in response to isolation from the mother when compared to wild type mice, to indicate that lack of MOR may induce resilience to isolation (16). Moreover, it is possible that the lack of MOR prevents the establishment of an association between maternal stimuli and feelings of reward, as transgenic mice do not show a marked preference for a familiar environment over an unfamiliar one (16). Later on in their life, these mice display reduced interest for interaction with other mice of the same sex and age (17) and they appear indifferent to ultrasounds emitted by mice of the opposite sex (18). A recent extensive behavioral characterization of *Oprm1*−/− mice confirmed that these animals display major autistic-like core symptoms and elegantly provided key neuroanatomical and neurofunctional correlates (19). In particular, *Oprm1*−/− mice display abnormal social behavior, as evidenced by a decreased time in close social contact and increased selfgrooming in the direct social interaction test, reduced sociability, and social novelty recognition in the three chamber test, accompanied by increased aggression, and impaired ability in building a nest. These mice also display increased perseverative and stereotyped behaviors such as increased rearing, grooming, circling, and head shaking associated with behavioral inflexibility, as evidenced by deficient pattern of exploration in a Y-maze test. Concerning the neurobiological substrates of such phenotype, of particular interest is the observation of changes in oxytocin (OXT) gene expression in specific areas of the brain: OXT transcripts were found to be reduced in the Nucleus accumbens (NAcc) but not in the Caudate–putamen (CPu) and Central amygdala (CeA) (19).

Together with the opioid system, the OXT system is a key regulator of all the aspects of social behavior, including those involved in reproduction and care of the offspring (20). In humans, OXT facilitates the processing of social information, improves cognitive emphatic abilities and increases interpersonal trust (21). As originally put forward by Modahl (22), a deficit in the OXT system linked to an altered opioid regulation may underline the social deficits in autism. Evidence of opioid–OXT interactions is indeed well established. Endogenous opioids are involved in the modulation of OXT release into the brain and the periphery via muand kappa-receptors expressed on OXT-secreting neurons (23– 26). Even though the opioidergic modulation of OXT release may account for many shared effects, the interplay of these two systems does not probably end with that. A social motivation circuitry in which OXT, vasopressin, endogenous opioids, and catecholamines were hypothesized to participate in a wide variety of affiliative behaviors was proposed more than 15 years ago (27) and has been more recently integrated into a network of neurobiological mechanisms, which include neuronal, neurotransmitters, and hormone systems whose alterations could underline the social impairment observed in autism (28).

To contribute to unravel the critical interactions between the opioid and OXT systems in the brain, we first reviewed the literature on oxytocin receptor (OXTR) and MOR distributions in the mouse brain. As shown in **Table 1**, we found an overlapped receptors' expression in several regions involved in social behavior.

This observation suggests that OXTR and MOR may reciprocally modulate each other even at the cellular and/or molecular level. To investigate, if alterations in MOR expression might induce changes in the OXTergic system we decided to evaluate the expression and distribution of OXTRs in the brain of *Oprm1*−/− mice.

Oxytocin receptors represent the pharmacological target of OXT, and OXT administration has been proposed as a potential treatment of social deficits in autistic patients (35). In particular, intranasal OXT administration is believed to circumvent the poor blood–brain barrier (BBB) permeability of this peptide. Even if the direct passage of intranasal OXT into the brain is still matter of debate (36, 37) acute and chronic intranasal OXT administration have been shown to exert behavioral effects in rodents (34, 38, 39). Even if it cannot be excluded that some of the behavioral effects of OXT are mediated via peripheral mechanisms, intranasal OXT administration in awake animals represents at present the most convenient and reproducible method to assess the therapeutic effects of this peptide on social behavior.We thus tested *Oprm1*−/− mice in a paradigm of social behavior and analyzed the effect of intranasal OXT treatment on their behavioral performances.

#### **MATERIALS AND METHODS ANIMALS AND HOUSING CONDITIONS**

*Oprm1*+/+ (WT) and *Oprm1*−/− mice were used in this study. *Oprm1*−/− mice were generated by disruption of exon 2 in the *Oprm1* gene as described elsewhere (40). The homozygotic parents (*Oprm1*+/+ and *Oprm1*−/−) were derived from heterozygous breeding pairs that were fully backcrossed on a C57BL6/J genetic background. The two homozygous lines were maintained separately. Animals were weaned when 28-day-old and maintained

in same sex/genotype groups of four to five subjects in transparent high-temperature polysulfone cages (27 cm × 21 cm × 14 cm) with water and food available *ad libitum* (2018 Teklad Global 18% Protein Rodent Diet, Harlan, Lyon, France). Room temperature (21 ± 1°C) and a 12:12 h light–dark cycle (lights on at 1900 h) were kept constant.

Two different groups of adultWT and *Oprm1*−/− male mice (3– 4 months old) were used: the first group (4 WT and 3 *Oprm1*−/− mice, one subject per litter) was used for autoradiography and histological examination; the second group of males (18 WT from 6 litters and 17 *Oprm1*−/− mice from 7 litters) underwent intranasal OXT treatment; USVs and behavior during exposure to a female partner were observed shortly after. The genotype of all animals used in this study was controlled by PCR at the end of the experiment, according to already described procedures (40). Every animal procedure used was in strict accordance with standard ethical guidelines (European Community Guidelines on the Care and Use of Laboratory Animals 2010/63/EU) and the Italian legislation on animal experimentation (D.Lvo 116/92).

#### **OXTR AUTORADIOGRAPHY**

Naïve WT and *Oprm1*−/− mice were sacrificed by cervical dislocation, the brains quickly removed and immediately frozen by immersion in cold isopentane at −25°C and subsequent storage at −80° C.

Coronal brain sections (14µm) were sliced with a cryostat, thaw-mounted on microscope slides pre-coated with chromealum–gelatin and kept at −80°C until further use.

Oxytocin receptor autoradiography was performed as described in Huang et al. (34). Briefly, sections were fixed with 0.2% paraformaldehyde in 0.1 M phosphate-buffered saline (pH 7.4) and rinsed twice with 0.1% bovine serum albumin in 50 mM Tris-HCl buffer (pH 7.4). OXT binding sites were detected by incubation (1 h at room temperature in a humid chamber) with the radioiodinated OXTR antagonist ornithine vasotocin analog ([125I]-OVTA, specific activity 2200 Ci/mmol; Perkin Elmer, MA, USA) at 0.02 nM in a medium containing 50 mM Tris-HCl, 0.025% bacitracin,5 mMMgCl2, and 0.1% bovine serum albumin. Sections immediately adjacent to the ones used for [125I]-OVTA binding were used to determine non-specific binding by addition of 2µM OXT to the incubation solution.

At the end of binding, the unbound excess of ligand was washed out by two rinses in ice-cold incubation medium and a final rinse in cold distilled water. The slides were quickly dried under a stream of cool air and exposed to Biomax MR Films (Kodak) in an autoradiographic cassette for 72 h. The final autoradiograms were digitalized by grayscale high-resolution scanning (600 × 600 dpi) and analysis of the optical binding density of the brain regions of interest (ROIs) was carried out using the ImageJ 1.47v software (NIH, USA). ROIs were identified by comparison with a reference mouse brain atlas (41) and manually delineated with the ROI manager tool of the software. Specific densitometric gray intensity was calculated by subtraction of the gray level of the respective section treated for non-specific binding. For each animal, the final gray intensities of each brain region were calculated by averaging two [for anterior olfactory nucleus (AON), LS, AHiPM, BLP, and PMCo] or three (for OB, NAcc, CPu, Hipp CA3, PV, Hb, BLA, MeA, and CeA) sections at different coronal planes from bregma. The regions of a limited rostro-caudal extension [medial AON (AONm) and BNST] were analyzed on a single coronal plane from bregma. Brain regions were selected on the basis of co-expression of OXTR and MOR receptors at medium-high level as resulting from a review of the data currently available in the literature and summarized in **Table 1**. Even though in literature neither OXTR nor MOR expressions in the hippocampus are reported to be high, we also included the CA3 field of this region in our analysis because it appeared intensely labeled in WT mice. Binding specificity was ensured by comparison with the adjacent sections incubated with an excess of OXT in order to displace any OVTA specifically bound to the OXTRs.

Autoradiographic 125I microscales (Amersham International, UK) also were exposed for 72 h and a reference standard curve was generated. Levels of gray intensity were then converted to

nanocurie per milligram tissue equivalent by interpolation with the standard curve.

In order to increase accuracy in identifying the brain regions within each brain section, the slides labeled for non-specific binding and the slides labeled for total binding were further colored with Nissl staining and acetylcholinesterase (AChE) staining, respectively (see below).

