PI: Massimo Pasqualetti, massimo.pasqualetti@unipi.it

Institute, Department of Biology, University of Pisa (Pisa, Italy)

University delivering the PhD diploma: University of Pisa

Secondments: Institut du Cerveau et de la Moelle (Paris France), Light4Tech (Florence, Italy)

Description of project

Serotonergic neurons communicate through either volume or wiring transmission. These two modalities appear early in development, but it is unclear whether they are uniformly distributed in the brain, and whether these two transmission modalities regulate distinct neuronal targets and behaviours. Preliminary results suggest the hypothesis that wiring transmission may regulate specific brain activities. The project aims to draw a map of the serotonergic wiring transmission connectome and to assess the topographic organization of wiring transmitting serotonergic ascending projections to the rostral brain of mice expressing a fluorescent marker in serotonin neurons (Migliarini et al, Mol Psychiatry 2013) (3D analysis of transparent brains, clarity). Additionally, we will also identify the role of wiring transmitting serotonin neurons in controlling behaviour, and investigate the role of these neurons in the activation of specific target neurons. This will be done by using state-of-the-art rabies viral tracing, chemogenetics (Giorgi et al, Cell Reports 2017; Cavaccini et al, Neuron 2018), and two-photon calcium imaging tools.

Methodology

The project will use retrograde tracing technique based on recombinant rabies virus, and chemogenetic strategies to modulate neuronal activity. Neuroanatomical, imaging and behavioral analyses will be performed on transgenic mouse models.

PI: Alberto Bacci/Patricia Gaspar; alberto.bacci@icm-institute.org

Institute : Institut du Cerveau et de la Moelle (Paris France)

University delivering the PhD diploma : Sorbonne Uniiversité

Secondments  : University of Pisa (Italy) Microcontrol Instruments (UK)

Description of project 

A specific subtype of deep-layer pyramidal neurons of the prefrontal cortex (PFC), transiently expressing the serotonin transporter SERT, is specifically affected by perinatal fluoxetine (PNFLX) treatment (Soiza-Reilly et al. Mol Psychiatry. 2019 ; Olusakin et al Neuropsychopharmacol. 2020) . This treatment during a critical period results in PFC hypoexcitability as well as cognitive deficits and depressive-like behaviors in adult animals. The project aims to characterize these SERT+ PFC neurons in detail, their dense bidirectional connectivity with specific thalamic nuclei during development, and to characterize the PNFLX-mediated dysfunctional phenotypes. We will couple anatomical labeling with in vivo and in vitro electrophysiology and use bidirectional chemogenetic approaches to establish a causal link between the dysfunctional excitability of the SERT+ PFC neurons with the in vivo network hypo excitability, and the behavioral changes resulting from the PNFLX-mediated early-life insult.

Methodology 

The project will use in vivo and ex vivo electrophysiology, coupled to chemogenetic alteration of neuronal activity in transgenic mice. It will also involve anatomical and transcriptomic approaches during development.

PI : Nicolas Renier / Patricia Gaspar (nicolas.renier@icm-institute.org)

Institute : Paris Brain Institute (France)

University delivering the PhD diploma: Sorbonne Université

Secondments : La Vision Biotec (Miltenyi)

Description of project

The brain is densely perfused by the vascular network, which provides nutrients and oxygen to support neuronal function. The architecture of the cerebral vasculature addresses specific constrains to support neuronal functions, including the near absence of energy storage and a very high metabolic demand. In the cortex, the topology of the vascular network has region-specific specializations to support the different type of neuronal computations done in prefrontal, motor, sensory or integrative areas. While serotonin plays a crucial role in the organization of some axonal projections in the cortex, its role on the development of the vasculature is yet unknown. The main reason this fundamental question has not been elucidated is the lack of methodology to study the vascular topology at a large scale in 3D. The goal of this project will be first to develop cutting-edge methodologies to study the development of the organization of the cortical network. Then, the role of serotonin released by thalamocortical inputs in the layering of the vascular network will be tested. Finally, we will describe how early SSRIs exposure can affect its structure.

Methodology/ Techniques

This project will make use of neuroanatomy, tissue clearing, light sheet microscopy and image reconstruction, as well as in vivo genetic mouse models. It will involve testing and applying novel strategies for high resolution light sheet imaging.

