Role of serotonin 3a receptor in early life stress and amygdala-medial prefrontal cortex networks during fear processing

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Thesis

Reference

Role of serotonin 3a receptor in early life stress and amygdala-medial prefrontal cortex networks during fear processing

ZEWDIE WONDIMU, Seblewongel

Abstract

In humans and rodents, the serotonin system interacts with early-life stress (ELS) through its various receptors, such as serotonin 3A receptor (HTR3A). Htr3a is exclusively expressed in a subset of interneurons (IN), where it has been shown to play a role in the establishment of developmental processes of cortical and subcortical circuit. Alterations in Htr3a impairs IN-driven homeostasis, leading to E-I imbalance, and may result in maladaptive anxiety-like behaviours. In rodents, deletion of the Htr3a leads to increased fear during cued fear memory retrieval and reduced attenuation during fear extinction, whereas its role in anxiety is equivocal. In my PhD project, I studied the role of HTR3A in ELS using genetic and epigenetic methods in humans. I also studied the role of its deletion on anxiety-like phenotypes and ELS in mice at a behavioural level, and further assessed its role in cued fear memory retrieval at a functional level.

ZEWDIE WONDIMU, Seblewongel. Role of serotonin 3a receptor in early life stress and amygdala-medial prefrontal cortex networks during fear processing. Thèse de doctorat : Univ. Genève et Lausanne, 2020, no. Neur. 289

DOI : 10.13097/archive-ouverte/unige:150398 URN : urn:nbn:ch:unige-1503986

Available at:

http://archive-ouverte.unige.ch/unige:150398

Disclaimer: layout of this document may differ from the published version.

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DOCTORAT EN NEUROSCIENCES des Universités de Genève

et de Lausanne

UNIVERSITÉ DE GENÈVE FACULTÉ DES PSYCHOLOGIE

Professeur Alexandre Dayer, directeur de thèse Docteur Charles Quaireaux, co-directeur de thèse Professeur Denis Jabaudon, co-directeur de thèse

TITRE DE LA THÈSE

ROLE OF SEROTONIN 3A RECEPTOR IN EARLY LIFE STRESS AND AMYGDALA-MEDIAL PREFRONTAL CORTEX NETWORKS DURING FEAR

PROCESSING THÈSE Présentée à la Faculté de Médecine de l’Université de Genève

pour obtenir le grade de Docteure en Neurosciences

par

Seblewongel Zewdie WONDIMU D’Ethiopie

Thèse N° 289 Genève

Editeur ou imprimeur : Université de Genève 2020

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Acknowledgement

‘If you’re walking down the right path and you’re willing to keep walking, eventually you’ll make progress’. Barack Obama

This quote sums up my journey to get to this point. After years of working towards it, reality only struck me when I opened a blank page to reflect, not on the thesis, but on what really made it happen. So many people have supported me and guided me through the ups and downs of my thesis. I believe most students would agree with me when I say that we all fantasise about reaching the end of our PhD and write notes of gratitude to those who supported us. This makes writing this section emotional and bitter sweet as one of the people who helped me the most and I want to give thanks to is no longer with us.

This thesis would not have been possible without the unrelenting support from my supervisor, Prof. Alexandre Dayer. I would like to thank him for giving me the opportunity to pursue a PhD in his lab and for his invaluable guidance, for always being there to answer my questions and for all the support he has provided me throughout my PhD. May you rest in peace, Alex.

Thank you for Profs. Camilla Bellone, Anthony Holtmaat, and Johannes Graff for being part of the thesis jury and Denis Jabaudon for taking me to the finish line.

I am thankful to Charles for all his support throughout the years and for teaching me how to perform electrophysiological experiments. Thank you for always challenging my thinking, for your encouragement and unwavering support.

Thanks also to Greta for your friendship and emotional and scientific support though out my PhD career. Thank you for reviewing my thesis manuscript and for keeping me on the right track when I doubted myself. Everyone should have a friend like you!