#### **HISTOLOGICAL STAINING**

#### **Nissl staining**

The non-specific binding labeled slides were treated for Nissl staining. They were defatted by immersion in deionized water (2 min) followed by subsequent immersions in increasing concentrations of ethanol (EtOH 70% v/v, 95% v/v, and 100%, 2 min each). Sections were then rehydrated by 30 s immersions in decreasing concentrations of ethanol (EtOH 100%, 95% v/v, and 70% v/v) and after a final dip in deionized water they were leftfor 6 h in cresyl violet solution (0.1% cresyl violet, 0.65% sodium acetate trihydrate in 0.5% acetic acid, pH 3.3). Differentiation was obtained by two consecutive changes (3 s each) of deionized water, EtOH 70% (v/v) and EtOH 95% (v/v), and final dehydration was achieved by two changes (10 s each) in EtOH 100%. Sections were finally cleared by immersion in xylene and coverslips were mounted onto the slides with permanent mounting medium (Entellan®, Merck-Millipore, Germany) and left to dry overnight under a fume hood.

#### **Acetylcholinesterase staining**

The slides labeled for total binding in autoradiography were processed for AChE staining following the protocol described by Franklin and Paxinos (41). All the reagents used in this procedure were obtained from Sigma Aldrich (Italy) with the exception of the mounting medium. Sections were immersed over night at room temperature in an incubation solution (50 mM sodium acetate, 4 mM copper sulfate, 16 mM glycine, 4 mM S-acetylthiocholine iodide, 10 nM ethopropazine, and pH 5.0 with HCl 1 N) and developed the following day by incubation for 10 min at room temperature in a solution containing 1% sodium sulfide (pH 7.5 with glacial acetic acid). The colored precipitate from the reaction was fixed to the sections by an overnight incubation with formalin 10%. Finally, the slides were left to dry under a fume hood, dehydrated by subsequent immersions in ethanol 100% and xylene 100%, coverslipped with permanent mounting medium (Entellan®, Merck-Millipore, Germany) and left to dry overnight under a fume hood.

For both histological protocols, dried slides were digitalized at high resolution and the obtained images were used as guidance for the identification of brain regions within the sections.

### **BEHAVIORAL EFFECTS OF OXT INTRANASAL ADMINISTRATION**

#### **Oxytocin intranasal administration**

Adult males were gently handled during the 4 days before testing to progressively habituate to the intranasal administration protocol. The first-day they were simply handled, the second-day they were firmly kept, the third-day they were firmly kept in supine position with their back supported by the palm of the manipulator's hand, and the fourth-day a drop of saline was introduced in each nostril. After the last manipulation, they were isolated in clean cages for 24 h, treated with OXT or saline and their vocalizations and behavior were recorded.

Oxytocin (Sigma Aldrich, Italy) was dissolved in saline (0.9% NaCl) to a concentration of 0.6 mg/10 ml. A total volume of 5µl of the OXT solution was administered intranasally by gently placing drops in each nostril, that were taken in when the mice reflexively inhaled (600 ng OXT/mouse). The dosage of OXT was based on data from the literature (34, 38, 39). Control mice received an equal volume of saline (Veh). A 20-µl Eppendorf pipette with gel-loading tips was used for administration. Administration was rapid (<15 s) and handling was consistent across treatment groups.

#### **Male–female social interaction test**

Adult WT and *Oprm1*−/− males were isolated for 24 h in clean cages. A total of 17 *Oprm1*−/− (8 OXT and 9 Veh) and 18 WT (9 OXT and 9 Veh) males was used as subjects and an equivalent number of females of the two genotypes were used as partners. Each animal was used only once and administered with OXT or saline. The estrous cycle of the female partners was not considered, as irrelevant on USV quantitative performance during the first minutes of interaction in sexual naïve males facing unfamiliar females (42–44). Five minutes after the male's intranasal drug/saline administration, a female of the same genotype was introduced into the male's home cage and left for 5 min. The delay of only 5 min was chosen according to previous studies analyzing the behavioral effects of OXT after intranasal administration (34, 39). *Oprm1*−/− males were exposed to *Oprm1*−/− females and WT males to WT females. The cage was placed in front of a video-camera and under a microphone for the recording and subsequent analysis of behavior and ultrasonic vocalizations. The videos were analyzed with the Observer software (Noldus Technology, The Netherlands) and the following males' behaviors were considered for the statistical analysis: locomotion, exploration (sniffing, rearing, digging, and climbing), social investigation (following, sniffing nose, body, and ano-genital region of the partner), and self-grooming. Ultrasonic vocalizations were recorded using an UltraSoundGate Condenser Microphone (CM16, Avisoft Bioacoustics, Berlin, Germany) lowered 5 cm above the top of the cage, and analyzed by SasLab Pro (version 4.40; Avisoft Bioacoustics). Details concerning USVs recording and analysis can be found in previous papers (17, 45). According to previous literature, USVs recorded during the first minutes of male–female social interaction, are considered to be mainly uttered by the male (46).

#### **STATISTICAL ANALYSIS**

In autoradiography, we quantified the intensity of the binding signal in *Oprm1*−/− mice and compared it with the binding showed by WT mice. Average nanocurie per milligram of tissue equivalents were compared by Student's *t*-test with GraphPad Prism 5.0 (GraphPad Software, USA). Results were deemed statistically significant when *p* < 0.05. The effect of the genotype and treatment on behavioral measures was first analyzed by multiple analysis of variance (MANOVA) followed by univariate ANOVAs and,in cases of significance (*p* < 0.05), Tukey HSD *post hoc* tests.

#### **RESULTS**

#### **QUANTITATIVE ANALYSIS OF OXTR DISTRIBUTION IN WT MICE BRAIN**

To analyze the pattern of OXTR distribution in *Oprm1*−/− mice, we first analyzed [125I]-OVTA binding data in WT mice on which *Oprm1*−/− animals are backcrossed. **Figure 1** shows representative autoradiographic sections for the selected area and associated Nissl staining of adjacent slices; the reported distances from bregma of the different coronal planes are deduced from a reference mouse brain atlas (41).

The use of a commercial 125I microscales standard allowed us to quantify the levels of [125I]-OVTA binding in the brain of our mice. Quantification of OXTR expression in several areas of the brain in WT mice is reported in **Figure 2**. In WT mice, we found the highest levels of OXTR (here in the text reported as mean nanocurie per milligram tissue equivalent ± SD) in the olfactory bulb (OB, 0.91 ± 0.25), the AON (0.88 ± 0.22), and in some posterior nuclei of the amygdala, specifically the amygdalohippocampal area (AHiPM, 0.94 ± 0.16), the posteromedial cortical amygdaloid area (PMCo, 1.06 ± 0.16), and the posterior part of the basolateral amygdaloid nucleus (BLP, 0.87 ± 0.16). Medium level of OVTA binding was detected in lateral septum (LS, 0.41 ± 0.06), the bed nucleus of the stria terminalis (BNST, 0.34 ± 0.06), the CA3 field of the hippocampus (Hipp CA3, 0.30 ± 0.06), the paraventricular thalamic nucleus (PV, 0.32 ± 0.06), and in the medial amygdaloid nucleus (MeA, 0.26 ± 0.04). In the other anterior nuclei of the amygdala analyzed, the CeA nucleus (0.16 ± 0.05) and the anterior part of the basolateral amygdaloid nucleus (BLA, 0.18 ± 0.04), and in the NAcc (0.10 ± 0.04) OXTR expression was rather low. Interestingly, Dölen et al. (33) report the basolateral nucleus of the amygdala as a whole to display "average" OXTR expression, however, from observation during analysis we noticed a clear difference in OXTR expression between the anterior and the posterior parts of this nucleus, therefore, we deemed it more appropriate to analyze the two subregions separately, distinguishing them into BLA and BLP. Finally, we found minimal expression of OXTR in the CPu (0.02 ± 0.02) and the habenula (Hb, 0.04 ± 0.02) of WT mice. Overall, the expression and the distribution of OXTR in the brains of WT mice, at least in relation to the regions analyzed, is consistent with what has been previously reported (see **Table 1**).

The regions with the lowest OXTR binding signal (CPu and Hb) presented a 20-fold lower signal compared to the regions with the highest OXTR expression such as the PMCo. The NAcc also showed a very low level of OXTR expression, being it around a tenth of the PMCo's OXTR expression. All the remaining regions, such as the anterior nuclei of the amygdala, the LS, the BNST, and

#### **FIGURE 1 | Schematic representation of the brain regions of interest analyzed in the study**. Representative sections of autoradiographic labeling of OXTR with [125I]-OVTA 20 pM are displayed in grayscale, flanked by corresponding Nissl stained sections. Red contours outline the regions of interest analyzed. A sagittal schematic representation of a mouse brain is reported in the central panel. Lines indicate the antero-posterior localization of the coronal sections taken into account, defined by the reported distances (millimeter) from bregma (according to the Franklin and Paxinos (41) mouse

brain atlas). AHiPM, amygdalohippocampal area, posteromedial part; AON, anterior olfactory nucleus; AONm, anterior olfactory nucleus, medial part; BLA, basolateral amygdaloid nucleus, anterior part; BLP, basolateral amygdaloid nucleus, posterior part; BNST, bed nucleus of the stria terminalis; CeA, central amygdaloid nucleus; CPu, caudate–putamen; Hb, habenular nucleus; Hipp CA3, hippocampus, field CA3; LS, lateral septum; MeA, medial amygdaloid nucleus; NAcc, nucleus accumbens; OB, olfactory bulb; PMCo, posteromedial cortical amygdaloid area.

the PV, showed instead an average OXTR expression, comprised between one-fifth and one-half of the PMCo's levels.