PI: Klaus-Peter Lesch (kplesch@mail.uni-wuerzburg.de)

Institute: University Hospital Würzburg (Würzburg, Germany)

University delivering the PhD diploma: Graduate School of Life Sciences, University of Würzburg

Secondments: MDC (Germany), ASCENION (Germany)

 Description of project

Multiple lines of evidence link allelic variation of the mediators of serotonin (5-HT) system function to a wide spectrum of neuropsychiatric disorders characterized by compromised social cognition and emotion regulation. The project aims at generating iPSCs and iNSCs from human individuals with allelic variation of serotonergic genes. Transgene-free iPSCs will be generated with the help of non-integrative Sendai viruses. The classical Yamanaka-type reprogramming protocol will be used to induce multipotent neural stem cells (iNSCs) from fibroblasts. We will generate iNSC lines from fibroblasts of volunteer donors carrying allelic variants of serotonergic genes. In addition, isogenic control lines with serotonergic gene haploinsufficiency will be produced by CRISPR/Cas9 genome editing. Their abilities to differentiate in vitro to serotonergic neurons will be assessed using mRNA expression. To record electrophysiologic activity signatures of serotonergic neurons derived from multipotent precursors, 2D and 3D models will be used. These neurons will be co-cultured with glutamatergic and GABAergic cortical neurons to elucidate the effects of the genetic alterations in these cells on neurite outgrowth and axon pathfinding. The models will be validated by transcriptional profiling, histological and immunohistochemical and electrophysiological (MEA) analysis. These iPSC models will finally be compared with mouse models carrying corresponding genetic modifications.

 Methodology

The project will combine generation of iPSC models carrying human genetic variation, CRSPR-Cas9 genome editing and phenotypic comparison with corresponding genetically modified mouse models.

PIs: Natalia Alenina / Michael Bader (alenina@mdc-berlin.de / mbader@mdc-berlin.de)

Institute The Max Delbrück Center for Molecular Medicine (MDC, Berlin, Germany)

University delivering the PhD diploma CHARITÉ

University and graduate program: CHARITÉ and MDC Graduate School (https://www.mdc-berlin.de/de/node/71417)

Secondments:

METRIS (Netherlands)

Radboud University Medical Centre (Netherlands)

Description of project

We have previously shown that brain serotonin deficiency affects early postnatal growth and survival of the pups (Alenina et al. Proc Natl Acad Sci U S A. 2009;106:10332-7), and induces behavioural alterations, such as hyperactivity and social communication deficits, prominent in adolescent animals lacking brain serotonin (Mosienko et al. Mol Autism 2015;6:13). This project aims to elucidate the causal link between low pre- and postnatal serotonin levels and growth retardation, PFC development and behavioural alterations in early postnatal and adult life using a knockout rat model lacking brain serotonin synthesis, TPH2-KO rats (Brivio et al., Mol Neurosci. 2018;11:389).

We will monitor the growth rate and PFC development in male and female TPH2-KO rats across developmental stages (P7, 14, 21, 28, 35) using histological techniques and magnetic resonance imaging (MRI). Using MRI we will also assess the connectivity of the PFC with other brain regions. To determine if these alterations are of prenatal developmental origin, or are dependent on postnatal serotonergic signaling, we will treat animals postnatally with 5-hydroxytryptophan (5-HTP), which can be converted to serotonin in TPH2-KO rats. The effects of early postnatal serotonin deficiency and its complementation by 5-HTP will be studied in adult animals by analysis of marker gene expression, epigenetic signatures (Sbrini et al., Front Cell Neurosci. 2020;14:128) and behavior profiling. Moreover, to dissect the causes of growth retardation we will analyze the metabolic profile of blood and liver in TPH2-KO rats, measure the energy expenditure using a TSE Phenomaster system, assess hypothalamus-pituitary-adrenal axis function, evaluate functions of the enteric nervous system, and correlate these parameters to the growth rate.

Methodology/ Techniques

In this project we will combine molecular biological, histological, magnetic resonance imaging, metabolic, and behavioral approaches. The PFC-steered cognition will be assessed using touchscreen homecages in collaboration with METRIS Radboud University Medical Centre.

PI : Sharon Kolk/Judith Homberg (s.kolk@donders.ru.nl/ Judith.Homberg@radboudumc.nl)

Institute: Donders Center for Neuroscience, Radboud University Nijmegen (Nijmegen, The Netherlands)

University delivering the PhD diploma: Radboud University Nijmegen

Secondments: Lavision BioTec GmbH (Germany) and Institut du Cerveau et de la Moelle (Paris France)

Description of project

Prenatally, both high and low serotonin levels affect the outgrowth of raphe neurons to the PFC and cortical layering. It is unclear whether these changes persist throughout life, and how they relate to behaviour and cognition. It is also unclear to what extent the developmental consequences of lifelong high serotonin levels (5-HTT-KO) are different from those associated with lifelong low serotonin (TPH2-KO) levels. This project aims to causally delineate how lifelong increases and decreases in serotonin levels affect PFC microstructure, behaviour and cognition. Prefrontal migration of glutamatergic pyramidal neurons and serotonergic fiber innervation can be tested as well as PFC-steered behaviour. To causally link this, we will manipulate 5-Htt gene expression in the PFC using in utero electroporation. Studies can be extended by investigation of synapses and branching of axons using iDISCO and 3D light microscopy.

