Newborn neurons enter an extended maturation stage, during which they acquire excitability characteristics crucial for development of presynaptic and postsynaptic connectivity. In contrast to earlier specification programs, little is known about the regulatory mechanisms that control neuronal maturation. The Pet-1 ETS (E26 transformation-specific) factor is continuously expressed in serotonin (5-HT) neurons and initially acts in postmitotic precursors to control acquisition of 5-HT transmitter identity. Using a combination of RNA sequencing, electrophysiology, and conditional targeting approaches, we determined gene expression patterns in maturing flow-sorted 5-HT neurons and the temporal requirements for Pet-1 in shaping these patterns for functional maturation of mouse 5-HT neurons. We report a profound disruption of postmitotic expression trajectories in Pet-1 Ϫ/Ϫ neurons, which prevented postnatal maturation of 5-HT neuron passive and active intrinsic membrane properties, G-protein signaling, and synaptic responses to glutamatergic, lysophosphatidic, and adrenergic agonists. Unexpectedly, conditional targeting revealed a postnatal stage-specific switch in Pet-1 targets from 5-HT synthesis genes to transmitter receptor genes required for afferent modulation of 5-HT neuron excitability. 5-HT 1a autoreceptor expression depended transiently on Pet-1, thus revealing an early postnatal sensitive period for control of 5-HT excitability genes. Chromatin immunoprecipitation followed by sequencing revealed that Pet-1 regulates 5-HT neuron maturation through direct gene activation and repression. Moreover, Pet-1 directly regulates the 5-HT neuron maturation factor Engrailed 1, which suggests Pet-1 orchestrates maturation through secondary postmitotic regulatory factors. The early postnatal switch in Pet-1 targets uncovers a distinct neonatal stagespecific function for Pet-1, during which it promotes maturation of 5-HT neuron excitability.

Central serotonergic signaling influences many physiological processes but a requirement for reproductive success has not been demonstrated. Using dams with a specific disruption in serotonin neuron development we show that serotonergic function is required for nurturing and survival of offspring. Full rescue of survival depends on the mother's expression level of the upstream serotonergic transcriptional cascade. Thus, intrinsic transcriptional programming of maternal serotonergic activity determines the quality of nurturing and organism survival.Serotonergic signaling in the CNS is critical for proper maturation and homeostatic modulation of neural circuits that shape emotions and many physiological responses 1, 2 . Perturbations in the level of serotonergic gene expression have significant impact on behavior and have been implicated in the pathogenesis of several neuropsychiatric diseases including disorders of anxiety, mood and appetite 1, 2 . Although serotonin (5HT) has been implicated in the regulation of female sexual behavior 3 , present evidence does not demonstrate an essential role for the central serotonergic transmitter system in reproductive success.Expression of the Pet-1 ETS transcription factor in the brain is restricted to 5HT neurons 4 . Normal numbers of serotonergic precursors are generated in Pet-1 −/− midbrain but expression of serotonergic gene expression and 5HT synthesis are greatly reduced 5 . We used Pet-1 deficient dams to investigate the impact of their arrested 5HT neuron development on offspring viability. Virgin wild type and Pet-1 −/− females were bred with wild type or Pet-1 +/− males. Birth rates and offspring body weights were normal for primiparous Pet-1 −/− dams (Supplementary Table 1, online; Supplementary Fig. 1, online). Yet, while 99% of pups survived when born and nurtured by wild type dams, no pups survived when born to Pet-1 −/− dams and the majority of these offspring were found dead without placentas and not cannibalized 3-4 days after birth (Fig. 1a). Cross fostering at postnatal day 1 showed that pups born to Pet-1 −/− dams survived when nurtured by wild type dams (Fig. 1a The profound deficit in survival of offspring born to Pet-1 −/− dams suggested deficient maternal behavior. Successful nurturing requires the coordinate expression of several discrete behaviors including nest building, pup retrieval, cleaning, and nursing 6 . Pups born to Pet-1 −/− dams were consistently observed to have milk in their stomachs each postnatal day before death suggesting normal lactation (Fig. 1b, inset). However, the percent time crouching was significantly less for Pet-1 −/− dams compared to wild type dams (Pet-1 −/− dams': 53%, stdev 8.7%, n=6; wild type dams': 73%, stdev 10.1%, n=13; p<0.05). Moreover, Pet-1 −/− dams often would not build suitable nests (Fig. 1b, c). Organizing pups in a huddle is essential for neonate survival as it facilitates feeding and maintains pup body temperature. Pups born to wild type dams were always found organized in huddles (Fig 1b, d) ...

The primary center of serotonin (5-HT) projections to the forebrain is the dorsal raphe nucleus (DR), a region known for its role in the limbic stress response. The ventromedial subregion of the DR (vmDR) has the highest density of 5-HT neurons and is the major target in experiments that involve the DR. However, studies have demonstrated that a variety of stressors induce activation of neurons that is highest in the lateral wing subregion (lwDR) and includes activation of lwDR 5-HT neurons. Despite the functional role that the lwDR is known to play in stress circuits, little is known about lwDR 5-HT neuron physiology. Whole cell patch clamp electrophysiology in mice revealed that lwDR 5-HT cells have active and passive intrinsic membrane properties that make them more excitable than vmDR 5-HT neurons. In addition, lwDR 5-HT neurons demonstrated faster in vitro firing rates. Finally, within the vmDR there was a positive correlation between rostral position and increased excitability, among several other membrane parameters. These results are consistent with stressor induced patterns of activation of 5-HT neurons that includes, in addition to lwDR neurons, a small subset of rostral vmDR neurons. Thus increased intrinsic excitability likely forms a major part of the mechanism underlying the propensity to be activated by a stressor. The membrane properties identified in lwDR recordings may thereby contribute to a unique role of lwDR 5-HT neurons in adaptive responses to stress and in the pathobiology of stress-related mood disorders.

Clozapine, by virtue of its absence of extrapyramidal side effects and greater efficacy, revolutionized the treatment of schizophrenia, although the mechanisms underlying this exceptional activity remain controversial. Combining an unbiased cheminformatics and physical screening approach, we evaluated clozapine's activity at 42350 distinct molecular targets. Clozapine, and the closely related atypical antipsychotic drug olanzapine, interacted potently with a unique spectrum of molecular targets. This distinct pattern, which was not shared with the typical antipsychotic drug haloperidol, suggested that the serotonergic neuronal system was a key determinant of clozapine's actions. To test this hypothesis, we used pet1 À/À mice, which are deficient in serotonergic presynaptic markers. We discovered that the antipsychotic-like properties of the atypical antipsychotic drugs clozapine and olanzapine were abolished in a pharmacological model that mimics NMDA-receptor hypofunction in pet1 À/À mice, whereas haloperidol's efficacy was unaffected. These results show that clozapine's ability to normalize NMDA-receptor hypofunction, which is characteristic of schizophrenia, depends on an intact presynaptic serotonergic neuronal system.