#### **ABSENCE OF MOR INDUCES SPECIFIC INCREASES OF OXTR EXPRESSION IN THE AONm, IN THE ANTERIOR AMYGDALOID NUCLEI AND IN NACCS**

In order to reveal possible interaction(s) betweenMOR and OXTR, we looked for alterations in oxytocinergic binding levels in the brains of *Oprm1*−/− mice.

Our data indicate that the expression of OXTR did not differ significantly between WT and *Oprm1*−/− mice in the OB

{*t*-test; [*t*(4) = 0.139, *p* = 0.897, n.s.]}, AON {coronal planes corresponding to its central part; *t*-test; [*t*(5) = 0.577, *p* = 0.589, n.s.]}, CPu {*t*-test; [*t*(4) = 1.238, *p* = 0.284, n.s.]}, LS {*t*-test; [*t*(5) = 0.046, *p* = 0.965, n.s.]}, BNST {*t*-test; [*t*(5) = 0.315, *p* = 0.766, n.s.]}, Hipp CA3 {*t*-test; [*t*(5) = 0.160, *p* = 0.880, n.s.]}, PV {*t*-test; [*t*(5) = 1.269, *p* = 0.260, n.s.]}, and Hb {*t*-test; [*t*(5) = 0.219,*p* = 0.835, n.s.]} (**Figure 3A**). Similarly, the posterior nuclei of the amygdala (AHiPM, BLP, and PMCo), did not present alterations in OXTR expression linked to the lack of MOR {*t*-test; [*t*(5) = 1.891, *p* = 0.117, n.s.], [*t*(5) = 1.442, *p* = 0.209, n.s.], and [*t*(5) = 1.087, *p* = 0.327, n.s.], respectively} (**Figure 3B**).

On the contrary, in the anterior nuclei of the amygdala, and in particularly in the CeA and MeA, the absence of MOR led to an up-regulation of OXTR {*t*-test; [*t*(5) = 3.102, *p* < 0.05] and [*t*(5) = 4.299, *p* < 0.01], respectively, **Figure 3C**}. A trend (*p* = 0.07) toward an increase in OXTR was also found in the anterior basolateral amygdala (**Figure 3C**).

Another region in which we found an up-regulation of OXTR is the AONm in the posterior part of AON {*t*-test; [*t*(7) = 3.590, *p* < 0.01]} (**Figure 4A**). Because discriminating between the AON and NAcc is particularly tricky in this area of the brain, we performed AChE staining on the same slices processed for autoradiography. AChE selectively labels the NAcc and the olfactory tubercle but not the AON, thus allowing unambiguous identification of the two structures. **Figures 4C,D** show examples of brain sections labeled with [125I]-OVTA and AChE. In the blow-ups of the merged images it is evident that the strong autoradiographic signal observed in this area does not overlap with the AChE staining, but it is adjacent to it. This allowed us to assign the strong specific [125I]-OVTA labeling of this region to the AONm. Given

**in the anterior nuclei of the amygdala**. Average regional expression of OXTR defined by [125I]-OVTA binding in Oprm1−/− mice in comparison with WT mice. **(A)** No differences in OXTR expression were detected in Oprm1−/− mice in several brain regions reported to co-express medium-high levels of

the posterior nuclei of the amygdala whereas **(C)** an increase of OXTR is detectable in the anterior nuclei of the amygdala CeA and MeA. Data expressed as mean and SEM of three to four animals. \*p < 0.05, \*\*p < 0.01 compared to WT controls.

the difficulty, in this region, to discriminate between AONm and NAcc in absence of AChE staining, we cannot exclude that the area identified as NAcc in (34) may, instead, correspond to the AONm.

Finally, in the NAcc of *Oprm1*−/− mice, sampled at three different coronal planes, we found an OXTR level twice as high as that observed in WT animals {*t*-test; [*t*(5) = 2.767, *p* < 0.05]} (**Figure 4B**), suggesting that the NAcc is an anatomical field of compensative interaction(s) between the oxytocinergic and the opioidergic systems.

#### **BEHAVIORAL EFFECTS OF INTRANASAL ADMINISTRATION OF OXYTOCIN IN Oprm1**−**/**− **MICE**

To evaluate the pro-social effects of OXT, we tested sexually naive males during courtship, before the initiation of sexual behavior, during the first minutes of interaction with a female. The behavioral profile of males interacting with females is shown in **Figure 5**. The MANOVA indicated no significant effect of main factors (genotype: λ = 0.90, *F*(5/27) = 0.58, n.s.; treatment: λ = 0.89, *F*(5/27) = 0.61, n.s.), but a significant genotype × treatment interaction (λ = 0.62, *F*(5/27) = 3.56, *p* < 0.05), suggesting that OXT had different effect on mice

behavioral profile according to the genotype. Univariate results confirmed significant genotype × treatment effect for locomotion [*F*(1/31) = 5.01, *p* < 0.05], social investigation [*F*(1/31) = 4.22, *p* < 0.05], and USVs [*F*(1/31) = 15.29, *p* < 0.001]. *Post hoc* analysis showed a significant difference at basal conditions between *Oprm1*−/− and WT mice only in ultrasonic vocalizations (*Oprm1*−/− +Veh vs. WT +Veh: *p* < 0.01), and an increase in the number of USVs due to OXT treatment, selectively in the *Oprm1*−/− line (*Oprm1*−/− +Veh vs. *Oprm1*−/− + OXT: *p* = 0.01). By contrast, OXT did not to significantly modify WT males' vocalizations (*p* = 0.13, n.s.) (**Figure 5B**). The deficit shown by *Oprm1*−/− mice in USVs emission in response to social cues does not depend on a general impairment in their capacity to emit high levels of ultrasonic calls or in their sensitivity to olfactory stimuli, in infancy (16) as well as in adulthood (data presented here), but suggests lower motivation to interact with conspecifics.

The low impact of OXT on mutant males' general social behavior (**Figure 5A**) can be related to the role of their mutant (untreated) females that may prefer to avoid their partner. In this respect, the USVs test will result more susceptible to treatment, as

it only relies on the male behavior, independently on the female receptivity. Adult male mice emit USVs in the presence of female conspecifics and these vocalizations, promoting proximity, facilitate close social interactions, and sexual behavior. The advantage of testing males in this context stands in the consistent amount of ultrasonic calls that characterizes their first approach to the partner, which is independent from female's receptivity and closely associated to the male's motivational state (42–44). As a matter of fact, males emit USVs also when exposed to female's cage bedding or urine (47). When in close proximity, mice can acquire information on the partner's individual characteristics (e.g. sex, rank, sexual experience, and estrous condition) modulating their social behavior, andfacilitating or avoiding social interactions.Moreover, the lack of strong deficits during sexual encounters in this mutant line confirms the entirety of their reproductive capabilities. Finally, the absence of significant effects of the intranasal OXT treatment in control male mice is in line with the Huang et al. study, showing only little changes in social investigation toward a female partner in C57BL/6J mice (34).

#### **DISCUSSION**

The aim of this study was to investigate, in *Oprm1*−/− mice, the altered molecular mechanisms underlying the well documented deficits in social behavior presented by this animal model (16–19). In particular, we focused on alterations in the expression of brain OXTRs because they are the target of intranasally administered OXT, a promising new treatment for social deficits in autism (35, 48, 49). Our data show an OXTR up-regulation in specific brain regions of MOR-null mice, such as AONm, CeA, MeA, and NAcc. All these regions are involved in the regulation of social behavior, but, in our study, not all regions involved in social behavior that express OXTR were found to up-regulate the receptor. Furthermore, no correspondence between a high expression level of the OXTR and its up-regulation was observed. In particular: (1) regions that display very high levels of OXTR such as LS and BNST did not show any OXTR up-regulation and (2) of the regions where we observed OXTR up-regulation, three (AON, MeA, and

CeA) have a high level of OXTR expression, and one (NAcc) has a medium-low level. Interestingly, a region-selective OXTR upregulation was also observed in a recent paper in which OXTR expression levels were analyzed in the mouse brain after morphine treatment and withdrawal (50). However, the regions found in the aforementioned pharmacological manipulation study did not overlap with those described in the present study, an observation consistent with multiple patterns of receptor modulation linked to specific genetic, developmental, and pharmacological conditions. AONm, MeA, CeA, and NAcc are, nevertheless, all characterized by a medium-high MOR expression in WT mice (**Table 1**), suggesting a local interplay between the two receptor systems, which may involve neuroanatomical, cellular, or even subcellular interactions.

