Acute neonatal oxytocin impacts hippocampal network development and restores adult social memory deficits in a mouse model of autism spectrum disorder

Several studies on rodent models with an Autism Spectrum Disorders-like (ASD) phenotype, notably Magel2-deficient mice, have shown a rescue of deficits in adult social behavior after neonatal administration of oxytocin. However, the neurobiological alterations responsible for the social deficits and the mechanism by which oxytocin-administration in infancy has a rescue effect in adulthood remain unclear. Here we show that Magel2-deficient adult mice exhibit a deficit in social memory that is corrected by neonatal oxytocin administration. We studied hippocampal regions known to be associated with social memory engrams involving the OT-system. At critical stages of development, we characterized cellular, physiological and biochemical alterations of these hippocampal regions in Magel2-deficient mice, alterations present in several ASD models. Overall we demonstrate a strong impact of oxytocin-administration at key stages of postnatal hippocampal neurodevelopment, shedding light on the role for oxytocin in treating neurodevelopmental disorders characterized by deficits in social memory.


INTRODUCTION
Oxytocin (OT) and its signaling pathway, the OT-system, are disrupted in several animal models of neurodevelopmental disorders characterized with autism-like phenotypes (1,2).
Indeed, knock-out mouse models of Ot (3,4), oxytocin receptor (Oxtr) (5-7), or ADP-ribosyl cyclase (Cd38) (8,9) genes show changes in social behavior. In addition, several rodent models of autism spectrum disorders (ASD) endophenotypes due either to the inactivation of genes such as Fmr1, Cntnap2, Magel2, Oprm1, or to environmental valproic acid exposure (VPA), exhibit a disruption of the brain OT-system (1). Many of these models have in common at least two constant phenotypes (10). First, pups isolated from the dam and littermates present a decrease in the number of ultrasonic vocalizations (USVs), the earliest behavioral test that can be performed in mice to measure social deficits. A second robust phenotype is a social recognition memory deficit in adulthood.
OT is thought to regulate aspects of social recognition, social novelty and social memory via interactions with OT-receptors (OXTR) in a number of key brain regions (11). 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) (12)(13)(14)(15).
In the aCA2/CA3d region, OXTRs are expressed in glutamatergic pyramidal neurons (which are also CCK positive) and in GABAergic interneurons, which account for over 90% of OXTR positive cells in the hippocampus (16). Notably, both types of neuron are necessary for the formation of stronger synapses that mediate LTP and social memory (12,13).
Although most studies of OT-dependent social behaviors have been conducted in adulthood, compelling data support key roles for OT in shaping various behaviors and social traits in infancy (17-21). Genetic mutations and stressful early-life social environments can impair social behaviors via changes in the OT-system (22, 23). On the other way, OT-dependent systems may be particularly vulnerable around the neonatal period where OXTR expression is dynamic, with a strong expression in the first 2 weeks postnatal followed by a decreased expression thereafter (24, 25), and involved in critical functions such as the excitatory-toinhibitory developmental GABA switch (26-28), a delay in which has been implicated as a driver of several rodent ASD models (27,29). It has also been demonstrated that, in mouse pups, sensory experience influences OT production and OT shapes neuronal circuits by modulating spontaneous and evoked activity in the cortex (25, 30, 31). Thus, the modulation of GABAergic activity throughout life appears to be an important key factor in the function of OT. In addition, OT acts also on glutamatergic neurons (32) and drives dendritic and synaptic refinement in immature hippocampal glutamatergic neurons (33). Collectively, these studies confirm a role early infancy. We confirmed that vehicle or OT-treatment had no effect on vocalization in WT pups and did not significantly increase the number of USVs recorded in mutant animals ( Figure   1C). Thus, OT-treatment did not exert immediately measurable effects on neonatal social behaviors in Magel2 KO pups.
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) (46) (Figure 2A). Magel2 KO males showed levels of sociability and social discrimination similar to WT males but exhibited a significant deficit in social memory ( Figure 2B Thus, the loss of Magel2 causes a deficit in USV calls in pups and a deficit in social memory in male Magel2 KO adults. While the USV deficit was not affected by post-natal OT-treatment, the deficit in social memory 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 (12,13).

