Oxytocin administration in neonates shapes the hippocampal circuitry and restores social behavior in a mouse model of autism

Oxytocin is a master regulator of the social brain. In some animal models of autism, notably in Magel2 tm1.1Mus -deficient mice, peripheral administration of oxytocin in infancy improves social behaviors until adulthood. However, neither the mechanisms responsible for social deficits nor the mechanisms by which such oxytocin administration has long-term effects are known. Here, we aimed to clarify these oxytocin-dependent mechanisms focusing on social memory performance. We showed that Magel2-deficient mice present a deficit in social memory and studied the hippocampal circuits underlying this memory. We showed a co-expression of Magel2 and oxytocin-receptor in the dentate gyrus and CA2/CA3 hippocampal regions. Then, we demonstrated: an increase of the GABAergic activity of CA3-pyramidal cells associated with an increase in the quantity of oxytocin-receptors and of somatostatin interneurons. We also revealed a delay in the GABAergic development sequence in Magel2-deficient pups, linked to phosphorylation modifications of KCC2. Above all, we demonstrated the positive effects of subcutaneous administration of oxytocin in the mutant neonates, restoring neuronal alterations and social memory. Although clinical trials are debated, this study highlights the mechanisms by which peripheral oxytocin-administration in neonates impacts the brain and demonstrates the therapeutic value of oxytocin to treat infants with autism spectrum disorders.


INTRODUCTION
The nonapeptide oxytocin (OT) and its signaling pathway, the OT-system, is a master regulator in the development of the social brain suggesting that OT plays a role in both childhood and adult neuropsychiatric disorders characterized by social cognition impairment (1). OT-system is disrupted in several animal models of neurodevelopmental disorders (2,3). Indeed, knockout mouse models of oxytocin (4,5), oxytocin-receptor (Oxtr) (6)(7)(8), or ADP-ribosyl cyclase (Cd38) (9, 10) genes show changes in social behavior reminiscent of autism spectrum disorders (ASD). On the other way, several rodent models of ASD due either to the inactivation of genes such as Fmr1, Cntnap2, Magel2, Oprm1, Shank3, Nlgn-3 or to environmental valproic acid exposure (VPA), exhibit indirectly a deficit of the brain OT-system (2). MAGEL2 is a gene that is involved in Prader-Willi (PWS) (11) and Schaaf-Yang (SYS) syndromes (12) and is classified as one of the highest relevant gene to ASD risk (SFARI ranking). Both of these genetic neurodevelopmental disorders have in common autistic features with alterations in social behavior and deficits in cognition that persist over the lifespan (13). Magel2 tm1.1Musdeficient mouse model is a pertinent model for both syndromes (13), mimicking alterations in social behavior and learning abilities in adulthood (14, 15). Magel2 is expressed in the developing hypothalamus until adulthood and Magel2 tm1.1Mus -knockout (KO) neonates display a deficiency of several hypothalamic neuropeptides, particularly OT (16). Daily administration of OT in Magel2-KO neonates during the first week of life improves social behavior and learning abilities beyond treatment into adulthood (14). Comparable long term effects have also been reported in other genetic rodent models such as the VPA-induced rat model (17), the Cntnap2 and Fmr1 KO mice (18,19), and following maternal separation (20). However, the neurobiological alterations involving the OT-system and responsible for social behavior deficits in these models are not known. Similarly, the mechanisms by which OT-treatment in infancy exerts its long-lasting beneficial effects, remain mysterious.
At adulthood, OT is thought to regulate aspects of social behavior via interactions with OXTRs in a number of key brain regions (21). Social recognition memory deficit in adulthood is the most robust phenotype, present in all the models with an alteration of the . With regard to social memory, a critical role has been ascribed to hippocampal OXTR expression in the anterior dentate gyrus (aDG) hilar and anterior CA2/CA3distal regions (aCA2/CA3d) (23-26). In the aCA2/CA3d region, OXTRs are expressed in glutamatergic pyramidal neurons and in GABAergic interneurons, which account for over 90% of OXTR positive cells in the hippocampus (27). Notably, both types of neuron are necessary for the formation of stronger synapses that mediate long term potentiation and social memory (23, 24, 28).
In the first two postnatal weeks, OT neuron projections set up and the expression of OXTRs is extremely dynamic followed by a decreased expression thereafter (29, 30). However, during this developmental period, the mechanisms by which the OT-system structures various behaviors are little studied. One study reports that OT is involved dendritic and synaptic refinement in immature hippocampal glutamatergic neurons (31).
Five clinical trials (phase 1 or 2) of OT administration in patients with PWS have been conducted and positive or no effects have been reported but no adverse effects (32). However, each of the studies is fairly empirical and uses different timings, durations and doses of OT, since we do not yet understand clearly how OT works. Based on our previous preclinical studies (14, 16), a phase 1/2 clinical trial with OT-treatment of infants with PWS significantly improves early feeding and "social skills" (33), supporting the translational relevance of our study. More research is needed to demonstrate and validate our hypothesis that the administration of OT in early infancy might be the most beneficial treatment for PWS/SYS. Thus, to build a strong scientific rationale, it is necessary to elucidate the PWS/SYS neuronal alterations and the mechanisms underlying the long-lasting effects of OT-administration in neonates.
Here, we aimed to clarify the physiological and cellular mechanisms related to the OT-system that are disturbed in Magel2 tm1.1Mus -deficient mice and those responsible for the long-term 7 Western blotting experiments were performed on hippocampal tissue and specific bands were visualized with secondary HRP-conjugated antibodies using ChemiDoc™ Imaging Systems (Bio-Rad). The relative intensities of immunoblot bands were determined by densitometry with ImageJ software. See Supplemental Information for details.