An open question regards, which is the neurobiological mechanism at the basis of the OXTR up-regulation. It is difficult to answer to this question as the OXT content and capability of OXT release in the hypothalamus of *Oprm1*−/− mice has not been evaluated so far. If reduced, an up-regulation of the OXTR in target sites of the neuropeptide's action may represent a compensatory mechanism to normalize the neuronal responses to reduced levels of secreted OXT. Becker et al. (19) actually found a reduced level of OXT transcript in *Oprm1*−/− mice, but this impairment was restricted to the NAcc, leaving the overall levels of OXT undetermined. A second possibility implies direct molecular interactions between OXTR and MOR. G-protein coupled receptors such as OXTR and MOR, can form homo- (with another molecule of the same receptor) or hetero- (with receptors from other families) dimers and it has been shown that this physical association can modulate receptor binding and function (51). Interestingly, in mice lacking the alpha2A-adrenergic receptor, morphine has a reduced analgesic effect, suggesting that alterations in the noradrenergic system induce alterations in the opioid system (52). Molecular dimerization of MOR and alpha2A-adrenergic receptor has been demonstrated in both cell lines and primary neuronal cultures (53) and it has been shown that, in MOR–alpha2Aadrenergic receptor dimers, the activation of MOR by morphine

inhibits the adjacent alpha2A-receptor by blocking its ability to activate the G-proteins even in the presence of noradrenaline (54). If dimeric interactions occur between MOR and OXTR, the lack of MOR may result in specific alterations of OXTR pharmacology in the brain regions in which the two receptors physically interact. Even if only speculative at present, this hypothesis will deserve further investigation.

A second question is: what are the neuroanatomical basis of the specific OXTR up-regulation in NAcc, CeA, MeA, and AONm of MOR-null mice?

As the NAcc is involved in the processing of social motivation and reward, it is not surprising that alterations in OXTR levels in NAcc have been found to be linked to altered social behaviors. First of all, natural variability in OXTR expression in the NAcc has been associated to social organization and mating behavior in voles, being the level of OXTR in the NAcc higher in monogamous voles displaying greater maternal care (55). Many other studies associated OXTR density in the NAcc to the degree of maternal and affiliative behavior and partner preference formation (56– 60). Regardless of whether differences in OXTR expression are intrinsic (due to individual or species-related variability) (55, 57, 58), exogenously induced by viral-mediated over expression (35, 59, 60), or physiologically caused by labor and lactation (56), an higher OXTR level in these brain regions correlates with more pronounced social behavior. On the contrary, reduced levels of OXTR binding in the NAcc were observed in voles after paternal deprivation (61). At the neurophysiological level, presynaptic OXTRs have been recently shown to be required for social reward in the NAcc (33). In particular, OXTRs located on axonal terminals of serotoninergic projections coming from the dorsal raphe have been found to elicit Long-Term Depression (LTD) in medium spiny neurons by activation of serotoninergic 5HT1B receptors (33). NAcc neurons express high levels or MOR, but the neuronal populations that mediate all the distinct opiate effects remain elusive; recently, a subpopulation of striatal direct-pathway neurons was shown to support opiate reward-driven behaviors (62), and specific "hotspots" involved in the modulation of hedonic "liking" responses in which opioid receptors might function as hedonic enhancers have been identified (63). Furthermore, activation of MORs within the dorsal striatum appears to be critical to partner preference formation by generating socially motivated behavioral responses (9). OXTR may thus be postulated to act on specific MOR positive NAcc subpopulation(s) to regulate specific aspects or components of social reward, possibly via a presynaptic mechanism similar to that observed for the OXT induced LTP at 5HT1B receptors.

The different nuclei of the amygdala are responsible for the processing of emotional content of both positive and negative experiences. The CeA, as part of the brain's reward circuitry, is important in processing positive emotions and, particularly, in the learning process of stimulus–reward association (64, 65)*.* Moreover, the CeA is strongly implicated in the expression of fear. In mice, a neural circuit within the CeA that gates fear responses activating active strategies of risk assessment or passive expression of fear (freezing) has been described and, in this circuit, the OXTR was found to be a key player (66). Interestingly, as it has been observed that excessively aggressive animals

show highest OXTR binding in the CeA (67), it is tempting to speculate that the increased aggressive behavior observed in *Oprm1*−/− mice (19) may be linked to the selective up-regulation of OXTR in the CeA. The MeA is highly activated during social encounters and recognition, thus representing a critical neuroanatomical substrate of social cognition. The recognition of conspecifics is an initial and crucial condition for the establishment of social and sexual behavior. Odor, scent, and pheromones mediate sexual and competitive interactions, and are important in individual and kin recognition and mate selection. In rodents, odor signals are processed by two systems: the main olfactory pathways and the vomeronasal pathway, both directly and indirectly projecting to the MeA, a key region for the processing of chemosensory information (68). OXT-deficient mice are not able to recognize a previously encountered, familiar conspecific, and this social recognition behavior can be restored with a direct microinjection of OXT into the MeA of OXT KO mice, indicating that OXT in the MeA is both necessary and sufficient for social recognition (69, 70). Furthermore, male social interest correlated positively with OXTR in the MeA, while female social interest correlated negatively with OXTR expression in the same region (71). Finally, it has recently been shown that OXT-mediated LTD in the AOB–MeA pathway is directly involved in long-term social recognition memory formation (72). The up-regulation of OXTR in MeA may thus be involved in the pharmacological rescue of intranasal OXT on the social interaction test used in our study.

The up-regulation of OXTR in AONm of *Oprm1*−/− remains of difficult interpretation. The *pars medialis* of the AON can be distinguished from the rest of the AON by cytoarchitectural features and projection patterns (73, 74), suggesting a different role than a station along the main olfactory and the vomeronasal pathways, but the role of OXTR in this region is still undefined.

Finally, what is the pathophysiological meaning of the OXTR up-regulation in *Oprm1*−/− mice? As *Oprm1*−/− mice do have severe defects in social interaction and social memory (19), the upregulation of OXTR binding sites observed in *Oprm1*−/− mice is apparently unable to normalize social cognition in these animals. Nevertheless, the up-regulation of OXTR may contribute to the rescue effects observed with intranasal OXT in MOR-null animals, as these KO animals completely recovered from communication deficits during the first minutes of social interaction, not differing from wild type animals anymore. Indeed, the present study confirms the efficacy of OXT treatment in reverting social deficits in a mouse model in which the oxytocinergic system underwent a compensatory modulation. These findings are of high relevance in the autism field as they further validate the use of OXT in ASD regardless of the primary etiopathogenetic cause(s) but solely on the basis of the social endophenotype. To fully understand the role of OXTR compensatory roles, it will be very interesting to investigate in detail the time-course of OXTR up-regulation in crucial periods of prenatal and post-natal life, including birth, weaning, and sexual maturity. Similarly, we only investigated male animals, but, due to the sexual dimorphism of OXTR expression in different species and brain areas (75, 76), similar analysis in female animals is warranted, as this may have a profound impact on the design of therapeutic schemes and doses.

In conclusion, the OXT system may act as a downstream effector toward social behaviors, compensating and/or bypassing genetic alterations in upstream circuits that produce social deficits. Such a hypothesis would justify the use of this neuropeptide in a wide variety of human conditions characterized by defects in social cognition and/or reward, such as ASDs, upon demonstration of OXT/OXTR modification(s) in the brain of affected individuals. Moreover, the results presented here further corroborate the hypothesis that alterations in the OXTergic system are involved in the pathophysiology of autism and that administration of OXT to such altered system might restore a balance in the neural circuitries involved in social behavior.

#### **AUTHOR CONTRIBUTORS**

Valentina Gigliucci: performed experiments (autoradiography, Nissl staining, and AChE) and data analysis; wrote the manuscript. Marianna Leonzino: performed data analysis and wrote the manuscript. Marta Busnelli: performed experiments (autoradiography and AChE staining) and data analysis. Alessandra Luchetti: performed experiments (pharmacological treatment and behavioral tests). Viola Stella Palladino: performed experiments (animal genotyping and behavioral tests). Francesca R. D'Amato: conceived and supervised behavior experiments and wrote the manuscript. Bice Chini: conceived and supervised the study and wrote the manuscript.

#### **ACKNOWLEDGMENTS**

Thanks are given to Dr. Diego Oddi for projecting and discussing preliminary data. This study was partly supported by Regione Lazio FILAS for "Sviluppo della Ricerca sul Cervello."