Methodology/ Techniques

ESR7 will use TPH2-KO (low serotonin) and 5-HTT-KO (high serotonin) rats and combine in utero electroporation-mediated 5-Htt gene knockdown within the PFC with immunostainings and PFC-related behavioural and cognitive tests.

PI: Trevor Sharp, trevor.sharp@pharm.ox.ac.uk

Co-PI: Simon Butt, simon.butt@dpag.ox.ac.uk

Institute: Department of Pharmacology and Department of Physiology, Anatomy and Genetics, University of Oxford

University delivering the PhD diploma: University of Oxford

Secondments: European Molecular Biology Laboratory, Rome; Microcontrol Instruments Ltd, UK

Description of project

Recent discoveries of serotonin-glutamate co-transmission offers the potential for a new perspective on the role of serotonin neurons in the developing and adult brain. This project will focus on the influence of serotonin and co-released glutamate on early cortical microcircuits, with mechanisms being characterized using pharmacological and genetic tools in conjunction with electrophysiology. A key aim is to understand how early life SSRI treatment alters the impact serotonin and glutamate co-transmission on cortical microcircuitry. This work will be complimented by studies of the effect of early life stress. The successful applicant will be trained in cutting-edge optogenetic approaches pioneered in the host lab. They will use these technologies to activate serotonin neurons and assess their effect on cortical circuits in both in vitro and in vivo preparations. The overall objective is to better understand the impact of serotonin signaling on early network activity in the context of normal and dysfunctional higher order cognitive function.

Methodology/ Techniques

Whole cell patch clamp electrophysiology combined with optogenetic-assisted circuit mapping, high density electrophysiological recordings, multiphoton imaging of early network activity.

PI: Cornelius Gross

Institute: European Molecular Biology Labratory

University delivering the PhD diploma: Ruprecht-Karls-Universitat Heidelberg (UHEI)

Secondments: Light4Tech SRL (Italy), Kobenhavns Universitet (Denmark)

Description of project

Increasing global activity of serotonin neurons during daily social defeat imparts resilience to the development of social avoidance behavior in mice, and serves as a model for understanding the action of antidepressants in stress resilience in humans. Our pilot data extend these studies by demonstrating that increasing serotonin levels selectively in PFC during daily social defeat confers resilience to social defeat. This project will use celltype specific KO of serotonin receptors coupled with in-vivo electrophysiology to map the PFC cell-types that mediate the neuromodulatory effects of serotonin on social defeat-induced avoidance behaviour and synaptic plasticity and then investigate how these circuits mature during  adolescence in males and females. We have shown that social defeat is associated with a weakening of PFC inputs to dorsal periaqueductal grey that suppress avoidance and this is driven by a weakening of glutamatergic PFC inputs from mediodorsal thalamus (MD) as measured by in-vivo evoked local field potential responses.

Methodology

Firstly, we will test the hypothesis that boosting serotonin in PFC blocks the weakening of MD-PFC synapses during defeat and promotes resilience ESR9 will be acquainted with calcium imaging at L4T, and thereafter will use fiber photometry calcium imaging of serotonin terminals in PFC to monitor serotonin activity and test the hypothesis that it is lowered during social defeat. We will use serotonin receptor (ant)agonists (WAY100635, ritanserin, psilocybin) to narrow down the responsible receptor and viral-mediated in vivo CRISPR/Cas9 KO of individual receptors in selected cell types (Camk2a::Cre, Sst::Cre, Pvalb::Cre) to identify the receptor/cell-type involved (serving as potential treatment targets). Subsequent ChR2-assisted circuit mapping using ex vivo electrophysiology will identify the precise serotonin-dependent synaptic plasticity changes following social defeat. Secondly, we will determine how PFC synaptic connections and their modulation by serotonin mature during adolescence. The capacity of PFC to inhibit brainstem targets first appears at this developmental stage and we hypothesize that serotonin modulation comes on board during this period. To measure this, we test the effects of serotonin receptor antagonists on executive functioning and depression-like behaviour. At SKU ESR9 will learn about 5-HT receptor PET imaging to validate data and translate them to humans.

