Caratterizzazione della linea knock-in di topo FloxedTph2 come modello per indagare il ruolo della serotonina nel Sistema Nervoso Centrale

La serotonina, 5-idrossitriptamina (5-HT), è un neurotrasmettitore monoaminergico con una vasta distribuzione sia nei tessuti periferici che nel Sistema Nervoso Centrale (SNC) dei mammiferi. A livello del SNC i neuroni che sintetizzano la serotonina si originano precocemente durante lo sviluppo embrionale, 11 giorni di gestazione (days post coitum, dpc) nel topo, e vanno a costituire i nuclei del raphe. Nonostante il loro esiguo numero rispetto ai neuroni totali presenti nel cervello, i neuroni serotoninergici innervano l'intero SNC. In questi neuroni la serotonina è sintetizzata a partire dall'L-triptofano, grazie all'attività catalitica della triptofano idrossilasi 2 (Tph2), enzima limitante per la sintesi di 5-HT. In linea con la vasta distribuzione delle innervazioni serotoninergiche, la 5-HT è implicata nella regolazione di numerosi processi fisiologici come sonno, umore, stress, appetito e comportamento sessuale. Evidenze sperimentali sempre più numerose, tra cui l'importanza dell'apporto di 5-HT materna per il corretto sviluppo embrionale e la comparsa precoce dei recettori per la serotonina nell'embrione, corroborano l’ipotesi che la serotonina, ancora prima di svolgere la sua funzione di neurotrasmettitore, sia implicata nella regolazione di specifici processi durante lo sviluppo del SNC. In aggiunta molti dati suggeriscono che alterazioni della neurotrasmissione serotoninergica potrebbero essere alla base di patologie neuropsichiatriche con origine nello sviluppo come schizofrenia, autismo, disturbi emotivi, ansia, depressione e ritardo mentale. Grazie a modelli animali in cui la neurotrasmissione serotoninergica è alterata, è stato possibile osservare come il mantenimento dell'omeostasi della serotonina durante lo sviluppo embrionale sia essenziale per la corretta costituzione dei circuiti neuronali. Ad esempio, la generazione, nel nostro laboratorio, di una linea di topo knock-in (KI) Tph2::eGFP (Enhanced Green Fluorescent Protein), in cui la sintesi di 5-HT cerebrale è abrogata, ha permesso di dimostrare che la serotonina è necessaria per il corretto sviluppo delle fibre serotoninergiche. Al fine di dissezionare il ruolo della serotonina in distinte finestre temporali durante lo sviluppo del SNC e durante la vita adulta, nel nostro laboratorio è stata recentemente generata una linea knock-out (KO) condizionale per il gene Tph2, in cui il terzo esone di Tph2 è posto tra due siti loxP (FloxedTph2). Pertanto, incrociando questa linea con linee di topo che esprimono la ricombinasi Cre in maniera tempo- e spazio-specifica, è teoricamente possibile promuovere l'escissione del terzo esone che, in quanto composto da un numero di basi non multiplo di tre, causa la perdita della cornice di lettura del trascritto del gene Tph2 e la produzione di una proteina priva del dominio catalitico. Durante il mio internato di tesi, mi sono occupata della caratterizzazione della linea FloxedTph2 per verificare che possieda i requisiti necessari per essere utilizzata in esperimenti di knock-out (KO) condizionale. Per effettuare questo tipo di analisi ho incrociato topi FloxedTph2 con topi che esprimono la ricombinasi Cre sotto il controllo di un promotore ubiquitario ottenendo una linea, che è stata denominata “Tph2Null”, in cui il terzo esone di Tph2 è stato rimosso. I mutanti Tph2Null mostrano, rispetto ai wild-type (wt) ed ai Tph2Null +/-, un'aumentata mortalità nelle prime due settimane di vita, ed una visibile riduzione del tasso di crescita; questi aspetti del fenotipo sono in linea con quanto osservato in animali depleti di serotonina nel SNC. Esperimenti di immunoistochimica condotti su topi Tph2Null -/- hanno mostrato che la rimozione del terzo esone del gene Tph2 risulta in una drastica riduzione della quantità di serotonina sintetizzata dai neuroni del raphe, nonostante un’analisi approfondita riveli la presenza di un esiguo quantitativo di 5-HT residua. Questo potrebbe essere spiegato ammettendo la presenza di un enzima Tph2, la cui attività catalitica sia estremamente ridotta ma non totalmente abrogata. Al fine di indagare questa ipotesi i trascritti del gene Tph2 presenti nel raphe di topi Tph2Null -/- sono stati analizzati mediante un approccio di RT-PCR e successiva analisi di sequenza. È stata così rilevata la presenza di un'isoforma di splicing del trascritto del gene Tph2, priva sia del terzo sia del quarto esone, la quale potrebbe codificare per una proteina Tph2 con attività catalitica residua. Al fine di mettere in evidenza le fibre serotoninergiche nel topo FloxedTph2, ho generato la linea tras-eterozigote FloxedTph2/Tph2::eGPF +/- +/-. Questi animali consentono sia di ottenere una drastica riduzione tempo- e/o tessuto-specifica della sintesi di serotonina, sia di evidenziare i neuroni serotoninergici indipendentemente dall'immunoreattività della 5-HT. Per verificare se in topi FloxedTph2/Tph2::eGPF +/- +/- l'escissione del terzo esone del gene Tph2 risulta in una riduzione della sintesi di 5-HT analoga a quella osservata nei mutanti Tph2Null e se tale riduzione produce alterazioni nello sviluppo delle fibre serotoninergiche, ho generato la linea Tph2Null/Tph2::eGPF +/- +/-. In questi animali, nonostante la presenza di un quantitativo residuo di serotonina, si osserva un evidente aumento delle innervazioni serotoninergiche a livello dell'ippocampo, in linea con il fenotipo evidenziato in topi completamente privi di 5-HT cerebrale. L'insieme di queste evidenze ci indica che la riduzione della sintesi di serotonina centrale ottenuta in seguito alla rimozione del terzo esone del gene Tph2 è tale da produrre un fenotipo per molti aspetti in linea con quello dei KO convenzionali del gene Tph2 e ci suggerisce che la linea FloxedTph2 possa essere utilizzata in esperimenti di KO condizionale. Pertanto la linea FloxedTph2/Tph2::eGFP è stata impiegata in associazione con il sistema inducibile CMV-CreERT2/Tamoxifen per ottenere una drastica riduzione dei livelli di 5-HT centrale a stadi post natali ed in animali adulti. La messa a punto di questo sistema, consentirà di studiare se l'abrogazione della sintesi di serotonina in topi adulti, in cui i circuiti serotoninergici si sono già costituiti, comporti alterazioni a carico del fenotipo delle fibre serotoninergiche. In aggiunta, sarà possibile indagare a quali stadi durante lo sviluppo, l'apporto di un adeguato quantitativo di 5-HT neuronale sia essenziale per la corretta costituzione dei circuiti serotoninergici