Thanks to Ludwig, who at the start of my PhD career held my hand and thought me so much with his never-ending patience and passion. Thank you to Alain Malafosse for giving me my first break to joining his lab and set me on this path. Thanks also to Prof. Corinne Charbonnel for mentoring me and for giving me the confidence to complete my thesis.

Thank you for all the members of the Dayer lab, past and present, for making my time in the lab a memorable one. To Ugo, thank you for your genuine friendship, emotional support and for the many interesting discussions, some of which are otherworldly. To Aude, for your friendship and emotional support throughout my PhD. Your passion towards your work has

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influenced me. To Christelle, your smile and upbeat personality made my time in the lab enjoyable. To Wafae and Julien, we have travelled longer together and thank you for the support you have given me. To Mathieu and Nico for all your help, advice and for always making me laugh. To Giuliana for all your help and advice with pupilometry and fear conditioning. To Marta for your friendship and support. To Lucia, Joan, and Aurelie for making the lab a fun place. Also to Esther and Subashika for your friendship and emotional support.

Thanks to Carmen Sandi and Jocelyn Grosse for all the help with the behavioural experiments and to Thomas Stefanelli for his help with fear conditioning. Thanks to Arianne Giacobino and Christelle Stauder for helping me with pyrosequencing experiments. Thanks also to Nicolas Liaudet for his help with pupilometry data analyses, and to Gwenael Birot for his support in LFP data analyses and for always being present to answer my questions and give me a helping hand; to Sebastien Pellat for his technical support.

Thanks to the member of the Michel lab, past and present. To Laurent and Florian for helping me with LFP recordings and analyses early on and to Guru and Abbas for your advice. Thanks to members of the Huber lab (Karin, Claudia, Ozge, Gregorio, Ali, and Daniel) for their technical and emotional support throughout my PhD and for keeping me company during late night experiments when Aurora was still living inside of me.

Of course, a big thank you to my mother, father and sister who have given me love and support and for instilling in me the desire to pursue a PhD and to never shy away from challenging situations. An immense thank you to my dearest friend and honorary-sister Betè Yilma to all that you have done for me. This journey would have been tougher without you.

Most of all, I would like to give special thanks to my dear husband who gave me the confidence and emotional support throughout my PhD. I would not have done it without you, Ståle. To my beautiful daughter, Aurora Abyssinia, you have been a shining star in my life. You give me so much love and stamina to keep me going. I hope you’ll be proud of your mommy one day!

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“Derrière le bleu de ce ciel cristallin cherche encore parmi les gravats la fraîcheur de l’anguille la célérité du crabe ce temps où tout restait à naître

ô matin clair de nos vies » Alexandre Dayer, Dans cet espace entre ciel et rêve, juin 2020!

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Abstract

In humans and rodents, the serotonin system interacts with early-life stress (ELS) through its various receptors, such as serotonin 3A receptor (HTR3A). Single nucleotide polymorphisms (SNPs) and cytocine-guanine dinucleotide (CpG) methylation in the regulatory region of the HTR3A are associated with depression and anxiety disorders as well as with altered activity in frontal cortices after ELS. Htr3a is exclusively expressed in a subset of interneurons (IN), where it has been shown to play a role in the establishment of developmental processes of cortical and subcortical circuit. Alterations in Htr3a impairs IN-driven homeostasis, leading to E-I imbalance, and may result in maladaptive anxiety-like behaviours. The effect of ELS on HTR3A through epigenetic mechanisms, may contribute to such imbalance. In rodents, deletion of the Htr3a leads to increased fear during cued fear memory retrieval and reduced attenuation during fear extinction, whereas its role in anxiety is equivocal. In my PhD project, I studied the role of HTR3A in ELS using genetic and epigenetic methods in humans. I also studied the role of its deletion on anxiety-like phenotypes and ELS in mice at a behavioural level, and further assessed its role in cued fear memory retrieval at a functional level.