#### **REFERENCES**

mu-opioid receptor gene (OPRM1) in adult healthy volunteers and psychiatric patients. *Soc Neurosci* (2011) **6**(1):88–97. doi:10.1080/17470919.2010.482786

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 31 May 2014; accepted: 13 August 2014; published online: 01 September 2014. Citation: Gigliucci V, Leonzino M, Busnelli M, Luchetti A, Palladino VS, D'Amato FR and Chini B (2014) Region specific up-regulation of oxytocin receptors in the opioid Oprm1*−*/*− *mouse model of autism. Front. Pediatr. 2:91. doi: 10.3389/fped.2014.00091 This article was submitted to Child and Neurodevelopmental Psychiatry, a section of the journal Frontiers in Pediatrics.*

*Copyright © 2014 Gigliucci, Leonzino, Busnelli, Luchetti, Palladino, D'Amato and Chini. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# PEDIATRICS

### Mapping pathological phenotypes in reelin mutant mice

#### **Caterina Michetti 1,2† , Emilia Romano3,4† , Luisa Altabella5 , Angela Caruso1,6, Paolo Castelluccio1 , Gaurav Bedse2 , Silvana Gaetani 2 , Rossella Canese5\*, Giovanni Laviola3§ and Maria Luisa Scattoni 1\* §**

1 Neurotoxicology and Neuroendocrinology Section, Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Rome, Italy

#### **Edited by:**

Yuri Bozzi, University of Trento, Italy

**Reviewed by:**

Elizabeth M. Powell, University of Maryland, USA Emanuel DiCicco-Bloom, University of Medicine and Dentistry of New Jersey, USA

#### **\*Correspondence:**

Rossella Canese, Molecular and Cellular Imaging Section, Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena 299, I-00161 Rome, Italy e-mail<EMAIL_ADDRESS>Maria Luisa Scattoni, Neurotoxicology and Neuroendocrinology Section, Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena, 299, I-00161 Rome, Italy

e-mail<EMAIL_ADDRESS>† These authors contributed equally to

this work. \$ These authors also contributed

equally to this work.

Autism Spectrum Disorders (ASD) are neurodevelopmental disorders with multifactorial origin characterized by social communication deficits and the presence of repetitive behaviors/interests. Several studies showed an association between the reelin gene mutation and increased risk of ASD and a reduced reelin expression in some brain regions of ASD subjects, suggesting a role for reelin deficiency in ASD etiology. Reelin is a large extracellular matrix glycoprotein playing important roles during development of the central nervous system. To deeply investigate the role of reelin dysfunction as vulnerability factor in ASD, we assessed the behavioral, neurochemical, and brain morphological features of reeler male mice. We recently reported a genotype-dependent deviation in the ultrasonic vocal repertoire and a general delay in motor development of reeler pups. We now report that adult male heterozygous (Het) reeler mice did not show social behavior and communication deficits during male–female social interactions. Wildtype and Het mice showed a typical light/dark locomotor activity profile, with a peak during the central interval of the dark phase. However, when faced with a mild stressful stimulus (a saline injection) only Het mice showed an over response to stress. In addition to the behavioral studies, we conducted high performance liquid chromatography and magnetic resonance imaging and spectroscopy to investigate whether reelin mutation influences brain monoamine and metabolites levels in regions involved in ASD. Low levels of dopamine in cortex and high levels of glutamate and taurine in hippocampus were detected in Het mice, in line with clinical data collected on ASD children. Altogether, our data detected subtle but relevant neurochemical abnormalities in reeler mice supporting this mutant line, particularly male subjects, as a valid experimental model to estimate the contribution played by reelin deficiency in the global ASD neurobehavioral phenotype.

**Keywords: autism spectrum disorders, reeler mice, ultrasonic vocalizations, social interaction, stress response, dopamine, glutamate, circadian cycle**

#### **INTRODUCTION**

Autism Spectrum Disorders (ASD) are neurodevelopmental disorders with multifactorial origin characterized by persistent deficits in social communication and interaction and restricted and repetitive patterns of behavior, interests, or activities (1). Several studies showed that abnormal reelin expression in the brain is involved in a number of neuropsychiatric disorders including lissencephaly, schizophrenia, and autism (2–8).

Clinical studies have shown reduced levels of reelin protein in blood serum and in post-mortem brain of ASD patients (9–12). Genetic variants in RELN have been investigated as risk factors of ASD in numerous epidemiologic studies but with inconclusive results (13–19). However, recent data collected on much larger samples and with more advanced genetic approaches indicated a relationship between *reelin* gene mutation and increase risk of autism, suggesting that *reelin* deficiency may be a vulnerability factor in the etiology of this neurodevelopmental disorder (20–27).

Animal models in which reelin expression is reduced or absent, provide important information about the role of *reelin* deficiency in the onset of neurodevelopmental disorders such as ASD. Homozygous reeler mice show decreased brain volume, increased ventricles volume, (28–30), a non-foliated cerebellum (30), reduced number of Purkinje cells (31), deficits in lamination of the hippocampus (Hip), and disorganization of the amygdala (30). Some of these abnormalities are comparable with the ones found in post-mortem studies on autistic brain such as: increased ventricle volume, altered cortical lamination, heterotopias, dysplastic changes, and reduced number of Purkinje cells (32–39). These morphological changes in homozygous reeler mice are also associated with serious physical impairments and for this reason these mice are not considered as a reliable animal model for basic behavioral research but their use has been so far limited to the study of neuronal migration and of etiology of human lissencephaly (4, 5).

2 Department of Physiology and Pharmacology "V. Erspamer", Sapienza University of Rome, Rome, Italy

3 Behavioural Neuroscience Section, Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Rome, Italy

4 Bambino Gesù Children's Hospital, Istituto Di Ricovero e Cura a Carattere Scientifico, Rome, Italy

5 Molecular and Cellular Imaging Section, Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Rome, Italy

6 Department of Psychology, School of Behavioural Neuroscience, Sapienza University of Rome, Rome, Italy

Heterozygous reeler mice, which exhibit the 50% reduction in reelin expression, do not display a reeler phenotype but express a number of abnormal traits including loss of Purkinje cells of the cerebellum (40, 41) and decrease in the number of dendritic spines in cortical and hippocampal neurons (42). Reduced levels of reelin are also associated with an increased anxiety profile (43, 44), cognitive deficits in the operant conditioning (44, 45), executive functions (46), fear conditioning learning (47, 48), olfactory conditioning learning (49), latent inhibition (50), and attentional set-shifting (51).

Surprisingly, only limited studies have investigated the contribution of *reelin* deficiency to the establishment of the social/communicative deficits, first ASD core symptom as indicated in the DSM 5 (1). Adult social responses in heterozygous (Het) reeler mice have been tested so far in two studies assessing either direct male–male and female–female social interactions (52) or performance in a modified version of the three-chamber sociability test (51). In both studies, only social behavioral performances have been assessed but a detailed evaluation of the ultrasonic vocalizations (USVs) emitted during the interaction was missing. To this aim, we deeply investigated the social and vocal repertoire of wildtype (Wt) and Het reeler mice during courtship (53), to evaluate the presence of qualitative alterations in social interaction and communication in this mutant line. In addition, we evaluated the baseline circadian locomotor activity in the home-cage as well as the response to a mild stressful stimulus represented by a saline injection (54–57) to check for abnormalities in the spontaneous locomotor activity that could affect the behavioral performances.

#### **MATERIALS AND METHODS**

#### **ANIMALS AND HOUSING**

Breeding pairs were originally purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and bred in our laboratory at ISS. About 2 weeks after pairing for breeding (15 Het × Het crosses), the females were individually housed and subsequently inspected daily for pregnancy and delivery. After weaning on postnatal day (pnd) 25, mice were housed by sex in mixed genotype groups (B6C3Fe Wt and Het) of two to three per cage. All mice were housed in a colony room with temperature maintained at 21 ± 1°C and humidity at 60 ± 10% with food (Enriched standard diet purchased from Mucedola, Settimo Milanese, Italy) and water available *ad libitum*. The colony room was maintained on a 12:12 light/dark cycle with lights on at 18.30 h. Mice genotype was determined at pnd 21 by polymerase chain reaction (PCR) analysis on tale samples and the animals were marked by an ear punching for identification. Consistent with the higher prevalence of autism in human males, only male mice were tested. Homozygous reeler mice were not tested due to their serious physical impairments after weaning. The same cohort of adult male mice was tested for male–female reciprocal social interaction with concomitant USVs (3 months of age), locomotor activity in the home-cage

(6 months), and HPLC (7 months). A separate cohort of mice was subjected to *in vivo* quantitative MRI and spectroscopy at 4 months of age. All procedures were conducted in strict compliance with the European Communities guidelines (EC Council Directive 86/609), Italian legislation on animal experimentation (Decreto L.Vo 116/92).

#### **ADULT MALE–FEMALE SOCIAL INTERACTIONS**

Three-month-old male mice (*N* = 9 Wt, *N* = 21 Het) were evaluated in the male–female social interaction test as in Ref. (53). Each male subject was isolated 1 h before testing and the vaginal estrous condition of each stimulus female was assessed as in Ref. (58). Only females in estrous were selected for the test. The unfamiliar stimulus C57BL/6J female mouse was placed into the home-cage of the isolated male mouse and behaviors and USVs were recorded for a 3-min test session. Stimulus mice (C57BL/6J females) were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and maintained in our colony room in social groups of three per home-cage. Each female was used only twice and were matched to the subject mice by age and body weight.

Social interaction test was conducted between 09.00 and 13.00 h, during the dark phase, under red light. In addition to the isolated mouse, the cage contained litter (1.5-cm deep) and the lid was removed during the test. For video recordings, the videocamera (Panasonic monochrome charge-coupled device camera) was mounted facing the side of the cage and the subsequent scoring of social investigation parameters was conducted with Noldus Observer 10XT software (Noldus Information Technology, Leesburg, VA, USA).