Fluoxetine Induces Morphological Rearrangements of Serotonergic Fibers in the Hippocampus.

Serotonin (5-HT)-releasing fibers show substantial structural plasticity in response to genetically induced changes in 5-HT content. However, whether 5-HT fibers appear malleable also following clinically relevant variations in 5-HT levels that may occur throughout an individual's life has not been investigated. Here, using confocal imaging and 3D modeling analysis in Tph2GFP knock-in mice, we show that chronic administration of the antidepressant fluoxetine dramatically affects the morphology of 5-HT fibers innervating the dorsal and ventral hippocampus resulting in a reduced density of fibers. Importantly, GFP fluorescence levels appeared unaffected in the somata of both dorsal and median raphe 5-HT neurons, arguing against potential fluoxetine-mediated down-regulation of the Tph2 promoter driving GFP expression in the Tph2GFP mouse model. In keeping with this notion, mice bearing the pan-serotonergic driver Pet1-Cre partnered with a Cre-responsive tdTomato allele also showed similar morphological alterations in hippocampal 5-HT circuitry following chronic fluoxetine treatment. Moreover 5-HT fibers innervating the cortex showed proper density and no overt morphological disorganization, indicating that the reported fluoxetine-induced rearrangements were hippocampus specific. On the whole, these data suggest that 5-HT fibers are shaped in response to subtle changes of 5-HT homeostasis and may provide a structural basis by which antidepressants exert their therapeutic effect