In the first part of this study, I aimed to identify the interaction between childhood maltreatment, severity of psychiatric disorders and HTR3A genetic and epigenetic alterations. Using a clinical cohort consisting of borderline personality disorder (BPD), attention deficit hyperactivity disorder (ADHD) and bipolar disorder (BP) patients, I showed that different subscales of ELS differentially influenced epigenetic changes, which in turn were associated with a diverse range of clinical outcome. Furthermore, a functional SNP rs1062613 was associated with the methylation of a neighbouring CpG site, which in turn was influenced by childhood physical abuse. Overall, these results suggest that ELS interacts with HTR3A through genetic and epigenetic mechanisms, which might contribute to the heterogeneity of clinical outcomes observed.

In the second part of my thesis, I studied the role of Htr3a in anxiety related-behaviours and its interaction with ELS in a mouse model. Here, I showed that constitutive deletion of the Htr3a led to anxiolytic phenotype in certain conditions but not in others, adding to the already equivocal evidence in the literature. Furthermore, Htr3a-ko mice with ELS exposure showed increased aggressive behaviour, showing that Htr3a mediates ELS-related behavioural outcomes in mouse.

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In the third and final part of my thesis, I investigated the role of Htr3a in fear memory retrieval.

I found that Htr3a-ko mice showed a protracted attenuation of fear-induced freezing during cued fear memory retrieval, while wild-types attenuated fear-induced freezing after the first 3 presentations of the conditioned stimulus (CS). This delay in attenuation of fear during fear memory retrieval could be linked to the impaired extinction behaviour previously described in these mice. In my data, such behavioural observations were accompanied by deficit in fear- induced theta power during cued fear memory retrieval in prelimbic (PrL), infralimbic (IL), and basolateral amygdala (BLA) and by deficit in medial prefrontal cortex (mPFC)-BLA synchronisation. Furthermore, within the BLA, theta modulation of gamma power, previously reported as enhanced during fear, was impaired in the Htr3a-ko. Overall, these results indicate that Htr3a may be necessary for maintaining proper network activity in the mPFC-BLA circuit and facilitating effective attenuation of fear memory.

Taken together, findings in this PhD proposes a possible role of HTR3A in effective network synchronisation facilitating fear memory reduction and that its dynamic interaction with ELS and clinical outcomes may underscore deficits in maintaining such network balance.

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Résumé

Chez l'homme et le rongeur, le système sérotoninergique est impliqué dans le syndrome de stress précoce (SSP) via divers récepteurs tels que le récepteur de la sérotonine 3A (HTR3A).

Des polymorphismes mononucléotidiques et la méthylation du dinucléotide cytosine-guanine dans la région régulatrice du gène Htr3a ont été associés à la dépression et aux troubles anxieux ainsi qu'à l’altération de l’activité des cortex frontaux dans le syndrome de stress précoce. Le gène Htr3a s’exprime exclusivement dans un sous-ensemble d'interneurones pour lesquels son rôle dans les processus de développement de leurs circuits corticaux et sous-corticaux a été démontré. Les altérations du gène Htr3a perturbent l'homéostasie des réseaux normalement régulée par les interneurones, ce qui déséquilibre l’équilibre excitation- inhibition (E-I) et peut entraîner une augmentation des comportements anxieux inadaptés. En agissant sur le gène Htr3a par des mécanismes épigénétiques, le stress précoce pourrait contribuer à un tel déséquilibre. Chez les rongeurs, la suppression du gène Htr3a entraîne une augmentation de l’expression de la peur induite par un stimulus conditionnel mémorisé et diminue l’atténuation des comportements de peur par les protocoles d’extinction. Le rôle de ce gène dans l'anxiété est cependant plus équivoque. Pendant mon projet de doctorat, j'ai étudié le rôle du gène HTR3A dans le syndrome du stress précoce en utilisant des méthodes d’analyses génétiques et épigénétiques chez l'homme. J’ai également étudié au niveau comportemental le rôle de la suppression de ce gène sur les phénotypes anxieux et dans le syndrome de stress prénatal chez la souris. Au niveau fonctionnel, j’ai ensuite évalué son rôle dans l’expression de la peur induites par des stimulus conditionnels mémorisés.

