• fluoxetine
• medial prefrontal cortex
• perinatal treatment
• serotonergic fiber
• serotonin

Serotonin (5-HT) is a neurotransmitter synthesized by serotonergic neurons whose cell body is located in the rhombencephalon at the level of the raphe nuclei (B1-B9). From these nuclei originate axons that projected anteriorly to innervate the entire central nervous system (CNS) and caudally to the spinal cord. The serotonergic system is one of the earliest neuronal systems to develop. In mice serotonergic neurons start to differentiate at embryonic day 10.5 (E10.5), at E11.5 they begin to synthesize 5-HT, and subsequently the axons reach the target regions at the end of gestation and continue to develop in the postnatal period. This neurotransmitter is involved in several functions, as the regulation of different developmental and physiological processes including mood, sleep, aggression, appetite, and sexual behaviour.
In order to better study the serotonergic system, in our laboratory was generated the Tph2::eGFP knock-in mouse line, whose serotonergic neurons are labelled independently of serotonin immunoreactivity. Through the generation of Tph2 knock-out animals in which 5-HT synthesis is completely abrogated, it was possible to observe an alteration in serotonergic innervation in some anatomical areas of the CNS. In particular, areas of serotonergic hypo-innervation were identified, like the suprachiasmatic nucleus and the paraventricular nucleus of the thalamus, or areas of hyper-innervation such as the hippocampus and nucleus accumbens. Similar alterations have also been obtained using the conditional knock-out of Tph2, in which 5-HT depletion is induced in adulthood, thus highlighting a response of serotonergic circuitry following the alteration in serotonin levels.
In addition, proper innervation could be restored by recovery of serotonergic neurotransmission, thus demonstrating a high degree of the system plasticity. Subsequently in our lab was investigated the possibility of remodelling serotonergic circuitry following an increase in 5-HT levels in adult animals. To this aim, a pharmacological approach was used to maintain 5-HT fluctuation within peri-physiological levels. In particular, a specific inhibitor of serotonin reuptake, fluoxetine, one of the most widely used antidepressant drugs, was used. Thanks to these experiments, it was possible to demonstrate the possibility of remodelling the serotonergic system of adult mice even following small fluctuations in neurotransmitter level.
Among antidepressant drugs, fluoxetine is one of those most widely prescribed to prevent postpartum depression or to treat depression and anxiety even during pregnancy. However, it is known that this drug crosses the placental barrier and is released into breast milk. Based on this evidence, in our laboratory, Tph2::eGFP females were treated with fluoxetine throughout pregnancy and lactation, thus exposing the offspring to the drug during development and the period of maximum CNS plasticity.
Then it’s possible to investigate the effect caused by fluctuating 5-HT levels on behaviour and serotonergic innervations. This treatment caused an increase in depressive- and anxiety-like behaviours in adult offspring ("behavioural paradox") and a decrease in hippocampal serotonergic innervation density detectable starting at birth to adulthood.
With this Thesis work, I have studied the development of serotonergic innervation in the medial prefrontal cortex (mPFC) following perinatal fluoxetine exposure. The mPFC is a region structurally connected to the hippocampus and associated with anxiety-related disorders. The structure of the serotonergic system was analysed at postnatal day 7 (P7), P14 and P90 in two mice cohorts: the control cohort and the fluoxetine cohort, exposed to the drug throughout development.
First of all, after an immunohistochemistry, serotonergic fibers were acquired with confocal microscope at low magnification (10X) to assess the relative optical density (ROD) in all the layers of the mPFC.
Results showed that perinatal treatment with fluoxetine causes a reduction in serotonergic fibers density at P7 at the level of layers I and V-VI. In contrast, in the same layers at P14 and P90 we observed an increase in fibers density.
Subsequently, to understand the nature of the observed alterations, we analysed the morphological parameters of the serotonergic fibers. To this aim, confocal images of all different mPFC layers were acquired at a higher magnification (60X). The images of the serotonergic fibers were three-dimensionally reconstructed through the use of IMARIS software. The reconstructions results demonstrate that perinatal fluoxetine induce an hyperinnervation at P7 in all layers of the mPFC, in contrast, at P14 and P>90 causes an hyperinnervation in most layers of the mPFC.