Perturbation of Serotonin Homeostasis during Adulthood Affects Serotonergic Neuronal Circuitry

Growing evidence shows that the neurotransmitter serotonin (5-HT) modulates the fine-tuning of neuron development and the establishment of wiring patterns in the brain. However, whether serotonin is involved in the maintenance of neuronal circuitry in the adult brain remains elusive. Here, we use a Tph2(fl)°(x) conditional knockout (cKO) mouse line to assess the impact of serotonin depletion during adulthood on serotonergic system organization. Data show that the density of serotonergic fibers is increased in the hippocampus and decreased in the thalamic paraventricular nucleus (PVN) as a consequence of brain serotonin depletion. Strikingly, these defects are rescued following reestablishment of brain 5-HT signaling via administration of the serotonin precursor 5-hydroxytryptophan (5-HTP). Finally, 3D reconstruction of serotonergic fibers reveals that changes in serotonin homeostasis affect axonal branching complexity. These data demonstrate that maintaining proper serotonin homeostasis in the adult brain is crucial to preserve the correct serotonergic axonal wiring

Bioelectronic Medicine: a multidisciplinary roadmap from biophysics to precision therapies

Bioelectronic Medicine stands as an emerging field that rapidly evolves and offers distinctive clinical benefits, alongside unique challenges. It consists of the modulation of the nervous system by precise delivery of electrical current for the treatment of clinical conditions, such as post-stroke movement recovery or drug-resistant disorders. The unquestionable clinical impact of Bioelectronic Medicine is underscored by the successful translation to humans in the last decades, and the long list of preclinical studies. Given the emergency of accelerating the progress in new neuromodulation treatments (i.e., drug-resistant hypertension, autoimmune and degenerative diseases), collaboration between multiple fields is imperative. This work intends to foster multidisciplinary work and bring together different fields to provide the fundamental basis underlying Bioelectronic Medicine. In this review we will go from the biophysics of the cell membrane, which we consider the inner core of neuromodulation, to patient care. We will discuss the recently discovered mechanism of neurotransmission switching and how it will impact neuromodulation design, and we will provide an update on neuronal and glial basis in health and disease. The advances in biomedical technology have facilitated the collection of large amounts of data, thereby introducing new challenges in data analysis. We will discuss the current approaches and challenges in high throughput data analysis, encompassing big data, networks, artificial intelligence, and internet of things. Emphasis will be placed on understanding the electrochemical properties of neural interfaces, along with the integration of biocompatible and reliable materials and compliance with biomedical regulations for translational applications. Preclinical validation is foundational to the translational process, and we will discuss the critical aspects of such animal studies. Finally, we will focus on the patient point-of-care and challenges in neuromodulation as the ultimate goal of bioelectronic medicine. This review is a call to scientists from different fields to work together with a common endeavor: accelerate the decoding and modulation of the nervous system in a new era of therapeutic possibilities

Characterization and use of a Tph2 conditional knockout mouse line to investigate the role of serotonin in the adult CNS: unveiling the impact of serotonin homeostasis perturbation on serotonergic wiring