Dans la première partie de cette étude, j'ai cherché à identifier l'interaction entre la maltraitance infantile, la gravité des troubles psychiatriques et les altérations génétiques et épigénétiques du gène HTR3A. En utilisant une cohorte clinique composée de patients atteints de troubles de la personnalité borderline, de trouble du déficit de l’attention avec d'hyperactivité (TDAH) et de troubles bipolaires (TA), j'ai montré que les niveaux de stress précoce influencent l’importance des changements épigénétiques, qui sont à leur tour associés à des troubles cliniques de sévérité grandissante. De plus, le polymorphisme fonctionnel rs1062613 est associé à la méthylation d'un site cytosine-guanine voisin, qui à son tour est influencé par la soumission à des violences infantiles. Dans l'ensemble, ces résultats suggèrent que le stress précoce interagit avec le gène HTR3A par voie génétique et par des mécanismes épigénétiques, et que la variabilité de ces derniers pourrait contribuer à

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Dans la deuxième partie de ma thèse, j'ai étudié le rôle du récepteurs Htr3a dans les comportements liés à l'anxiété et son interaction avec le stress précoce dans un modèle de souris. J'ai pu démontré que la suppression constitutive du récepteur Htr3a conduit à un phénotype anxiolytique dans certaines conditions mais pas dans d'autres, ce qui va dans le sens des observations équivoques rapportées dans la littérature. En outre, les souris Htr3a- ko exposées à un stress précoce montrent une augmentation du comportement agressif, suggérant que le récepteur Htr3a influence le comportement chez la souris.

Dans la troisième et dernière partie de ma thèse, j'ai étudié le rôle du récepteur Htr3a dans la récupération de la mémoire associée à la peur. J'ai constaté que les souris Htr3a-ko n’atténuait pas efficacement l’expression de la peur lorsque le stimulus conditionnel était répété plusieurs fois sans association avec le stimulus inconditionnel douloureux. Chez les souris contrôles en revanche, l’expression de la peur induite par le stimulus conditionnel diminue fortement après seulement trois présentations non associées au stimulus inconditionnel. Ce retard dans l'atténuation de la peur mémorisée pourrait être liée au comportement d'extinction qui a été précédemment décrit comme étant altéré chez ces souris knockout. J’ai pu montrer également que ces modifications comportementales étaient accompagnées d'un déficit dans l’augmentation de l’activité thêta des neurones normalement associée avec l’expression de la peur induite par le stimulus conditionnel mémorisé dans le cortex pré-limbique (PrL) et infralimbique (IL) et dans l’amygdale basolatérale (BLA). De plus, on observe un déficit de la synchronisation des activités du cortex préfrontal médian (mPFC) et de la BLA. Enfin, dans la BLA, la modulation de la puissance du gamma par thêta, qui a été rapportée comme étant augmentée pendant l’expression de la peur dans la littérature, est altérée chez les souris Htr3a-ko. Dans l'ensemble, ces résultats indiquent que le récepteur Htr3a est nécessaire pour maintenir une activité des réseaux neuronaux appropriée dans les circuits mPFC-BLA et pour faciliter l’atténuation efficace de la mémoire de la peur.

Pour conclure, les résultats de cette thèse proposent un rôle possible du récepteur Htr3a dans la synchronisation de l’activité des réseaux de neurones, facilitant l’atténuation de la peur induite par les stimulus associés, et suggèrent que l’interaction dynamique du gène de ce récepteur avec le stress précoce et les troubles cliniques serait la conséquence de déficits dans le maintien de l’équilibre de ces réseaux.