Social interactions were scored from the videotapes for the frequencies and durations of the following behavioral responses performed by the subject mouse: *anogenital sniffing* (direct contact with the anogenital area), *body sniffing* (sniffing or snout contact with the flank area), *head sniffing* (sniffing or snout contact with the head/neck/mouth area), *locomotor activity*, *rearing* up against the wall of the home-cage, *digging* in the bedding, and *grooming* (self-cleaning, licking any part of its own body). No observations of *mounting*, *fighting*, *tail rattling*, and *wrestling* behaviors were observed. Scoring was conducted by two investigators uninformed of the genotype. Inter-rater reliability was 98%.

For audio recordings, the ultrasonic microphone (Avisoft UltraSoundGate condenser microphone capsule CM16, Avisoft Bioacoustics, Berlin, Germany) was mounted 20 cm above the cage and the USVs recorded using Avisoft RECORDER software version 3.2. Settings included sampling rate at 250 kHz; format 16 bit. The ultrasonic microphone was sensitive to frequencies between 10 and 180 kHz. For acoustical analysis, recordings were transferred to Avisoft SASLabPro (version 4.40) and a fast Fourier transformation (FFT) was conducted as previously described (59). Start times for the video and audio files were synchronized. Parameters analyzed included number and mean duration of calls, qualitative and quantitative analyses of sound frequencies measured in terms of frequency, and amplitude at the maximum of the spectrum. Waveform patterns of calls [a total of 17195 (Wt) and 8454 (Het) calls] were examined in depth in the sonograms collected from every mouse tested. Each call was identified as one of eight distinct categories, based on internal pitch changes, lengths, and shapes, as in our previously published studies (53, 59, 60).

Inter-rater reliability in scoring the call categories was 98%. Scoring was conducted by two investigators blind to the mouse genotype. Call category data were subjected to two different analyses: (1) Genotype-dependent effects on the probability of producing calls (proportion of calls in each category for each subject) from each of the eight categories of USV, as described below under statistical analysis; (2) a descriptive analysis that included genotype-dependent effects on the percentage of calls emitted by each subject in each of the eight categories of USV.

#### **LOCOMOTOR ACTIVITY IN THE HOME-CAGE**

At 6 months of age, male mice (*N* = 9 Wt, *N* = 10 Het) were individually housed in standard cages (33 cm × 13 cm × 14 cm) and assigned to a continuous monitoring of spontaneous locomotor activity. The assessment of daily spontaneous activity in the home-cage was carried out by means of an automatic device using small passive infrared sensors positioned on the top of each cage (Activiscope system, see the website: www.newbehavior.com) (61–63). The system operated continuously for 13 days and after 2 days of acclimation the experimental procedure began. The sensors (20 Hz) detected any movement of mice. Data were recorded by an IBM computer with dedicated software. No movements were detected by the sensors when mice were sleeping, inactive, or performed moderate self-grooming. Scores were obtained during 30-min intervals and expressed as counts per minute (cpm). The 24-h profile of activity was obtained by averaging 7 days of continuous registration. The position of Wt and Het mouse cages in the rack was equally distributed in rows and columns. Animals were provided with tap water and food pellets *ad libitum*. After the first 7 days of spontaneous activity, all animals were subjected to an injection of saline (a mild stressful stimulus), at 11 h (dark phase), and locomotor activity monitored up to 3 days later. The analysis of the locomotor profile over a period of 7 h (11–18) after saline injection was performed to evaluate the immediate stress response.

#### **MONOAMINES AND THEIR METABOLITES: HPLC DETERMINATION**

Subsequently to behavioral studies, male mice (*N* = 9 Wt, *N* = 10 Het) were sacrificed, their brains removed and rapidly dissected on ice to obtain the olfactory bulb, frontal cortex, striatum, Hip, and cerebellum for HPLC analysis. All samples were immediately flash frozen on dry ice, and then stored at −80°C until further processing. HPLC was performed according to Ref. (64). In particular, each brain region was weighed, ultrasonicated in 0.1 M perchloric acid, centrifuged for 20 min at 15,000 g (4°C) and the supernatant was used for monoamine neurotransmitters and their metabolites detection. The endogenous levels of 5-HT and 5-HT metabolite (5-hydroxyindolacetic acid; 5- HIAA), dopamine (DA) and final DA metabolite (homovanillic acid; HVA), and norepinephrine (NA) and NA metabolite (4-hydroxy-3-methoxyphenyl-glycol, MOPEG) were assayed by HPLC using a SphereClone 150 mm × 2 mm column (3-µm packing). Detection was accomplished with a Unijet cell (BAS) with a 6-mm-diameter glassy carbon electrode at +650 mV versus an Ag/AgCl reference electrode, connected to an electrochemical

amperometric detector (INTRO, Antec Leyden, The Netherlands). For each analysis, a set of standards containing various concentrations of each compound (monoamines and their metabolites) was prepared in the perchloric acid solution, and calibration curves were calculated by a linear regression. The retention time of calibration standards was used to identify peaks, and areas under each peak were used to quantify monoamine levels. Results were normalized to the weight of wet tissue.

#### **MAGNETIC RESONANCE IMAGING AND SPECTROSCOPY**

At 4 months of age, a separate cohort of male mice (*N* = 7 Wt, *N* = 7 Het), was subjected to *in vivo* MRI and magnetic resonance spectroscopy (MRS). During the MR analyses, animals were anesthetized with 2.5–2.0% isoflurane in oxygen 1 l/min (Isoflo, Abbott SpA, Latina, Italy). An integrated heating system allowed maintaining the animal body temperature at 37.0 ± 0.1°C. All MRI and MRS experiments were conducted on a 4.7 T Varian/Agilent Inova animal system (Agilent Inc., Palo Alto, CA, USA), equipped with actively shielded gradient system (max 200 mT/m, 12 cm bore size). A 6-cm diameter volume coil was used for transmission in combination with an electronically decoupled receive-only surface coil (Rapid Biomedical, Rimpar, Germany). Spin-echo sagittal anatomical images (TR/TE = 3000/60 ms, 13 consecutive slices of 0.8 mm thickness, FOV = 20 mm × 20 mm, matrix of 128 × 128, 2 averages) were acquired for accurate positioning the voxel for the MRS study. Single voxel localized 1H MR spectra (PRESS, TR/TE = 4000/23 ms, ns = 256 or 512) were collected from relevant brain areas: Hip (11.7µl), striatum (STR, 10.4µl), thalamus (Th, 12.96µl), and cerebellum (Cb, 7.45µl), as shown in **Figure 1A** and defined in the mouse brain atlas (65). Quantitative MRS protocol, including water T2 measurements, was applied (66) and T2 measurements were performed on water signal in order to identify any change in the mutant mice. Unsuppressed water signal was used for metabolite quantification (assuming 79.9% for gray matter water content). Spectra were analyzed using LCModel (67). Only those metabolites that were estimated to have Cramer–Rao lower bounds (CRLB) <20%, which corresponded to an estimated concentration error <0.2µmol/g, were included into the quantitative analysis. In some cases, metabolites that have resonance overlapped or very close are also given as their sum. An example of spectra and its LCModel analysis is shown in **Figure 1B**.

Multislice fast spin-echo axial images (TR/TEeff = 3200/60 ms, ns = 4, slice thickness 0.6 mm, 24 slices, matrix 256 × 256, FOV = 25 mm × 25 mm, which correspond to voxel resolution of 98 × 98 × 600µm3 ) were also acquired for volumetric analyses.

Motor cortex thickness was measured at +1.32 from bregma as shown in **Figure 1C**. Volumetric analyses of the whole brain have been performed from olfactory bulb to cerebellum excluded. Ventricles and cerebellum volumes were also measured. Brains were manually segmented for forebrain, ventricles, and cerebellum using Varian/Agilent Imaging Browser, which perform a 3Dvolume calculation by summing the pixels areas on the center of each slices and interpolating the cross sectional areas between the center of the other slices (Agilent Inc., Palo Alto, USA) on MR images. Manual segmentation of the ventricles was facilitated by

the high contrast that cerebrospinal fluid has in the MR images. **Figure 1D** shows an example of segmentation (slice central to the brain in sagittal images).

#### **STATISTICAL ANALYSIS**

A mixed-model ANOVA with repeated measures was used to analyze: (1) sniffing of different body areas (anogenital, body, or head), (2) spontaneous locomotor activity in the home-cage, (3) number of USVs for each minute of interaction, and (4) probability of vocalizations in eight call categories with genotype as between-subject factor. Probability of vocalizations within each genotype was calculated as number of calls in each category for each subject/total number of calls analyzed in each subject and standardized by angular transformation.

Data relative to MRI and MRS were analyzed by a one way ANOVA with genotype as the independent factor and MRI/MRS parameters (values of water T2, metabolite levels in each brain region and volume of each brain region) as dependent factor. Differences between genotypes in each brain region with respect to serotoninergic, dopaminergic, and noradrenergic systems (5- HT, 5-HIAA, and 5-HT turnover for serotoninergic system; DA, HVA,DOPAC, and DA turnover for dopaminergic system; and NA, MOPEG, and NA turnover for noradrenergic system) were determined by a multivariate analysis of variance (MANOVA), due to the potentially high correlation between these dependent variables within each system. Pillai's statistic was used. Univariate ANOVAs were conducted for each variable (Statview II, Abacus Concepts, CA, USA).