The neurotransmitter serotonin (5-hydroxytryptamnine, 5-HT) has been implied in the modulation of a plethora of physiological, cognitive and behavioral processes as well as in the regulation of specific morphogenetic events during neurodevelopment. In the last decades, human genetics and animal studies reveled the importance of appropriate serotonin homeostasis maintenance in the developing and in the adult brain, suggesting that perturbation of 5-HT physiological levels during critical temporal windows may contribute to the onset of neuropsychiatric disorders. In line, cumulating evidences have shown that an altered serotonergic neurotransmission during brain development could affect the establishment of neuronal wiring. However the precise role of serotonin in age-dependent activities is only beginning to be elucidated and whether perturbation of the proper brain level of 5-HT during adulthood could affect neuronal circuits remains enigmatic. In this context, the use of suitable genetic tools to allow a time-specific depletion of brain serotonin levels is crucial to further dissect the roles of 5-HT in developing and adult brains. During my PhD I validated a newly generated Tph2flox conditional knockout mouse line and I used it to investigate whether serotonin depletion in the adult brain impacts on serotonergic wiring. Results showed that the Tph2flox mouse line could be efficiently used to conditionally abrogate serotonin synthesis in a time-specific manner, thus representing a valuable tool to dissect the role of serotonin in age-dependent activities. Using this line, I showed that the density of serotonergic fibers was increased in the hippocampus and decreased in the thalamic paraventricular nucleus as a consequence of brain serotonin depletion during adulthood. Nevertheless, these innervation defects were reduced when brain serotonin signaling was restored by chronic treatment with the serotonin precursor 5-hydroxytryptophan. Finally, 3D computer-based analysis of serotonergic fiber morphology revealed that an altered 5-HT homeostasis affected the complexity of axonal branching. Overall, these data unveiled an unexpectedly high plasticity of the adult serotonergic system and demonstrated that a correct serotonin homeostasis is life-long required to preserve the proper serotonergic wiring of the brain

Imbalance of serotonin homeostasis during adulthood affects serotonergic neuronal circuitry

Serotonin (5-hydroxytryptamnine, 5-HT) is a monoaminergic neurotransmitter orchestrating a broad array of cognitive and behavioral processes in the adult brain. The early expression of its receptors during development and the requirement of maternal and placental sources of serotonin to the foetus have led to the hypothesis that 5-HT could act as growth regulator in the fine-tuning of specific morphogenetic events during neurodevelopment. Outcomes from genetic mouse models in which brain 5-HT homeostasis has been perturbed by targeting genes necessary for serotonin reuptake, metabolism or synthesis such as SERT, MAO-A and Tph2, respectively, support this hypothesis. However, evidence of a role for 5-HT in adulthood as a growth regulator or its requirement for maintenance of the proper neuronal circuitry, which is known to be susceptible to 5-HT imbalance during early postnatal stages, is still missing. To bridge this gap we used the Tph2 conditional knock-out (cKO) allele that allows an efficient abrogation of 5-HT synthesis in the adult brain, in combination with the Tph2::GFP allele, in which GFP reporter expression highlights 5-HT neuron fibers and somata. Beside previously reported data showing that the lack of brain serotonin in Tph2 knock-out (KO) mice deeply affects serotonergic circuitry development with a brain region-specific effect, the abrogation of 5-HT synthesis during adulthood produces alterations of serotonergic innervation in rostral brain targets matching those observed in mice with a life-long depletion of brain serotonin. Remarkably, we reported that restoring brain 5-HT signaling in both Tph2 KO and cKO mice by chronic administration of the serotonin precursor 5-hydroxytryptophan (5-HTP) results in a significant reduction in the extent of serotonergic fiber innervation defects, thus demonstrating an unexpectedly high degree of plasticity of the adult serotonergic system in response to changes of 5-HT homeostasis. Moreover, 3D computer-based analysis of serotonergic axon terminal morphology showed that imbalances in brain 5-HT content exert their growth regulatory activity on 5-HT axon terminals by promoting sprouting. Altogether these data demonstrate that a correct 5-HT homeostasis is life-long required to maintain the proper serotonergic innervation of specific rostral brain regions

Serotonin depletion affects serotonergic neuronal circuits in adult mice

Serotonin is a neurotransmitter synthesized in two steps with tryptophan hydroxylase 2 (Tph2) as the rate-limiting enzyme and it is implicated in the modulation of numerous physiological processes including mood, sleep, aggressivity and sexual behavior. Serotonergic neurons provide a profuse innervation to the whole CNS. The synthesis of serotonin and the expression of its receptors early in embryonic development, as well as its maternal and placental sources to the foetus, have led to the hypothesis that serotonin could act as a growth regulator in specific developmental events such as neurogenesis, neuronal migration and circuitry formation. However, the precise role of serotonin in specific morphogenetic activities during CNS development remains poorly understood. To address the consequences of time-controlled serotonin depletion on CNS development, we have generated a Tph2 conditional (floxed) allele and used it in combination with a Tph2-GFP knockin mouse line allowing the visualization of serotonergic neurons and fibers (Migliarini et al., 2013). Besides the severe abnormalities in the serotonergic circuitry formation observed in Tph2 knockout mice, serotonin depletion in adult animals induces an increase of the density of serotonergic fibers projecting to the hippocampus. These results show that the mature serotonergic system exhibits a previously unexpected plasticity and that appropriate serotonin homeostasis is crucial not only for proper development of the serotonergic neuronal circuit but also for its maintenance during adulthood. Migliarini et al. Mol Psychiatry 2013 18: 1106-18

