Generation of a Tph2 Conditional Knockout Mouse Line for Time- and Tissue-Specific Depletion of Brain Serotonin

Abstract

Serotonin has been gaining increasing attention during the last two decades due to the dual function of this monoamine as key regulator during critical developmental events and as neurotransmitter. Importantly, unbalanced serotonergic levels during critical temporal phases might contribute to the onset of neuropsychiatric disorders, such as schizophrenia and autism. Despite increasing evidences from both animal models and human genetic studies have underpinned the importance of serotonin homeostasis maintenance during central nervous system development and adulthood, the precise role of this molecule in time-specific activities is only beginning to be elucidated. Serotonin synthesis is a 2-step process, the first step of which is mediated by the rate-limiting activity of Tph enzymes, belonging to the family of aromatic amino acid hydroxylases and existing in two isoforms, Tph1 and Tph2, responsible for the production of peripheral and brain serotonin, respectively. In the present study, we generated and validated a conditional knockout mouse line, Tph2 flox/flox, in which brain serotonin can be effectively ablated with time specificity. We demonstrated that the Cre-mediated excision of the third exon of Tph2 gene results in the production of a Tph2 null allele in which we observed the near-complete loss of brain serotonin, as well as the growth defects and perinatal lethality observed in serotonin conventional knockouts. We also revealed that in mice harbouring the Tph2 null allele, but not in wild-types, two distinct Tph2 mRNA isoforms are present, namely Tph2Δ3 and Tph2Δ3Δ4, with the latter showing an in-frame deletion of amino acids 84–178 and coding a protein that could potentially retain non-negligible enzymatic activity. As we could not detect Tph1 expression in the raphe, we made the hypothesis that the Tph2Δ3Δ4 isoform can be at the origin of the residual, sub-threshold amount of serotonin detected in the brain of Tph2 null/null mice. Finally, we set up a tamoxifen administration protocol that allows an efficient, time-specific inactivation of brain serotonin synthesis. On the whole, we generated a suitable genetic tool to investigate how serotonin depletion impacts on time-specific events during central nervous system development and adulthood life.

Brainwide mapping of endogenous serotonergic transmission via chemogenetic-fMRI

“…Pgk-Neo/Kana (NEO) and Pgk-Difteric Toxin A cassettes (DTa) were used for positive and negative selection, respectively. PvuI-linearized pROSA26-1 Sor -LA-CAG-DIO-hM3Dq-NEO-RA-DTa was electroporated in E14Tg2a.4 embryonic stem cells (ESCs) as previously described (Pelosi et al, 2015). Positive recombinants identified by southern blot were microinjected in the host C57BL/6J blastocysts, which were injected in utero of pseudopregnant CD1 females (3.5 dpc) to give rise to chimeras (n=29).…”

Development of Serotonergic Fibers in the Post-Natal Mouse Brain

“…This ensures an easy and high fidelity visualization of the fine-grain anatomy of the whole 5-HT system, which already allowed us to study different aspects of 5-HT fiber dynamics, both in vitro and in vivo , as well as during mouse development and in adulthood (Migliarini et al, 2013; Pelosi et al, 2015; Pacini et al, 2017; Pratelli et al, 2017). We combined GFP immunofluorescence with confocal microscope imaging and 3D-reconstruction to quantitatively characterize the post-natal development of serotonergic fibers in selected brain regions.…”

Stress inhibits tryptophan hydroxylase expression in a rat model of depression

“…Biosynthesis of 5-HT is a two-step process; the first and rate-limiting step is conversion of the amino acid L-tryptophan into 5-hydroxytryptophan (5-HTP), which is catalyzed by tryptophan hydroxylase (TPH). Two TPH isoforms (TPH1 and TPH2) have been identified and share 71% homology in their protein sequence; The protein sequences of TPH1 and TPH2 differ at their N-terminal [ 11 ] and exhibit different spatial distribution patterns [ 12 ]. TPH1 is primarily located in a variety of non-neuronal cells, such as the enterochromaffin cells of the gut and the pineal gland [ 13 – 16 ], whereas TPH2 is predominantly expressed in the myenteric plexus [ 17 ] and in the serotonergic neurons of the raphe nuclei.…”