PI: Tansu Celikel

Institute: Radboud University

University delivering the PhD diploma: Radboud University

Secondments: Light4Tech SRL (Italy), University of Oxford (UK)

Description of project

Computational modeling allows the simulation of behaviour and brain mechanisms through mathematic analyses, and brings the advantage of providing greater explanatory depth than experimental work and theories on how behaviour is brought about. Computational models also improve the translational prediction of animal data. We have previously developed a spiking neural network for the effects of genetically targeted or transient 5-HTT inactivation in early life in rodents on sensorimotor computation, and found based on an archived database of whisker-mediated sensory exploration74 that these conditions impairs the emergence of adaptive motor control of whisker position based on recent sensory information47. A computational circuit model of adaptive versus non-adaptive (uniform) whisking showed that inactivation of the communication between primary somatosensory and primary motor cortices impairs adaptive whisking sensorimotor control. A change in sensorimotor control influences various behaviours, as behaviour depends on the processing and response to sensory information. This project aims to extend this model with detailed (electro)physiological data on prefrontal cortical and raphe interactions from UNIPI (ESR2), UOXF (ESR8), EMBL (ESR9) and UMR (ESR11), with the ultimate goal to model the role of serotonin (as function of sex) in controlling sensitivity to changes in the environment (“environmental sensitivity”). During a secondment at L4T ESR10 will learn about calcium imaging for its correct implementation in the computational models. During a secondment at UOXF ESR10 will test the predictions made by the model.

Methodology

ESR10 will develop a spiking neural network model to bring together the observations on the structural and functional organization of the serotonergic circuits as studied by ESR2, 8, 9, and 11. This model is an extension of the computational model46 that mechanistically describes the role of serotonin in integration of sensory information for motor control, and in controlling sensitivity to change in the environment (“environmental sensitivity”). By extending the model with detailed (electro)physiological data on prefrontal cortical and raphe interactions from UNIPI, UOXF, EMBL and UMR, and two-photon imaging (with L4T), the computational model will infer expected phenotypes

associated with altered serotonergic signaling. During a secondment at UOXF, the model will be experimentally tested.

PI: Markus Wöhr; (markus.woehr@staff.uni-marburg.de)

Institute: Faculty of Psychology, Philipps-University Marburg, Germany

University delivering the PhD diploma: Philipps-University Marburg, Germany

Secondments: University Hospital Würzburg (Germany); Micro Control Instruments Ltd (UK)

Description of project

The serotonergic system is strongly involved in regulating a wide range of behaviors. This includes social behavior and communication (Mosienko et al., Molecular Autism, 2015). Moreover, serotonergic genetic factors influencing perinatal brain development strongly interact with environmental factors, in particular social living conditions. Such social factors can either have protective effects or they may increase the risk for psychopathologies (Braun et al., Human Molecular Genetics, 2019). This project aims at assessing alterations in brain functioning implicated in cognitive deficits with relevance to autism and other neuropsychiatric disorders in genetically modified rats deficient for the serotonin transporter (5-HTT) and the rate-limiting enzyme in the synthesis of neuronal serotonin (TPH2). This will be combined with a gene x environment (GxE) interaction approach that allows to test the influence of the social environment through socially enriched housing versus social isolation for their efficacy to ameliorate or aggravate such deficits.

Methodology

The project will use behavioral assays for measuring cognitive performance in combination with in vivo electrophysiology (secondment: Micro Control Instruments Ltd; UK) in genetically modified rats. Epigenetic consequences of GxE interactions will be studied in collaboration with the laboratory headed by Klaus-Peter Lesch (secondment: University Hospital Würzburg; Germany).

PI: Henning Tiemeier/Hanan El Marrou; (tiemeier@hsph.harvard.edu)

Institute: Erasmus Medical Center (Rotterdam, the Netherlands)

University delivering the PhD diploma: Erasmus University Rotterdam

Secondments: SKU Donders Institute for Brain, Cognition, and Behavior, or KU Copenhagen University Hospital

Description of project

Serotonin is an important neurotransmitter in the brain, involved in many different processes including mood, cognition, learning and memory. Animal models have shown that serotonin also plays an important role in the prenatal brain development including neuronal proliferation, differentiation, migration, and synaptogenesis. The primary aim of this PhD project is to better understand the role of serotonin in neurodevelopment in children and adolescents. The student will investigate the role of prenatal antidepressant exposure (SSRIs, selective serotonin reuptake inhibitors) a and the serotonin genotypes on different brain developmental outcomes. Dr. Hanan El Marroun (co-promotor) and Professor Henning Tiemeier (promoter) will jointly lead this project.