aDG and aCA2/CA3d regions are activated by the social memory test in WT and Magel2
KO mice.
Cells in the aCA2/CA3d and aDG regions have been previously shown to express OXTRs and are involved in social memory (12,13). We therefore asked whether these cells could be 6 activated by the social memory test. WT and Magel2 KO mice were sacrificed 90 min after the end of three-chamber test (+SI, for Social Interactions) and their brains examined for cFos immunolabeling, a marker of neuronal activity ( Figure 3A). We found a significant increase (83%) in the number of cFos+ cells in the stratum pyramidale of aCA2/CA3d in tested WT (WT+SI) compared with untested WT (WT-SI) mice; in Magel2 KO+SI mice we observed a 25% significant increase of cFos+ cells compared with WT+SI. In the aDG, a significant increase of ~60% of cFos+ cells compared with WT-SI was observed in both WT+SI and Magel2 KO+SI, mainly in the hilus and stratum granulare. Thus, the activation of c-Fos is significant in both regions following social memory test in both genotypes (WT+SI and Magel2 KO+SI mice) with an increase of cFos activated cells observed in the aCA2/CA3d region in Magel2 KO+SI.
Overall, these data confirm a strong activation of neurons in aCA2/CA3d regions in WT and Magel2 KO mice following social interactions.

Expression profile of Magel2 and Oxtr in the aDG and aCA2/CA3d regions
Magel2 is known to be highly expressed in hypothalamus, while its expression in hippocampal regions is less well characterized. We investigated the developmental expression of Magel2 and Oxtr transcripts in the anterior hippocampus using RNAscope technique at two different time points (P7 and P21), taking in account the dynamic expression of OXTRs. At P7 ( Figure   4A), we detected Oxtr and Magel2 mRNAs in the aCA2/CA3d region with Magel2 more expressed in the deep layer of the stratum pyramidale, a fainter expression is also detected all over the layer. At this stage, Oxtr transcripts were more concentrated in the CA2 region, compared to Magel2. At P21 (Figure 4), 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 is detected in the hilus, some cells presenting a co-localization of both transcripts.
In adulthood, based on the in-situ hybridization maps of Allen Brain Atlas, we could confirm expression of Magel2 in the CA3 deep layer of the stratum pyramidale (not shown). Then, we consulted the public Allen brain RNAseq data from cortex and hippocampal samples (http://celltypes.brain-map.org/rnaseq/mouse/cortex-and-hippocampus) to define the cell types expressing Magel2 and Oxtr. Oxtr transcripts are expressed in GABAergic interneurons mainly in SST and also in parvalbumin (PV) and Calbindin expressing interneurons.
Expression is also detected in glutamatergic neurons of a CA2-CA3 region co-expressing CCK. Magel2 is faintly detected in the glutamatergic neurons only. Finally, from RNAseq data extracted from the atlas of cell types from Linnarsson Lab.
(http://mousebrain.org/genesearch.html) co-expression of Oxtr and Magel2 is detected in the CA3 excitatory neurons of adult mice and also in several interneuron sub-populations expressing SST or SST and PV such as the axo-axonic long-range projections interneurons and the basket bistratified cells. These sub-populations of interneurons represent a small number of cells that have a wide and diverse spectrum of actions. These data suggest that the lack of Magel2 can alter the Oxtr expressing excitatory neurons in the aCA2/CA3d region and also the SST or PV interneurons.