Statistical Analysis
Statistical analyses were performed using GraphPad Prism (GraphPad Software, Prism 7.0 software, Inc, La Jolla, CA, USA). All the statistical analyses are reported in a specific file. For details, see Supplemental Information.

Deficit of social memory in Magel2-KO males is rescued by neonatal OT-treatment
At adulthood, we focused on social behavior using the three-chamber test in order to assess social exploration (sociability), the preference for social novelty (social discrimination) and social memory (short-term social memory) ( Figure 1A). Magel2-KO males showed levels of sociability and social discrimination similar to WT males but exhibited a significant deficit in social memory ( Figure 1B, Figure 1-Supplement1). As reported (37), we observed a failure of the three-chamber test in revealing sociability in the cohort of female mice (Figure 1-Supplement2). As a consequence, we restricted all following studies to male mice.
First, the effects of neonatal vehicle and OT-treatment were assessed in WT male pups at adulthood in the three-chamber test. We found that neither treatment had any measurable effect on sociability, social discrimination or social memory: the amount of time spent sniffing in different compartments was similar to that recorded in untreated WT males ( Figures 1B-C). Then, neonatal vehicle and OT-treatment were administered in Magel2-KO pups.
Unsurprisingly, Magel2-KO-vehicle males presented a social memory deficit similar to untreated Magel2-KO males ( Figure 1B,D). However, this deficit was rescued by neonatal OT-treatment ( Figure 1D). Sociability and social discrimination indices were not affected by vehicle or OTtreatment (Figure 1-Supplement1).
Thus, the loss of Magel2 causes a deficit in social memory in male Magel2-KO adults. This deficit was rescued by a neonatal OT-treatment. Due to the robust effect observed on social memory, we focused our subsequent investigations on the hippocampal region, previously shown to be specifically involved in OT-mediated effects on social memory (23, 24). Neurons expressing the OXTRs in the CA2/CA3d and DG regions of the anterior hippocampus are involved in social memory (23, 24) therefore we first tested whether Magel2 and Oxtr are coexpressed in those regions.

aDG and aCA2/CA3d regions co-express Magel2 and Oxtr transcripts
Magel2 is known to be highly expressed in hypothalamus, while its expression in hippocampal regions is less well characterized. Taking in account the developmental and dynamic expression of Oxtr (29, 30), we looked at the expression of Magel2 and Oxtr transcripts in the anterior hippocampus at P7 and P28, using RNAscope technique. At P7, we detected Oxtr and Magel2 mRNAs in the aCA2/CA3d region with Magel2 more expressed in the deep layer of the stratum pyramidale (Figure 2A). At P28, the level of Magel2 transcripts was reduced but still present in the deep layer of aCA2/CA3d region and Oxtr transcripts were also strongly expressed in pyramidal cells. Expression of Magel2 and Oxtr was also detected in few cells of the stratum oriens and stratum radiatum where co-expression can be observed. In the DG, an expression of Oxtr and Magel2 was detected in the hilus, with co-localization of both transcripts. Then, in parallel, we extracted data from public RNAseq data libraries obtained in adult mice (Allen brain; Linnarson lab: http://celltypes.brain-map.org/rnaseq/mouse/cortexand-hippocampus; http://mousebrain.org/genesearch.html). It appears that Oxtr and Magel2 are co-expressed in CA3 excitatory neurons (expressing CCK) and also in several interneuron sub-populations expressing SST. Indeed, we observed a co-expression of Oxtr and Sst mRNAs in hippocampus ( Figure 2B).
We therefore tested the hypothesis that the aCA2/CA3d and DG regions are involved in the social memory deficit of Magel2-KO mice.