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Abbreviations

5HT Serotonin 5HTT 5HT transporter

AADC Aromatic L-amino acid decarboxylase Ach Acetylcholine

ADHD Attention deficit hyperactivity disorder AHP Hyperpolarizing phase

AP Action potential AuC Auditory cortex BA Basal amygdala BLA Basolateral amygdala BNST stria terminalis BP Bipolar disorder

BPD Borderline personality disorder CA1 Cornu ammonis 1

Ca2 Calcium

CCK Cholecystokinin CEA Central amygdala CEl Lateral CEl

CEm Medial CEA

c-fos Fos proto-oncogene CGE Caudal ganglionic eminence ChAT Choline Acetyltransferase CNS Central nervous system

CoupTFII COUP transcription factor 2 (NR2F2) CP Cortical plate

CpG Cytocine-guanine dinucleotiide CR Conditioned response

CRF Corticotropin-releasing factor CRH corticotropin-releasing hormone

CRHR1 Corticotropin Releasing Hormone Recep. 1 CS Conditioned stimulus

CSF Cerebro spinal fluid CTCF CCCTC-binding factor

CXCL (12) C-X-C Motif Chemokine Ligand (12) CXCR (4/7) Chemokine (C-X-C Motif) Receptor (4/7) Dbx1 Developing brain homeobox 1

dCGE Dorsal CGE DG Dentate gyrus dHPC Dorsal Hippocampus

Dlx (1) Distal-less homeobox gene (1) dMGE Dorsal MGE

DR Dorsal raphe DRL Dorsolateral raphe DRM Dorsomedial raphe

E (10) Embryonic (10)

EBS Electrical Burst Stimulation EEG Electroencephalogram E-I Exitatory-inhibitory ELS Early life stress En Engrailed

eNGC Elongated neuroglia form cells EPM Elevated plus maze

eQTLs Expression quantitative trait loci ERP Event related potential

Fezf2 FEZ Family Zinc Finger 2 Fgf8 Fibroblast Growth Factor 8 Foxa2 Forkhead Box A2

G Guanine

G x E Gene-enviromnment interaction GABA Gamma-Aminobutyric acid GAD65 Glutamate Decarboxylase 2 GAP43 Growth Associated Protein 43 Gata2 GATA Binding Protein 2 GE Ganglionic eminence GluR Glutamate receptor

GREs Glucocorticoid response element GTPase Guanosine triphosphate hydrolase GW Gestational week

H (3) Histone 3

Hip Hippocampus

Hmx3 Homeobox coding protein NKX5.1

HPC Hippocampus

HTR3A Serotonin 3a receptor HTR6 Serotonin 6 receptor

HYP Hypothalamus

IL Infralimbic IN Interneurons

IPC Intermediate progenitors

IPSCs Inhibitory post-sypnaptic currents IS Irregular spiking

ITC Intercalated cell masses IZ Intermediate zone

K Potasium

KCC2 K+/Cl- cotransporter 2

ko Knockout

L (1) Layer (1)

Lamp5 Lysosomal Associated Membrane Protein LFP Local field potential

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Lhx6 LIM Homeobox 6 Lhx6 LIM homeodomain (2)

Lmx1b LIM Homeobox Transcription Factor 1 Beta LS Late spiking

LTP Long term potentiation MAOA Monoamine oxidase A MAOB Monoamine oxidase B MBA Basal medial amygdala MC Motor cortex

MEIS2 Myeloid Ecotropic Viral Integration Site..

MGE Medial ganglionic eminence mPFC medial prefrontal cortex MRN Medial raphe neurons

MRS Maternal restraint and separation MS maternal separation

MWAS Methylome wide association

MZ Marginal zone

Na Sodium

nAChR Nicotinic acetylcholine receptors, Narp Neuronal activity–regulated pentraxin NDNF Neuron Derived Neurotrophic Factor NGC Neuroglia form cells

Nkx2.1 Thyroid transcription factor 1 (TTF-1) nNOS1 Nitric oxide synthase 1

NOS Nitric Oxide Synthase NPTX2 Neuronal Pentraxin 2 NPY Neuropeptide Y NR2B NMDAR2B

Nr2f2 Nuclear Receptor Subfamily 2 Group 2 NRP2 Neuropilin 2

OE Over expression

OF Open field

OHDA hydroxydopamine

OR Odd ratio

P(21) Postnatal day 21 PAG Periaqueductal grey PCPA Para-chlorophenylalanine Pdchα Protocadherinα