SEROTONIN DEPLETION AFFECTS DEVELOPMENT AND MAINTENANCE OF SEROTONERGIC NEURONAL CIRCUITS

Serotonin is a neurotransmitter implicated in the modulation of several behavioral and physiological processes within the central nervous system (CNS) including mood, control of sleep, appetite, aggressivity and sexual behavior. In neurons serotonin is synthesized in two main steps with tryptophan hydroxylase 2 (Tph2) as the rate-limiting enzyme. Serotonergic neurons are one of the most early born neuronal systems in the mammalian brain and provide a widespread innervation to the whole CNS. The synthesis of serotonin during embryonic development together with a dynamic expression of serotonergic receptors in the CNS has led to the hypothesis that serotonin could act as a growth regulator in specific neurodevelopmental events such as neurogenesis, neuronal migration and circuitry formation. Although recent discoveries from animal models and human genetic studies have highlighted the importance of serotonin homeostasis maintenance during CNS development, the precise role of this molecule in specific morphogenetic activities remains poorly understood. To address the consequences of time-controlled serotonin depletion on CNS development, we have generated a Tph2 conditional (floxed) allele and used it in combination with a Tph2-GFP knockin mouse line allowing the visualization of serotonergic neurons and fibers1. We were able to demonstrate that abrogation of serotonin synthesis in adult mice affects the proper serotonergic wiring, thus indicating that the serotonergic system exhibits a previously unexpected plasticity in response to serotonin signaling. Our results together with previous observations suggest that appropriate serotonin homeostasis is crucial not only for proper development of the serotonergic neuronal circuit but also for its maintenance during adulthood

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

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, Tph2flox/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 Tph2null 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 Tph2null 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 Tph2null/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

Generation of Pet1210-Cre transgenic mouse line reveals non-serotonergic expression domains of Pet1 both in CNS and periphery

Neurons producing serotonin (5-hydroxytryptamine, 5-HT) constitute one of the most widely distributed neuronal networks in the mammalian central nervous system (CNS) and exhibit a profuse innervation throughout the CNS already at early stages of development. Serotonergic neuron specification is controlled by a combination of secreted molecules and transcription factors such as Shh, Fgf4/8, Nkx2.2, Lmx1b and Pet1. In the mouse, Pet1 mRNA expression appears between 10 and 11 days post coitum (dpc) in serotonergic post-mitotic precursors and persists in serotonergic neurons up to adulthood, where it promotes the expression of genes defining the mature serotonergic phenotype such as tryptophan hydroxylase 2 (Tph2) and serotonin transporter (SERT). Hence, the generation of genetic tools based on Pet1 specific expression represents a valuable approach to study the development and function of the serotonergic system. Here, we report the generation of a Pet1210-Cre transgenic mouse line in which the Cre recombinase is expressed under the control of a 210 kb fragment from the Pet1 genetic locus to ensure a reliable and faithful control of somatic recombination in Pet1 cell lineage. Besides Cremediated recombination accurately occurred in the serotonergic system as expected and according to previous studies, Pet1210-Cre transgenic mouse line allowed us to identify novel, so far uncharacterized, Pet1 expression domains. Indeed, we showed that in the raphe Pet1 is expressed also in a non-serotonergic neuronal population intermingled with Tph2-expressing cells and mostly localized in the B8 and B9 nuclei. Moreover, we detected Cre-mediated recombination also in the developing pancreas and in the ureteric bud derivatives of the kidney, where it reflected a specific Pet1 expression. Thus, Pet1210-Cre transgenic mouse line faithfully drives Cre-mediated recombination in all Pet1 expression domains representing a valuable tool to genetically manipulate serotonergic and non-serotonergic Pet1 cell lineages. © 2014 Pelosi et al