Social memory test activates aDG and aCA2/CA3d in WT and Magel2-KO mice
WT and Magel2-KO mice were sacrificed 90 min after the end of social memory test (+SI, for Social Interactions) or without being tested (-SI) and their brains examined for cFos immunolabeling, a marker of neuronal activity, in the aDG and aCA2/CA3d regions ( Figure   3A,B). WT-SI and Magel2-KO-SI mice showed a similar quantity of cFos positive cells in both regions. In the aCA2/CA3d region, WT+SI versus WT-SI ( Figure 3C,D) showed a significant increase (x1.8) in the number of cFos+ cells; an increase (x2.2) was also observed in Magel2-KO+SI mice compared with Magel2-KO-SI ( Figure 3C,E), notably a 23% significant increase of cFos activated cells was observed in Magel2-KO+SI compared with WT+SI ( Figure 3D). In the aDG, mainly in the hilus and stratum granulare, a significant increase of ~60% of cFos+ cells was observed in both WT+SI and Magel2-KO+SI compared with untested (-SI) mice. Overall, these data confirm a strong activation of neurons in aDG and aCA2/CA3d regions following social memory test in both WT and Magel2-KO mice, with an increased activation in the aCA2/CA3d Magel2-KO region.

treatment.
We then looked at the distribution of OT-binding sites, reflecting the presence of functional OXTRs, in Magel2-KO-vehicle or Magel2-KO+OT compared with WT-vehicle hippocampi by autoradiography ( Figure 4). We observed a significant increase of OT-binding sites in the aCA2/CA3d (100%, Figure 4A,B) and aDG (80%, Figure 4C,D) regions but not in the ventral region ( Figure 4E,F). In Magel2-KO+OT we observed a normalization of the amount of OTbinding sites in the aDG, this amount is also decreased in the aCA2/CA3d region but remains high compared to the WT ( Figure 4A,B). This binding study indicates subregions specific modulation of OXTRs in the hippocampus of Magel2-KO compared with WT. We then wondered if this specific effect could be linked to changes in neuronal subpopulations in these sub-regions.

KO adult mice is normalized by neonatal OT-treatment.
In the anterior adult hippocampus OXTRs are expressed in pyramidal cells of aCA2/CA3d region and mainly in SST and/or PV interneurons of aCA2/CA3d and aDG (23, 27). In Magel2-KO adult mice, the number of SST+ cells was higher than in WT mice in both aCA2/CA3d (more 60%) and aDG (more 80%) regions ( Hippocampal brain slices of WT, Magel2-KO, WT+OT and Magel2-KO+OT male mice were analyzed using whole cell patch clamp to record the activity of aCA2/CA3d pyramidal neurons ( Figure 6A). Spontaneous activities analysis ( Figure 6B) revealed a reduced amplitude of postsynaptic glutamatergic currents (sGlut-PSCs) in Magel2-KO as compared to WT mice (x1.7 less, Figure 6D), while the frequency of sGlut-PSCs was not changed ( Figure 6C). The same Magel2-KO neurons presented a significant increase in GABAergic (sGABA-PSCs) frequency (x1.8, Figure 6E) while the amplitude of sGABA-PSCs was similar to that of WT ( Figure 6F). Patch clamp recordings of the glutamatergic and GABAergic miniature currents We next investigated the effects of OT-treatment on the GABA/Glutamate balance in WT and Magel2-KO neurons. Quite unexpectedly, the frequency (x3 less) and amplitude (x1.7 less) of sGlut-PSCs were significantly reduced in WT+OT mice compared with WT-vehicle ( Figure   6C,D). In Magel2-KO mutants, OT-treatment did not modify the amplitude but reduced the frequency (x2.9 less) of sGlut-PSCs compared with Magel2-KO mice ( Figure 6C,D). The GABAergic activity (amplitude and frequency) was not changed in WT mice after an OTtreatment ( Figure 6E,F). In Magel2-KO, OT-treatment decreased (x1,9 less) significantly the frequency of sGABA-PSCs restoring a frequency similar to WT ( Figure 6E); no effect was observed on the amplitude of sGABA-PSCs ( Figure 6F). These results show that OT administration in the first week of life normalized the frequency of spontaneous GABAergic activity in Magel2-KO neurons. Neonatal OT-treatment reduced strongly the frequency of glutamatergic activity in Magel2-KO and the reduction is even stronger on the amplitude and frequency in WT mice. Notably, WT mice had normal behaviors (Figure1-Supplement3).