Magel2 KO adult mice and normalized in the aDG following an OT-treatment
We then looked at the distribution of OT-binding sites in Magel2 KO-vehicle or Magel2 KO+OT compared with WT-vehicle hippocampi by autoradiography ( Figure 5). We observed a significant increase of OT-binding sites in the aCA2/CA3 (100%, Figure 5A-B) and aDG (80%, Figure 5C-D) regions but not in the ventral CA1/CA2/CA3 region ( Figure 5E-F). In Magel2 KO+OT we observed a normalization of the quantity of OT-binding sites in the aDG, but not in the aCA2/CA3 region where OT-binding sites level was decreased but remained elevated compared to WT ( Figure 5A-B). The binding study indicate subregions specific modulation of OXTR in the hippocampus. We then asked if this subregion specific effect could be linked to specific changes in neuronal subpopulations in the same regions.

The number of SST+ neurons is increased in the aCA2/CA3d and aDG regions of Magel2
KO adult mice and normalized following an OT-treatment.
In the anterior adult hippocampus OXTRs are expressed in pyramidal cells and in SST and/or PV interneurons of aCA2/CA3d region and in interneurons of aDG (see above, (12,16)). In control mice following social memory test, cFos expression was detected in 23 or 30% of SSTimmunopositive cells (SST+) and 22 or 28% of PV+ cells in the aCA2/CA3d or aDG regions following the social memory test, respectively ( Altogether, those results show that, in Magel2 KO pyramidal neurons of aCA3d, there is a significant increase in the GABA/Glutamate ratio with no change in their neuronal morphology.
We next investigated the effects of OT-treatment on the GABA/Glutamate balance in WT and Magel2 KO. Quite unexpectedly, the spontaneous glutamatergic activity was significantly reduced in WT+OT mice compared with WT: frequency was considerably reduced (x 2.7), being even lower than in OT-treated mutants ( Figure 7C) and the amplitude was reduced (x1.5), being similar to the one observed in mutant mice ( Figure 7D) . These findings indicate that OT-treatment reduced strongly the glutamatergic activity in WT juvenile mice without any apparent impact on the behavioral outcome measured (see above). In Magel2 KO, OTtreatment in the first week of life decreased significantly the frequency of sGABA-PSCs restoring a frequency similar to WT ( Figure 7E). There was no effect of OT-treatment on the amplitude of sGABA-PSCs ( Figure 7F). However, on the same pyramidal neurons, such treatment in Magel2 KO+OT reduces significantly the frequency of sGlut-PSCs (2x) compared with the frequency recorded in WT or in the Magel2 KO mice ( Figure 7C)  to progressive lowering of [Cl -]i that at P7 shifts GABA action from depolarizing to hyperpolarizing. As consequence, the activation of GABAAR produces neuronal Clinflux.
The 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 9 A-B). The ion-transport activity of KCC2 and its stability at the cellular plasma membrane also strongly depend on posttranslational modifications of multiple phosphorylation sites (50). We therefore applied in a next step phospho-site-specific antibodies, as they were previously shown to quantitatively monitor changes in KCC2 phosphorylation (51)(52)(53). Currently, a limited number of such phospho-specific antibody is available. They are directed against the well-known KCC2's phospho-sites Ser 940 (54) and Thr 1007 (52,53). 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, but albeit higher in Magel2 KO mice ( Figure 9A-B). Thus, in Magel2 KO mice there was significant decrease of P-Ser 940 (inactivation of function lee (54)) and no change of P-Thr 1007 whose progressive developmental de-phosphorylation is associated with increase of KCC2 activity Friedel (52). 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 show that Magel2 KO mice display two specific and constant phenotypes that are stereotypical of other models characterized by a deficit in OT-system: isolation-induced vocalizations in infancy and social memory at adulthood (10). OT administration in the first week of life rescued the latter but not the former. To understand how neonatal OT-treatment drives such long-term effect in Magel2 KO mice, we studied the hippocampal regions that contains social memory engrams, which also involve the OT-system, and unraveled several and successive temporal changes in the aDG and aCA2/CA3d hippocampal regions that might contribute to the social memory deficit in Magel2 KO mice. We showed that Magel2 deficiency alters the GABAergic developmental sequence probably resulting from post-translational biochemical modifications of KCC2, modifies the activity of pyramidal neurons and presents a higher number of SST-positive interneurons and an increase in the quantity of OT-binding sites at adulthood. Interestingly nearly all those changes are rescued by administration of OT in the first days after birth.