For all comparisons, data are expressed as mean ± SEM and significance was set at *P* < 0.05. *Post hoc* comparisons were performed using Tukey's test only when a significant *F*-value was determined.

#### **RESULTS**

#### **MALE–FEMALE SOCIAL INTERACTION TEST**

To assess the presence or absence of a social communication deficit in Het reeler mice, we evaluated the behaviors and the USVs emitted by a male mouse during the interaction with an estrus C57BL/6J female. Analysis of the social sniffing response on different body areas (head, body, and anogenital) did not reveal significant effects of genotype [frequency, *F*(2,56) = 0.15, *P* = 0.858, (data not shown); duration,*F*(2,56) = 1.74; *P* = 0.183,**Figure 2A**]. No genotype effect was detected on explorative behaviors such as *rearing* [frequency, *F*(1,28) = 0.27, *P* = 0.610 and duration, *F*(1,28) = 0.30,*P* = 0.589] and *digging* [frequency,*F*(1,28) = 0.75, *P* = 0.392 and duration, *F*(1,28) = 1,15, *P* = 0.292] (data not shown).

Analysis of the USVs emitted by male mice during the social interaction test did not detect significant differences between Het reeler and Wt mice: number of USVs [number of calls × genotype, *F*(2,56) = 0.89, *P* = 0.41, **Figure 2B**], mean duration [*F*(2,54) = 0.79, *P* = 0.457 (data not shown)], peak frequency [*F*(2,54) = 0.43, *P* = 0.650 (data not shown)], and peak amplitude [*F*(2,54) = 0.14, *P* = 0.863 (data not shown)]. As a whole, the pattern of sonographic structures did not differ between Het reeler and Wt mice indicating a comparable vocal repertoire in both genotypes (see pie graphs in **Figure 2C**).

#### **LOCOMOTOR ACTIVITY IN THE HOME-CAGE**

Sleep problems and irregular sleep–wake cycles have been identified in several ASD children (68–71). Alterations in circadian rhythm lead to anxiety-like, impulsive, and depressive behaviors both in humans and mice (72–74). In the present study, we evaluated baseline circadian locomotor activity in the home-cage as well as response to a mild stressful stimulus represented by a saline injection to check for abnormalities in the spontaneous locomotor activity that could affect the behavioral performances.

Analysis of spontaneous locomotor activity measured in the home-cage for 7 days revealed, as expected, an increased activity in mice of both genotypes during the dark phase of the light/dark cycle [phase effect, *F*(1,17) = 239.05, *P* < 0.001] (see **Figure 3**). No genotype differences were found [light phase: genotype, *F*(1,17) = 2.99, *P* = 0.102; dark phase: genotype, *F*(1,17) = 1.34, *P* = 0.263].

The analysis of the locomotor profile over a period of 7 h after saline injection (11–18, dark phase) was performed to evaluate the immediate stress response. Het reeler mice increased significantly their locomotor activity as compared to Wt mice [genotype, *F*(1,17) = 5.63, *P* = 0.029] thus revealing a genotypedependent increased sensitivity to mild stress challenge (see graph on the top right of **Figure 3**). After this 7-h-period, locomotor activity goes back to the normal activity profile [genotype effect, *F*(1,17) = 0.226, *P* = 0,64; genotype × hours, *F*(1,23) = 0.38, *P* = 0.99; data not shown].

**FIGURE 2 | Male–female social interaction test (3-min-session) performed in 3-month-old reeler mutant mice**. Parameters measured during a direct interaction between a male with a sexually receptive C57BL/6J female. **(A)** Sniffing duration. **(B)** Number of ultrasonic vocalizations. Data are expressed as mean + SEM. **(C)** Pie graphs show the percentages of the different call categories emitted by Wt and Het reeler mice. Percentages were calculated in each genotype as number of calls in each category for each subject/total number of calls analyzed for each subject. Number of calls analyzed: 17195 in Wt and 8454 in Het. N = 9 Wt and N = 21 Het.

N = 10 Het.

# for 1 week. The graph on the top right shows the spontaneous locomotor

**MONOAMINES AND THEIR METABOLITES: HPLC DETERMINATION** High performance liquid chromatography determination has been applied to investigate whether *reelin* mutation influences the different monoamine systems. MANOVA revealed a significant genotype effect on several components of the dopaminergic system in hypothalamus [Pillai's Trace: *F*(4,13) = 9.73, *P* < 0.001] and in Hip [Pillai's Trace: *F*(4,13) = 4.97, *P* = 0.005]. ANOVA showed a strong reduction of DA levels [genotype effect, *F*(1,16) = 5.29, *P* = 0.035] and a consequent increase in the DA turnover [genotype effect, *F*(1,16) = 7.43, *P* = 0.015] in the cortex of Het reeler as compared to Wt mice (see **Table 1**). Moreover, Het reeler mice showed an increase of DOPAC and HVA levels [genotype effect: DOPAC, *F*(1,16) = 18.88, *P* < 0.001; HVA, *F*(1,16) = 11.47, *P* = 0.003] in hypothalamus. ANOVA evidenced also a decrease of HVA levels [*F*(1,16) = 6.43, *P* = 0.022] and an increase of DA turnover [genotype effect, *F*(1,16) = 6.14, *P* = 0.025] in Hip of Het mice.

Heterozygous mice showed an higher DA levels [genotype effect, *F*(1,16) = 4.38, *P* = 0.052] and a lower DA turnover in olfactory bulb [genotype effect, *F*(1,16) = 6.87, *P* = 0.018] than Wt mice. No genotype related differences were found on noradrenergic and serotoninergic systems in each brain areas analyzed.

#### **MAGNETIC RESONANCE IMAGING**

To acquire deeper information into the functional state of brain areas involved in ASD, we assessed a 1H magnetic resonance in adult reeler male mice. Enlarged ventricles and reduced cerebellum are typical features of reeler mice (75). Volumetric analyses confirmed a cerebellum reduction [genotype effect, *F*(1,10) = 15.50, *P* = 0.002] and an enlargement of ventricles volume [genotype effect, *F*(1,11) = 8.01, *P* = 0.016] in Het reeler when compared to

Wt mice. No genotype differences were detected in volume [genotype effect, *F*(1,11) = 0.86, *P* = 0.374] and medial motor cortex (MC) thickness [genotype effect, *F*(1,11) = 3.62, *P* = 0.083] (see **Table 2**).

#### **MAGNETIC RESONANCE SPECTROSCOPY**

To investigate the possible alterations in brain metabolism of adult reeler male mice, we performed MRS. The high quality spectra allowed reliable quantification (%SD <20%) not only for the commonly observed *N*-acetyl-aspartate (NAA), total creatine (Cr + PCr) and total choline resonances (NAA + NAAG), but also for glutamine (Gln), glutamate (Glu), taurine (Tau), and inositol (Ins) in all the investigated brain regions.

Water T2 analyses confirmed that no changes between the genotypes occurred in the T2s in Hip [*F*(1,10) = 2.52,*P* = 0.143], Striatum [*F*(1,10) = 0.02, *P* = 0.883], Thalamus [*F*(1,10) = 1.15E-5, *P* = 0.977], and Cerebellum [*F*(1,10) = 0.32, *P* = 0.584] (data not shown).

Metabolic changes were detected in Hip while no differences have been found for any metabolite in thalamus, striatum, and cerebellum. Het reeler mice showed increased levels of Glu [genotype effect, *F*(1,11) = 4.61, *P* = 0.044], Tau [genotype effect, *F*(1,11) = 4.82, *P* = 0.050], PCr [genotype effect, *F*(1,7) = 18.08, *P* = 0.003], and total amount of PCr + Cr [genotype effect, *F*(1,11) = 24.68, *P* < 0.001] in Hip as compared to Wt mice (see **Figure 1E**).

#### **DISCUSSION**

Reelin is a glycoprotein playing a crucial role during development: it regulates neuronal migration and brain lamination (6, 8, 29, 30, 76, 77) and its reduced or complete lack of signaling impairs

Data are expressed as mean ± SEM. \*P < 0.05, between wildtype and heterozygous reeler mice.

neuronal connectivity and synaptic plasticity (43, 78). Moreover, recent data suggest that a defect in reelin signaling pathway confers greater susceptibility to autism (20–25, 27).

For these reasons,we consider Het reeler mice, haploinsufficient for*reelin* and sharing some neurochemical and behavioral features with autistic patients, a suitable animal model for studying the effects of *reelin* deficiency in determining social communication deficits and in changing brain monoamine and brain metabolites levels. Unfortunately, no comparison can be drawn with homozygous mutant mice, since adult knockout reeler mice did not survive longer than weaning (79–82).

#### **NO DEFICITS IN SOCIAL AND VOCAL REPERTOIRES DURING COURTSHIP**

To our knowledge, this is the first time that a detailed analysis of the adult male vocal repertoire has been performed in this mutant line. Only behavioral data on same-sex interactions or approaching/recognizing a conspecific have been collected (44, 51, 52).