hippocampal neurons is corrected by neonatal OT-administration.
Because Oxtr and Magel2 are co-expressed in aCA2/CA3d hippocampus in infancy (P7), and because in Oxtr KO mice (35), as in several models (38) of autism, the depolarizing to hyperpolarizing developmental GABA shift is delayed, we investigated the GABA shift timing involving primarily Clextrusion by potassium/chloride cotransporter type 2 (KCC2) whose expression increases progressively during neuronal maturation (40). In developing WT hippocampal neurons, the emerging activity of KCC2 contributes to progressive lowering of [Cl -] i that at P7 shifts GABA action from depolarizing to hyperpolarizing. As consequence, the activation of GABA A R produces neuronal Clinflux.
Quantitative western blot analysis of the total KCC2 protein expression in hippocampi of P7 mice did not reveal statistically significant difference of the amount of KCC2 between WT and Magel2-KO animals ( Figure 8A,B). However, the ion-transport activity of KCC2 and its stability at the cellular plasma membrane depend on post-translational modifications of multiple phosphorylation sites (41). We therefore used phospho-site-specific antibodies, previously shown to quantitatively monitor changes in KCC2 phosphorylation (42-44). Currently, a limited number of such phospho-specific antibody is available. They are directed against the wellknown KCC2's phospho-sites Ser 940 (45) and Thr 1007 (43, 44). Western blot analysis revealed that the Magel2-KO hippocampi (as compared to WT) were characterized by significantly decreased amount of the phosphorylated form of Ser 940 (P-Ser 940 ). The amount of phosphorylated Thr 1007 (P-Thr 1007 ) was not statistically different, although a small but not significant increase was observed in Magel2-KO mice ( Figure 8A,B). At P7, the decreased P-Ser 940 /P-Thr 1007 ratio in Magel2-KO mice may thus result in predominance of KCC2 internalization over surface expression. As a consequence of the decreased amount of surface expressed molecules, the Clextrusion ability of KCC2 is decreased, causing an increase of [Cl -] i and could induce a depolarizing shift of GABA described above ( Figure 8C).

DISCUSSION
Here we investigated, in hippocampus, the mechanisms by which peripheral administration of OT in neonates acts to restore normal social memory in Magel2-KO mice. Peripheral OTadministration in neonates permanently rescued almost all the hippocampal alterations (quantity of OT-binding sites, number of SST-positive interneurons and an increase in the GABAergic activity of pyramidal neurons) that we have characterized and that are associated with the increase of GABAergic activity observed in Magel2-KO adult mice; but a decrease of glutamatergic activity is still present. Those alterations are related with the OT-signaling pathway and relevant to explain the loss of social memory. However, a significant impact of OT-treatment, reducing the glutamatergic activity, was also observed in wild-type mice but all performed behavioral tests were normal. A significant effect of OT-administration on the delayed excitatory-to-inhibitory developmental GABA-shift (at P7), delays that are observed in various neurodevelopmental disorders, underlies the therapeutical use of neonatal oxytocin in these diseases.

