ORIGINAL ARTICLE Lack of brain serotonin affects postnatal development and serotonergic neuronal circuitry formation S Migliarini 1 , G Pacini 1,3 , B Pelosi 1,3 , G Lunardi 2 and M Pasqualetti 1 Despite increasing evidence suggests that serotonin (5-HT) can influence neurogenesis, neuronal migration and circuitry formation, the precise role of 5-HT on central nervous system (CNS) development is only beginning to be elucidated. Moreover, how changes in serotonin homeostasis during critical developmental periods may have etiological relevance to human mental disorders, remains an unsolved question. In this study we address the consequences of 5-HT synthesis abrogation on CNS development using a knock- in mouse line in which the tryptophan hydroxylase 2 (Tph2) gene is replaced by the eGFP reporter. We report that lack of brain 5-HT results in a dramatic reduction of body growth rate and in 60% lethality within the first 3 weeks after birth, with no gross anatomical changes in the brain. Thanks to the specific expression of the eGFP, we could highlight the serotonergic system independently of 5-HT immunoreactivity. We found that lack of central serotonin produces severe abnormalities in the serotonergic circuitry formation with a brain region- and time- specific effect. Indeed, we observed a striking reduction of serotonergic innervation to the suprachiasmatic and thalamic paraventricular nuclei, while a marked serotonergic hyperinnervation was found in the nucleus accumbens and hippocampus of Tph2HeGFP mutants. Finally, we demonstrated that BDNF expression is significantly up-regulated in the hippocampus of mice lacking brain 5-HT, mirroring the timing of the appearance of hyperinnervation and thus unmasking a possible regulatory feedback mechanism tuning the serotonergic neuronal circuitry formation. On the whole, these findings reveal that alterations of serotonin levels during CNS development affect the proper wiring of the brain that may produce long-lasting changes leading to neurodevelopmental disorders. Molecular Psychiatry (2013) 18, 1106–1118; doi:10.1038/mp.2012.128; published online 25 September 2012 Keywords: BDNF; neural development; neurodevelopmental disorders; serotonergic innervation; serotonin; Tph2 INTRODUCTION Serotonin (5-HT) is a neurotransmitter implicated in the modula- tion of numerous physiological processes including mood, sleep, aggressivity and sexual behavior. 1–4 Serotonergic neurons repre- sent one of the most early-born and widely distributed neuronal systems in the mammalian brain. These neurons collectively form the raphe system and cluster in distinct nuclei (B1-B9) providing a widespread serotonergic innervation to the whole central nervous system (CNS). 5,6 The synthesis of 5-HT and the expression of its receptors early in embryonic development, as well as its maternal and placental source to the fetus, has led to the hypothesis that serotonin could act as a growth regulator in specific develop- mental events. 7–10 Support for a role of 5-HT in brain development comes from the idea that dysregulation of serotonergic signaling is at the origin of disorders thought to have developmental bases, such as schizophrenia, affective disorders, anxiety, autism and mental retardation. 11–13 Genetic models in mice indicated that excess of brain 5-HT levels obtained by knocking-out the serotonin transporter (Slc6a4) or monoamine oxydase-A (MAO-A), genes involved in 5-HT re- uptake and degradation, respectively, prevents the development of topographically organized whisker-barrel fields in the mouse somatosensory cortex. 14,15 Further, recent findings showed that increased 5-HT transmission could also alter normal cortical development causing incorrect interneuron migration. 16 Taken together these observations indicated that normal 5-HT neurotransmission is required for a correct brain development. On the other hand, analysis of mouse models lacking most serotonergic neurons, obtained inactivating genes involved in the specification of 5-HT identity, such as Lmx1b and Pet1, failed to show brain malformations. 17,18 Recently, animal models with targeted inactivation of tryptophan hydroxylase 2 (Tph2), the rate- limiting enzyme for the synthesis of central 5-HT, have been generated. 19–23 In different genetic models, serotonin depletion consistently resulted in physiological and behavioral disturbances. 19,22–24 However, lack of serotonin in Tph2 knockout mouse lines did not produce any gross cellular or morphological alteration in the CNS. How can these results be reconciled with the developmental role ascribed to serotonin? On one side, maternal availability of serotonin during early fetal life to the developing brain may explain the absence of overt CNS morphological defects. 7,8 On the other, it can be hypothesized that lack of brain serotonin may selectively affect fine tuning modulation of developmental events, whose alterations would require subtle analysis at cellular level. In this regard, the present work aims to investigate whether constitutive depletion of brain serotonin might affect develop- ment of the CNS and/or the serotonergic system itself. To this purpose we genetically introduced the enhanced green fluores- cent protein (eGFP) into the Tph2 locus in a way to highlight the serotonergic neurons independently of serotonin immunoreac- tivity. Overall, we report that Tph2HeGFP À / À mice show a reduced body growth rate and a high mortality level and, most 1 Department of Biology, Unit of Cellular and Developmental Biology, University of Pisa, Pisa, Italy and 2 Department of Oncology, Sacro Cuore Hospital of Negrar, Verona, Italy. Correspondence: Professor M Pasqualetti, Unit of Cellular and Developmental Biology, University of Pisa, S.S.12 Abetone e Brennero 4, Pisa 56127, Italy. E-mail: mpasqualetti@biologia.unipi.it 3 These authors contributed equally to this work. Received 23 January 2012; revised 19 July 2012; accepted 23 July 2012; published online 25 September 2012 Molecular Psychiatry (2013) 18, 1106–1118 & 2013 Macmillan Publishers Limited All rights reserved 1359-4184/13 www.nature.com/mp