Recently, we characterized vocal and motor repertoires on homozygous and Het reeler pups (60) evidencing a general delay in vocal and motor development during the first 2 weeks of postnatal life, in line with the alterations in the same two systems observed in children with ASD. In addition, a preferential use of a specific call category (two-components) at pnd 2 and 6 was detected in both mutants (Het and homozygous), whereas an increased number of vocalizations characterized only Het pup's emission.

Contrary to what we found in pups, adult Het male mice did not show deficits on USVs emitted during courtship of a female in estrous. Social behaviors, generally associated to this vocal emission, were not affected either. These results are in contrast with the reduction in anogenital sniffing and/or the number of USVs found in other ASD animal models such as BTBR, En2, NMDA-Nr1, NLG3, NLg4, Dlg4, and FmR1 mice (53, 83–87), but in line with data collected on Shank3 mice, carrying a mutation strongly implicated in autism and Phelan-McDermid 22q13 deletion syndrome, where male knockout mice did not present alterations in social communication and interaction (88).

These data thus confirm that adult Het reeler mice present deficits on cognitive performances but not on social domains (44– 47, 49–52). It is worth of notice that intellectual disabilities are present in about 50% of autistic individuals. Due to the cerebellar alteration leading to death shortly after weaning, no data could be collected on mice with the complete deletion in the reelin gene, thus we cannot exclude an impairment of the social domain only related to the complete deletion of *reelin*.

#### **OVER RESPONSE TO A MILD STRESS STIMULI**

Previous studies indicated that Het reeler mice have several abnormalities in their brain architecture (40–42), but, at a first sight, their phenotype is absolutely"normal"(7,44,89). Some behavioral deficits become evident only after a "second hit" (7, 63, 90, 91) supporting the "double-hit" theory postulating a gene–environment interaction in the pathogenesis of several neurodevelopmental disorders such ASD (89). Depending on the features of environmental factors and the time-window of insult interacting with reelin expression, an individual could thus develop one neurodevelopmental disorder rather than another one (i.e., schizophrenia versus ASD).

Our previous evidence shows that either an environmental pollutant or, for example, an activated stress reactivity caused by repeated separation from the dams, elicits different responses as a function of the mouse genotype (91). In line with these data, in the present study, no significant genotype differences were found in basal activity levels of mice monitored in their home-cages for 1 week. By contrast, after a saline injection (a mild stressful stimulus), the Het male displayed a higher locomotor activity profile as compared to Wt male mice. Already in a previous study, our group showed a hyperactive profile in Het adolescent reeler mice following handling plus saline injections (63). Altogether, these data indicate that Het reeler mice show a different response to environmental stimuli, confirming the suitability of such mutant line for the study of gene–environment interactions (7, 92).

Moreover, a deficit in behavioral inhibition has been reported as a core alteration of Het reeler mice, associated with dysfunctions of mesolimbic DA transmission (93) and reduced GABAergic transmission in central nervous system (40, 94–96).

#### **IMPAIRMENT IN THE DOPAMINERGIC PATHWAY**

To correlate observed behavioral abnormalities to the neural systems reportedly affected by *reelin* mutation, we conducted HPLC analyses in different brain areas involved in autism, detecting impairments in the dopaminergic system. Specifically, Het reeler mice had decreased DA levels in cortex and increased levels in the olfactory bulb, whereas DA turnover was altered in cortex, bulb, and Hip.

A disruption of DA maturation in *reelin* haploinsufficient mice had been already suggested: a reduced locomotor activation by damphetamine in reeler mice was associated with an exaggerated drug-induced stereotyped behavioral syndrome (90). Moreover, Ballmaier et al. (93) reported alterations in the mesolimbic DA pathway of Het reeler mice. In particular, they found that Het mice exhibit a reduction in DA transporter immunoreactivity and D2 receptor mRNA in the limbic striatum and the ventral tegmental area (93). In agreement with our study, they did not find any significant alteration in the dopaminergic markers examined in the nigrostriatal pathway of Het reeler mice.

Alterations in DA levels and its turnover have been found in brain areas primarily associated to reward. Individuals with ASD show reduced responsiveness to reward stimuli, a feature that appears to be especially prominent with social reinforces such as facial expressions, spoken language, and gestures (97, 98). No effects have been found in the striatum where DA contributes to motor performances.

In addition, the neurotransmitter DA plays a pivotal modulatory role on executive functions and learning (99, 100), thus a dysfunctional DA system could underlying the cognitive deficits detected in Het mice.

#### **GLUTAMATE AND TAURINE INCREASED LEVELS IN HIPPOCAMPUS**

To gain deeper insights into the functional state of brain areas involved in ASD, we carried out a 1H MRI guided spectroscopy examination in adult reeler mice. MRS is a powerful, non-invasive tool for monitoring neurological diseases (101) and it is also used in clinical studies on autistic individuals (102). Abnormalities in neurotransmitter pathways have been associated to ASD, with evidence for a possible implication of glutamatergic, GABAergic, and serotonergic imbalances (102).

In the Hip, as compared to Wt in Het mice, MRS showed increased levels of glutamate, taurine, phospho-creatine, and of the total amount of phospho-creatine plus creatine. Glutamate is the main brain excitatory neurotransmitter involved in cognitive functions, although in excessive quantities can cause neuronal damages typical of neurodegenerative diseases (for example, Alzheimer's and Huntington's diseases) (103–105). The higher levels of glutamate in the Hip of Het reeler mice are in agreement with previous findings reporting an increase of glutamate in Hip (106) and cortex (107) of ASD patients; altogether these evidences support the hypothesis of an imbalance between excitatory and inhibitory (GABA) systems as one of the possible causes of autism (107).

Recently, clinical trials with glutamate antagonists have been initiated, since they have been proved to be effective in rescuing social deficits and repetitive behaviors in selected animal models of autism (108). Also the presence of high levels of taurine in the Hip could be correlated to high levels of glutamate. In fact, taurine appears to have a protective action against glutamate excitotoxicity (109) and it is widely considered a general index of neuronal functionality.

The largest meta analysis performed on ASD patients showed evidence that ASD is characterized by age-dependent fluctuations in metabolite levels across the whole brain. In particular, significant reduction in the level of a cerebral metabolites, NAA, a specific neuronal marker, in whole gray matter of ASD children as well as significant increase in the total pool of creatine (phosphocreatine plus creatine) in adult subjects were observed (110). The observed differences in creatine as a function of age and brain region, suggest caution in the use of Cr-based ratio measures of metabolites. For this reason, we adopt a quantitative approach for brain metabolites level determination,which has been validated on phantom (111) as well as on other animal models of psychiatric and neurodevelopmental diseases (51, 112–114).

#### **CONCLUSION**

In the literature, Het reeler mice are widely considered a reliable animal model of either autism or schizophrenia. Genetic and molecular evidences showed that reelin messenger-RNA and its protein are downregulated in cortical, hippocampal, and cerebellar neurons of patients suffering of schizophrenia and autism (3, 8, 10, 20, 115, 116). In particular, these mutant mice are characterized by decreased contextual fear conditioning (48), prepulse inhibition (43, 117), impaired executive functions (45), and associative

learning (48), all typical traits of schizophrenia. In addition, Het reeler mice yielded autistic-like deficits in social behavior and communication in the first two postnatal weeks of age (60) and perseverative (51) and hyperactive behaviors (44) at adulthood. Discordant evidences exist on this model, possibly associated with differences in the genetic background, age of mice, training and testing protocols, and rearing conditions (52, 89).

Overall, our results, together with data previously collected by our (Laviola and collaborators) and other groups suggest that Het reeler mice have common behavioral traits to both these neurodevelopmental disorders. Moreover, these studies indicate the suitability of this mutant line to investigate the role of *reelin* as vulnerability factor on the etiology of both disorders. In addition, Het reeler mutant mice may represent a useful animal model to develop novel treatment strategies for these devastating human disorders. For example, our HPLC and MRS results favor further studies to evaluate the effects of DA agonist or glutamate antagonist treatments on behavioral and neurochemical responses.

#### **ACKNOWLEDGMENTS**

Supported by the Italian Ministry of Health Grant (GR3), Young Researcher 2008,"Non-invasive tools for early detection of Autism Spectrum Disorders." We thank Flavia Chiarotti for the statistical advice.

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 07 June 2014; accepted: 21 August 2014; published online: 05 September 2014. Citation: Michetti C, Romano E, Altabella L, Caruso A, Castelluccio P, Bedse G, Gaetani S, Canese R, Laviola G and Scattoni ML (2014) Mapping pathological phenotypes in reelin mutant mice. Front. Pediatr. 2:95. doi: 10.3389/fped.2014.00095*

*This article was submitted to Child and Neurodevelopmental Psychiatry, a section of the journal Frontiers in Pediatrics.*

*Copyright © 2014 Michetti, Romano, Altabella, Caruso, Castelluccio, Bedse, Gaetani, Canese, Laviola and Scattoni. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*