The effect of peripheral OT-administration in Magel2-KO neonates
Here, with social memory study, in addition to our previous work, we have shown long-term and beneficial effects of a peripheral administration of OT in Magel2-KO neonates that rescues nearly all social and cognition deficits described in adult Magel2-KO mice (14). However, previously, we did not investigate neither the neurobiological causes of these deficits nor the effects of OT on those alterations. We focused this study on social memory because the mechanisms by which OT controls social memory via the OXTR-expressing neurons in hippocampus are known. We clearly demonstrated that 1) the neurobiological alterations found in Magel2-KO mice involve the hippocampal brain OT-circuitry and 2) peripheral administration of OT in neonates impacts this circuitry. Thus, those results give a clear and positive response to the debated question on the action of peripheral administration of OT on the brain. Is it a direct or indirect effect? It might be both. Today, several studies converge to propose that peripheral OT goes through the Brain Blood Barrier via an active mechanism (RAGE transporters) and/or a passive mechanism, in neonates, when this barrier is more permeable.
The observed long-lasting OT effects could result from a strong impact of OT administration in key developmental hippocampal processes such as the developmental GABA-shift (as discussed below) and can also be achieved by epigenetic modifications that impact gene expression such as the Oxtr expression, as observed in prairie voles following a maternal OT administration (46). Transcriptomic and proteomic studies at different developmental ages would help to understand the life-long effect of an early OT-treatment in mutant and WT mice. KO mouse model, that elevated depolarizing GABA signaling is a precursor for the later E/I imbalance (in favor of inhibition) and social impairment. Similarly, we showed that, in a KCC2 mutant mouse, the GABA-shift delay is responsible for the E/I alteration (49).

The lack of Magel2 alters the OT-system: causes and consequences
Importantly, OT-treatment has an opposite action on the excitatory-to-inhibitory GABA-shift with a relative hyperpolarizing effect at P7 in Magel2-KO and WT pups compared with WTvehicle animals. This effect of OT-treatment might modify the maturation of the hippocampal circuitry.

The E/I ratio and social behavior
Reductions in synaptic signal-to-noise ratio in cortical and hippocampal pyramidal neurons, driven by a change in the ratio of dendritic excitatory and inhibitory synapses, are widely thought to contribute to reduced efficiency of signal processing in ASD, a mechanism known as the E/I ratio hypothesis (57). We confirm E/I imbalance characterized by increased GABAergic activity and lower glutamatergic activity in CA3 neurons in Magel2-KO mice, consistent with observations made in some ASD models (58-61). Furthermore, we report that perinatal OT administration restored normal GABAergic activity in Magel2-KO mice without improving glutamatergic transmission. Unexpectedly, perinatal OT treatment has a significant impact on the WT neurons inducing a strong reduction of glutamatergic activity without affecting GABAergic activity. This is a significant observation, because it shows that, although the ASD-like behavior Magel2-KO animals is correlated with a change in E/I ratio, E/I imbalance in OT-treated WT animals was not sufficient to drive detectable changes in social behavior or cognitive performance. We therefore propose that E/I imbalance characterized by isolated decreased spontaneous glutamatergic transmission is unlikely to underlie the ASD traits investigated here, and suggest that an upper threshold of GABAergic or glutamatergic activity, but not the E/I ratio per se, may be important for normal development.

Role of oxytocin receptors and somatostatin neurons
In adult Magel2-KO mice we observed increased OT-binding in the DG and CA2/CA3 regions of the anterior hippocampus compared to WT mice. OT administration in Magel2-KO neonates normalized hippocampal OT-binding sites in adulthood, suggesting that the increased expression of OXTR observed in Magel2-KO hippocampus may be a consequence of the reduced OT production reported in these animals (16). This observation supports the idea that life-long OXTR expression is to some extent determined by early life OT binding, described as a "hormonal imprinting" effect (46, 62).
Since DG and CA2/CA3 hippocampal OXTRs are expressed in PV and SST interneurons, we

Conclusions
Oxytocin deficiency, present in the Magel2-KO mouse model and in PWS, has also been frequently described in rodent models of ASD (2). Recently, evidences for a unifying role of oxytocin in pathogenic mechanisms responsible for social impairments across abroad range of autism etiologies have been provided (67,68). Thus, our results demonstrate that peripheral OT-administration in a critical period of time, after birth, represents a viable therapeutic strategy for patients with SYS or PWS and possibly other neurodevelopmental disorders.             These indexes report for the social exploration: the sniffing time with S1/ sniffing time with S1 + time in empty room x 100; for the discrimination: the sniffing time with S2/ sniffing time with S1 + sniffing time with S2 time x 100 and for short term memory: the sniffing time with S3/ sniffing time with S1 + sniffing time with S3 time x 100.  Empty S1 S2 S1 S3 * WT females B S1 S1 S2 S1 S3 5' 10' 10' 10' 30' 5' 5'

Habituation
Social exploration Social discrimination Short term social memory A F i g u r e 1 s u p p l e me n t 3 .   Figure 8C). Histograms report mean ± SEM. Unpaired t test with Welch's correction: *P<0-05, **P<0.01, ****P<0.0,001. Statistical analysis is reported in Supplemental Table 7  replaced with another novel object, which was of similar size but differ in the shape and color with the previous object (white and blue lego bricks). Then, the same mouse was placed in the arena and allowed to explore the two objects (a new and an "old" familiar object) for 10 min.

