DEVELOPMENTAL NEUROLOGY Response to Comment on “Oxytocin-mediated GABA inhibition during delivery attenuates autism pathogenesis in rodent offspring”

W e are very grateful to Bambini-Junior et al. (1) for raising the issues of (i) the long-lasting effect on behavior of bumetanide pretreatment in rodent models of autism, (ii) the long-lasting effect on behavior of blocking oxytocin signaling in naïve mothers during the delivery period, and (iii) the sex-dependent response to bumetanide treatment. We are glad to respond with additional experimental observations. To evaluate whether maternal pretreatment with bumetanide has long-term behavioral effects, we have now evaluated adult behavior in two animal models of autism: rats exposed in utero to valproate (VPA rats) andmice carrying the fragile X mutation (FRX mice). Because VPA rats have been shown to display altered behavior (2–4), using the social approach-avoidance paradigm (2) we show that VPAmale adult rats that received bumetanide 1 day before birth (maternal pretreatment) (5) display improved sociability (Fig. 1A), spending significantly more time in the social chamber than agematched nontreated VPA rats. Conversely, it has been shown that FRX mice have similar sociability to wild-type (WT) mice in the three-chamber social test (6), thereby precluding the use of this paradigm to test the effects of bumetanide. Therefore, we evaluated stereotypical behavior (7), and adult FRXmale mice displayed a significantly higher number of grooming events (bouts) than WT littermates (Fig. 1B). Maternal pretreatment with bumetanide of FRX mice restored the grooming behavior (Fig. 1B). Therefore, in the valproate and fragile X rodentmodels of autism, maternal pretreatmentwith bumetanide 1 day before birth restores control behavioral phenotypes in adult offspring. We further evaluated the effect of blocking oxytocin receptors with maternal pretreatment of naïve mothers 1 day before delivery with SSR126768A (SSR) (5) on adult social behavior in mice and rats (2, 8). We observed that adult rat (Fig. 1C) and mouse (Fig. 1D) offspring exposed to SSR display lower sociability than controls, extending our earlier observations (5) to adulthood. Thus, blocking oxytocin signaling in naïvemothers during the delivery period produced adult offspring with altered social behavior. In addition to our behavioral tests in adult animals, we wanted to verify the presence of network alterations in the developing brain of FRX mice, as we did for the VPA rats (5). We therefore performed extracellular intracranial electroencephalographic (EEG) recordings in vivo in the hippocampal CA3 area of head-restrained P14 and P15 WT and FRX mice. We show prominent differences in the oscillatory activity of FRX mice compared with WT (Fig. 1E1). There was a significant decrease in slow oscillatory activity (0.1 to 4 Hz) in comparison with age-matched WT mice. In contrast, fast EEG activity in a wide band of frequencies from 4 to 800 Hz was increased. The most pronounced differences in EEG were observed at the d (0.1 to 4 Hz) and low g-band (25 to 40 Hz) frequencies (Fig. 1E2). Maternal pretreatment with bumetanide of FRX mice partly reduced these aberrant oscillations and significantly decreased low g and fast and ultrafast oscillatory activity in offspring (Fig. 1E1 and 1E2). Hence, FRX mice display an enhanced network oscillation in the hippocampus not observed in age-matched WT mice, and this is restored to physiological values aftermaternal pretreatment with bumetanide. Collectively, these results extend our earlier observations suggesting that the priming effects of the polarity of g-aminobutyric acid (GABA) during delivery are long-lasting, being present in adults, and concern also social behavior components. They also validate our physiological recordings with the behavioral manifestations and the priming effects of bumetanide and oxytocin during the delivery period. The underlying mechanisms remain to be investigated, although it has been suggested that insults during delivery and the early postnatal period lead to long-termbehavioral and molecular consequences. Bambini-Junior et al. also criticize the use of males and females in our analysis, highlighting the higher prevalence of autism spectrum disorders in males in humans. However, this male/ female difference is one of incidence and not of treatment, because in our clinical trials girls are as efficiently treated as boys by bumetanide (9), which suggests elevated intracellular chloride in both. In addition, behavioral differences between males and females are neither systematically investigated nor essential because they do not constitute a condition to identify the autistic syndrome and their underlying mechanisms remain to be determined. Inourpresent experiments, onlymales were used to test adult behavior (Fig. 1, A and B), and bumetanide pretreatment was efficient in reducing autistic features. Furthermore, our in vivo experiments in FRXmicewere performed in a similar ratio of males and females in each group tested, and we observed no difference regarding the sex, suggesting that independently of their sex bumetanide pretreatment restores control phenotypes. Finally, if we assume that bumetanide is less effective in female than in male rodents (or vice versa), we see an overall significant positive effect of bumetanide treatment. In sum, our study has shown that a pretreatment restricted to the delivery period of autistic rodents attenuates the severity of the syndrome in young and adults, and this is controlled by the polarity of GABA and oxytocin signals at that stage. This raises questions concerning the role of delivery in the GABA excitatory/inhibitory shift and in relation to the emergence of autism. Our priority is to understand these links and the alterations in brain patterns stemming from excitatory GABA.