The effect of neonatal OT-administration on Magel2 KO mice
With regard to social behavior, the consequences of postnatal administration of OT in Magel2 KO mice are consistent with those described previously (38). We have shown a rescue of all social deficits described in adult Magel2 KO mice without an effect on USVs (here and (38).
The "USV test" measures early communication behavior in rodent pups with an ASD-like phenotype and in particular in those with a deficit in the OT-system; however, the role of OTsystem in USV calls remains unknown. We cannot explain yet why the OT-administration in the first week of life does not improve the number of USVs in Magel2 KO pups (P8).
One surprising finding of the current study is the relatively mild behavioral phenotype of the homozygous mutant, which we expected to be more profoundly affected than heterozygous animals, previously reported to exhibit a social deficit (38). This difference may reflect differences in behavioral assays used in the two studies (3-chamber test versus open field) or the fact that heterozygous mice grew up with WT littermates, whereas homozygous -/-mutants constitute a homogeneous population. Indeed, it has been shown that transgenic mice and their WT littermates can modify each other's behavior (55). The behavior of the mother can also be modified in the presence of one or two genotypes in the same litter and thus impact the behavior of her offspring differently. The modulation of the severity of the social behavior deficits based on the genotype of the Magel2 KO pups and their environmental context open a number of interesting translational opportunities that will need to be deeply investigated.
Converging evidence suggests that the neurobiological alterations described here may be related to a deficiency in the OT-system of Magel2 KO mice. Noticeably, Ates et al. (56) showed, ex vivo, that lack of Magel2 expression is associated with significant suppression of the overall activity of oxytocin neurons, suggesting that dysregulation of the OT-system goes beyond oxytocin expression. Furthermore, given the role of MAGEL2 in ubiquitination, actin regulation and endosomal sorting processes (57) (62)), intrinsic membrane properties (e.g. input resistance) and synaptic connectivity (63).
Altogether those data suggest that the absence of Magel2 delays neuronal maturation during this critically vulnerable period of brain development, resulting in a distinct adult phenotype.
Whether the delay of the GABA-shift alone is sufficient to derail neurotypical developmental trajectory remains a key question for future study: notably, similar or longer GABA-shift delays have been observed in several models of autism (64,65) and in Oxtr KO mouse models (28).
Recently, Kang et al. (66) showed in Disc1 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 (60).
Importantly, OT-treatment has a relative hyperpolarizing effect at P7 in Magel2 KO and WT pups compared with WT-vehicle animals that might modify the maturation of the circuitry.

The E/I ratio and social behavior
Reductions in synaptic signal-to-noise 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 (67). 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 other ASD models (68)(69)(70)(71). Furthermore, we report that perinatal OT administration restored normal GABAergic activity in Magerl2 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 ASDlike behavior Magel2 KO animals 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 conclude 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/CA3d 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 (37). 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 (58,72). SST interneurons have recently been shown to play a role in the modulation of social behavior (73,74) and a reduction in the number of PV or SST interneurons has been reported to be associated with social deficits or ASD. Noticeably, a link between altered social memory and an increase in SST cell number has been recently reported in LPS-treated female neonates (75). It is tempting to speculate that OXTR-transmission regulates the activity of SST hippocampal interneurons and the production/release of mature SST and impacts social memory. Further work is needed to fully characterize the role of OXTRs on SST interneurons in relation with social memory.