Methodology

The project will be embedded in the population-based Generation R Study, a population-based cohort from fetal life onwards. Information on behaviour and cognition, as well as neuroimaging has been repeatedly collected in over 5000 children.

PI : Vibe G. Frokjaer/Gitte M. Knudsen; (vibe@nru.dk)

Neurobiology Research Unit, Copenhagen University Hospital, Denmark.

University delivering the PhD diploma: University of Copenhagen.

Secondments: Universitaetsklinikum Wuerzburg (Germany), European College of Neuropsychopharmacology (The Netherlands).

Description of project

Environmentally-induced epigenetic modifications are potent drivers of adaptive changes in brain function and structure not only early in brain development but also later in life. Such processes may be key triggers of risk mechanisms for psychopathology, such as depression. Our work indicates that environment-dependent depressive responses are coupled to increased PFC 5-HTT binding (Frokjaer et al. Biol. Psychiatry 2015;78,534-43). This project aims to extend this finding and determine if environment-induced epigenetic modifications of the 5-HTT gene are associated with key pre- and postsynaptic markers of serotonergic neurotransmission and brain structure in the adult brain in a large cohort of male and female healthy individuals (n=628) (Knudsen et al. Neuroimage 2016;124,1213-9). This project also will assess whether epigenetic changes in serotonin signalling affect responsivity to antidepressant SSRI treatment in a depressed population followed from 0 to 12 weeks of treatment (n=100), (Kohler-Forsberg et al. Front Psychiatry. 2020 Jul 23;11:641). Correlation analyses between environmental factors, psychological health status, brain imaging data and 5-HTT epigenetic status will be performed.

Methodology/ Techniques

The project will apply statistical tools to use existing human data that combine molecular and structural brain imaging, questionnaire data on negative and positive environmental factors, mental health, and biomarkers. In particular, the project involves hands-on experience with determining gene transcript and DNA methylation of 5HTT related genes.

PI: Judith Homberg/Jan Buitelaar; (Judith.Homberg@radboudumc.nl/Jan.Buitelaar@radboudumc.nl)

Institute: Radboud University Medical Centre (Nijmegen, Netherlands)

University delivering the PhD diploma: Radboud University Medical Center

Secondments: Erasmus Medical Center (Netherlands), LIPIDOMIX (Germany), Autism Europe (Belgium)

Description of project

Beyond classical inheritance effects of serotonin genes, the serotonin genotype of the mother can influence offspring brain development and behaviour (Van der Knaap et al., J. Neurosci 2014). We hypothesize that the gut microbiome may play a role in the maternal effects. In this combined human cohort and rat experimental study you will investigate how the genotype of the mother affects prefrontal cortex and behavioural and cognitive correlates in offspring. In humans we make use of existing cohort data concerning parent and offspring genotype, behavioural and cognitive outcomes, and neuroimaging readouts. Furthermore, stool samples are available. In animals we make use of rats lacking the serotonin transporter, TPH1 and TPH2 (serotonin synthesizing enzymes), which will be subjected to neuroanatomical analyses, tests for behaviour and cognition, tryptophan and serotonin measurements in various biosamples and gut microbiome composition analyses.

Methodology

The project will combine statistical analyses and bioinformatics with animal experimental research employing behavioural and cognitive testing, and neuroanatomical and neurochemical analyses.

PI : Ronald Bulthuis

Company :METRIS B.V.

University delivering the PhD diploma: Radboudumc

Secondments : Radboudumc  (the Netherlands), Erasmus Medical Center (the Netherlands)

Description of project

To assess how serotonin-mediated changes in the development of the PFC affects cognition, cognition is ideally investigated during defined developmental time windows, such as the juvenile phase and adolescence. However, currently available methods to assess cognition in rodents require weeks to months of training, such that measurements can only be done during adulthood. This project aims to optimize a prototype touchscreen homecage system and to assess cognitive milestones in 5-HTT and TPH2-KO rats. METRIS has recently built in a touchscreen into their automated Laboras homecage system. ESR15 will work with METRIS to upgrade the software allowing synchronization of behavioural and cognitive data. When developed, ESR15 will set-up a protocol allowing young animals to acquire cognitive tasks most optimal. The protocol will be validated in serotonergic knockout rat models, and compared to cognitive measures of children genotyped for serotonergic polymorphisms in the Generation R cohort (secondment EMC).

Methodology/ Techniques

This project combines engineering and computer science (e.g. programming) with behavioural neuroscience.