Pet1 FEV Transcription Factor, ETS Family Phox2b Paired Like Homeobox 2B

Pir piriform cortex PN Projection neurons

PNN Perineuronal net POA Preoptic area PoN Preoptic nucleus

PP Preplate

PrL Prelimbic

Prox1 Prospero homeobox 1 PTC1 Type 2C protein phosphatase PTSD Post traumatic stress disorder

PV Parvalbumin

R (1) Rhombomere (1) RGC Radial glial cells RS Restraint stress

SCR Skin conductance response SEMA Semaphorin

Serpinf1 Serpin Family F Member 1 SERT Serotonin transorter Shh Sonic hedgehog

Sncg Synuclein, Gamma (Breast Cancer-Specific Protein 1) SNP Single nucleotide polymorphism

SOM Somatostatin

Sox6 SRY-Box Transcription Factor 6

SP Subplate

Sp8 Specificity protein 8

SSRIs Serotonin reuptake inhibitor SST Somatostatin

T Thymine

Tatb1 Sec-independent protein translocase protein TatB TF Transcription factors

TPH Tryptophan hydroxylase TSS Transcriptional start site US Unconditioned stimulus VC Visual cortex

Vgut3 Vesicular glutamate transporter 3 VIP Vasoactive Intestinal Peptide vlPAG Ventrolateral PAG

VMAT2 vesicular monoamine transporter 2 VTA Ventral tegmental area

VZ Ventricular zone WM White matter

WT Wild-type

α7 Alpha 7

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List of figures

Introduction

Figure 1. Genetic codes controlling the development and specification of INs... 23!

Figure 2. Molecular diversity of interneurons. ... 27!

Figure 3. Diversity and classification of neocortical INs ... 28!

Figure 4. L1 HTR3A+ IN subtypes. ... 33!

Figure 5. Embryonic origin and morphological diversity of cortical IN. ... 35!

Figure 6. Direct or indirect GABAergic IN inhibition of PNs. ... 38!

Figure 7. Critical period is characterised by sequential plasticity in the mouse brain. ... 39!

Figure 8. Changes in the E/I balance during stress and development. ... 42!

Figure 9. Placental contribution of 5HT to the foetal brain. ... 45!

Figure 10. Development of raphe 5HT neurons from specification to guidance. ... 49!

Figure 11. Distribution of 5HT neuronal types in the raphe and their projections. ... 52!

Figure 12. Distribution of Htr3a-GFP+ INs in the mouse brain at P21... 56!

Figure 13. Autonomic fear responses ... 57!

Figure 14. Micro and macro circuits involved in acquisition of fear memory. ... 63!

Figure 15. Fear expression and extinction involve distinct but overlapping networks. ... 65!

Figure 16. Macro-circuits involved in anxiety states. ... 67

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Results

Figure 17. ELS affects body weight in both WT and Htr3a-ko. ... 96!

Figure 18. Htr3a-ko mice show anxiolytic behaviour open-field anxiety test ... 99!

Figure 19. Htr3a-ko mice showed increased locomotion but unaltered anxiety-like behaviour on elevated plus maze ... 101!

Figure 20. Htr3a-ko mice with or without early life stress do not differ in social preference test ... 102!

Figure 21. Htr3a-ko mice with early life stress show increased aggressive behaviour ... 104!

Figure 22. : Fear acquisition and retrieval in freely moving WT and Htr3a-ko mice ... 106!

Figure 23. Conditional Stimulus (CS)-evoked pupil response in WT... 108!

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Figure 24. Fear conditioning increases pip-induced local field potential (LFP) in control but not in Htr3a-ko ... 110!

Figure 25. Deficit of the theta (4-12Hz) power increase during retrieval in Htr3a-ko. ... 112!

Figure 26. Gamma (40-100Hz) power did not significantly increase during fear memory retrieval in WT and Htr3a-ko mice ... 113!