Be h a v i o r a l t e s t s i n ma l e WT mi c e h a v i n g b e e n OT -t r e a t e d o r v e h i c l e -t r e a t e d i n n e o n a t e s .
The movement of the mice was video-tracked with Ethovision 11.5 software. Time of exploration of both objects (nose located in a 2 cm area around object) was automatically measured by the software.

Three-chamber social preference test.
The test was performed as described previously 68 .
The three-chamber apparatus consisted of a Plexiglas box (50x25 cm) with removable floor and partitions dividing the box into three chambers with 5-cm openings between chambers.
The task was carried out in four trials. The three-chambers apparatus was cleaned and wiped with 70% ethanol between each trial and each mouse.
In the first trial (habituation), a test mouse was placed in the center of the three-chamber unit, where two empty wire cages were placed in the left and right chambers to habituate the test mouse to arena. The mouse was allowed to freely explore each chamber. The mouse was video-tracked for 5 min using Ethovision software. At the end of the trial, the animal was gently directed to the central chamber with doors closed. In the second trial (social exploration), a 8weeks old C57BL/6J mouse (S1) was placed randomly in one of the two wire cages to avoid a place preference. The second wire cage remained empty (E). Then, doors between chambers were opened and the test mouse was allowed to freely explore the arena for 10 min. At the end of the trial, animal was gently directed to the central chamber with doors closed. A second 8-weeks old C57BL/6J mouse (S2) was placed in the second wire cage for the third trial (social discrimination). Thus, the tested mouse had the choice between a familiar mouse (S1) and a new stranger mouse (S2) for 10 min. At the end of the trial, the mouse was returned to home-cage for 30 min. For the fourth trial (short-term social memory), S2 was replaced by a new stranger mouse (S3), the familiar mouse (S1) staying the same. Then tested mouse was allowed to freely explore the arena for 10 min. Time spent in each chamber and time of contact with each wire cage (with a mouse or empty) were calculated using Ethovision software. The measure of the real social contact is represented by the time spent in nose-to-nose interactions with the unfamiliar or familiar mouse. This test was performed using grouped-house mice of 4 months old.

Primary hippocampal cultures
Embryonic day 18 dissociated hippocampal neurons were obtained from wild-type and Magel2-KO timed pregnant mice as previously described 69  Glutamate. This media was replaced with glutamate-free media after 5 hours. Neurons were then maintained at 37°C in humidified atmosphere (95% air and 5% CO 2 ), and half of the medium was refreshed twice a week.  trough single channels and current-voltage relationships were performed using Clampfit 9.2 (Axon Instruments) as described by 70 .

Morphological analysis
During electrophysiological recordings, biocytin (0.5%, Sigma, USA) was added to the pipette solution for post hoc reconstruction. Images were acquired using a Leica SP5 X confocal microscope, with a 40x objective and 0,5 µm z-step. Neurons were reconstructed treedimensionally using Neurolucida software version 10 (MBF Bioscience) from 3D stack images.
The digital reconstructions were analyzed with the software L-Measure to measure the number of primary branches and the total number of ramifications of each neuron 71 . Comparisons between groups were done directly in L-Measure.

Immunohistochemistry and quantification
WT and mutant mice were deeply anaesthetized with intraperitoneal injection of the ketamine/xylazine mixture and transcardially perfused with 0.9% NaCl saline followed by  OVTA (Perkin Elmer), at a concentration of 10 pM. After a 2h incubation under gentle agitation, the incubation medium is removed and slides are rinsed twice in ice-cold incubation medium and a third time in ice cold distilled water. Each slide is then dried in a stream of cool air, and placed in an X-ray cassette in contact with a KODAK film for 3 days.
ROIs were chosen and analyzed through ImageJ, using Paxinos' Mouse Brain Atlas as a reference to find the brain areas of interest. To remove background noise caused by nonspecific binding, each slide was compared with its contiguous one, which had been incubated in presence of an excess of "cold" oxytocin (2 M). Net grey intensity was quantified and then converted to nCi/mg tissue equivalent using a calibration curve. For each region, a minimum of 4 slices per brain were included in the analysis. Data plotted on graphs are the differences between the total and the nonspecific binding. Right and left hemispheres were kept separate.

Chromogenic In situ Hybridization
The