Conclusions
Short-term OT-treatment in infancy reduced behavioral traits associated with ASD in adulthood and permanently rescued functional and cellular hippocampal alterations that were identified in Magel2 KO mice. However, we do not know whether the deficient OT-system observed in KO are a consequence of dysfunction in a single sequential pathway, or whether they reflect parallel interconnected circuits in the developing hippocampus. Finally, and surprisingly, postnatal OT-treatment plays a role in many key processes, suggesting a pleiotropic action of oxytocin stimulating the maturation of hippocampal circuitry involved in social memory and possibly other behavioral deficits. A significant impact of such treatment was also observed in WT animals without any effect on social behavioral as tested.
Overall, we have shown that OT-treatment in infancy has a significant impact and rescues permanent specific hippocampal alterations in Magel2 KO mice at different developmental ages, many of these hippocampal alterations have been described in several models of neurodevelopmental disorders with ASD (see discussion). In addition, OT deficit has often been described in rodent models of ASD (see introduction). Taken together, our findings reinforce the idea that OT-treatment in early life may represent a viable therapeutic strategy for patients with SYS or PWS and possibly other neurodevelopmental disorders.

Animals
To perform functional studies, we chose to work with the    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.
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 (46).
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 homecage 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 (76)  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% CO2), and half of the medium was refreshed twice a week.

Calcium imaging recordings
Calcium imaging experiments were carried out as previously described (28 trough single channels and current-voltage relationships were performed using Clampfit 9.2 (Axon Instruments) as described by (77).

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 (78). 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

OT binding assay
Adult WT and mutant mice were sacrificed and non-perfused mouse brain were frozen in -25°C isopenthane and stored at -80°C until cut. 14 µm thick brain slices were cut using a cryostat (Frigocut-2700, Reichert-Jung) and collected on chromallume-coated slides and 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
Fresh-frozen brains from WT mice at post-natal days (P)7, P21, and P28 were sectioned in a cryostat in the coronal plane at 20μm thickness and mounted on Superfrost Plus slides and      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. They are measured in (  (A) Paradigm of the three-chamber test. Sniffing time between mice is measured in each test. (B) WT (N=11) females do present differences in social exploration but do not present significant differences in social discrimination and short-term social memory, suggesting that the three-chamber test is not relevant to assess the social behavior in females. Similarly, Magel2 KO (N=9) females do not present significant differences in all three steps of the paradigm. Data in histograms report interaction time (time of sniffing in seconds) as mean ± SEM.
Mann-Whitney test, *P<0.05. Statistical analysis is reported in Table S2-2. Behavioral analysis of male and female OT-treated versus vehicle-treated WT mice. (A) Novel object recognition (NOR) test allows to assess the non-social memory. Training simply involves visual exploration of two identical objects, while after one hour the test session involves replacing one of the previously explored objects with a novel object. Because mice have an innate preference for novelty, a mouse that remembers the familiar object (same object) will spend more time exploring the novel object (new object) conducting to a discrimination index significantly different from 50%. A similar discrimination index is observed in vehicle or OTtreated WT males and females. (B) Open field (OF) test measures locomotor activity and vertical activity (rearing) and anxiety-related behavior (time in zones and grooming): no significant differences have been detected in all those activities between OT-treated and vehicle-treated WT mice but WT+OT females present an approximately 15% reduction in the moved distance and a tendency to increase rearing.             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 Table S8. phosphorylation is significantly lower as compared to WT hippocampi whereas the amount of phosphorylated Thr 1007 remains unchanged. Respectively, the decreased P-Ser 940 /P-Thr 1007 ratio results in predominance of KCC2 internalization over surface expression. As consequence of the decreased amount of surface expressed molecules, the Clextrusion ability of KCC2 is decreased that causes increase of [Cl -]i and depolarizing shift of GABA. The model includes also important components that are known to control the level of Ser 940 and Thr 1007 phosphorylation. The Ser 940 is directly phosphorylated by kinase C (pkC) and dephosphorylated under pathology conditions by protein phosphatase type 1 (PP1). The Thr 1007 is directly phosphorylated by SPAK. It remains to be elucidated whether in Magel2 KO mice the decreased level of Ser 940 results from reduction of pkC activity or enhancement of PP1 action. Statistical analysis is reported in Table 9.