Figure 27. WTs and Htr3a-ko showed no difference in Prelimbic cortex-Basolateral Amygdala (PrL-BLA) theta (4-12Hz) amplitude coherence increase during fear memory retrieval. .... 115!

Figure 28. Medial prefrontal cortex-Basolateral amygdala (mPFC-BLA) theta (4-12Hz) phase- locking during fear memory retrieval is impaired in Htr3a-ko mice ... 117!

Figure 29. Basolateral amygdala (BLA) theta-fast-gamma coupling increase during retrieval in WT but not in Htr3a-ko. ... 118!

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Introduction

The cerebral cortex is a complex structure that plays an important role in higher cognitive functions, such as language, attention, and emotions. The cortex is composed of a wide diversity of cell types, such as neurons, astrocytes and oligodendrocytes. Cortical neurons can be broadly categorised into glutamatergic excitatory projection neurons (PNs) and GABAergic interneurons (INs) that generate a functional network by releasing glutamate and ϒ-aminobutyric acid (GABA), respectively. INs play a pivotal role in inhibitory control of PNs, thus maintaining cortical circuit homeostasis. Dysfunctions in the cortical inhibitory control has been associated with several neuropsychiatric disorders. The serotonin 3a receptor (HTR3A) is expressed in a portion of INs that provide direct and indirect inhibitory control of PNs, thus contributing to serotonin (5-hydroxytryptamine or 5HT) signalling and mediating cross-talk between different regions. Furthermore, Htr3a expression has been shown to be modulated by epigenetic factors as well as single nucleotide polymorphisms (SNPs), and has been shown to interact with ELS.

My thesis work aimed at investigating the functional and behavioural role of this receptor with a translational outlook. To this end, the first part of this work focused on the epigenetic effects of early-life adversity on HTR3A in a clinical cohort. The second part of my work aimed to assess the effect of Htr3a deletion in anxiety-related behaviours and its interaction with early life stress in mice. The third part this work utilised intra-cortical local field potential (LFP) and pupil recordings in head-fixed mice to understand the functional consequence of Htr3a deletion in medial prefrontal cortex (mPFC) – amygdala network during cued fear memory retrieval.

1. Generation of cortical neuron diversity

PNs and INs are generated in discrete germinal zones, from where they migrate out to reach and populate the different structures of the forebrain. In the neocortex, they organise into layers and columns, where they interconnect to each other to form intricately linked networks (Rojas-Piloni, Guest et al. 2017, Lin, Wang et al. 2018, Jiang, Guan et al. 2020). PNs within each cortical layer display similar features and pattern of connectivity, while diverse subtypes of INs distribute throughout the layers to partner with other INs or PNs (Tremblay, Lee et al.

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2016, Lim, Mi et al. 2018). Neurons within each column are stereotypically interconnected and share extrinsic connectivity that allows them to function as a basic unit underlying cortical operations (Mountcastle 1997, Jones and Rakic 2010). Thus, the balance between PNs and INs is crucial for the proper functioning of the neocortex (Hensch 2005, Turrigiano 2011).

1.1 Excitatory neurons

Excitatory glutamatergic PNs are present throughout layers 2 to 6 and are broadly classified depending on their distinct laminar position and projection patterns. They constitute about 80%

of neurons in the mammalian neocortex, making them the most abundant population. With the exception of spiny stellate neurons in layer 4 of the barrel cortex, PNs have a characteristic pyramidal shaped soma, from which a long axonal projection departs at the basal midline and contain lateral dendritic arborisation and an apical dendrite that branches out towards the pial surface (Molyneaux, Arlotta et al. 2007, Leone, Srinivasan et al. 2008).

PNs are generated from progenitor cells along the ventricular wall of the dorsal pallium (ventricular zone, VZ), between embryonic day E11.5 and E17.5 in the mouse. Along the VZ, radial glial cells (RGCs), which are pluripotent self-renewing cells, undergo several symmetric divisions before starting to differentiate into neuroblasts. The neurogenic potential of a RGC is progressively restricted depending on mitotic spindle cleavage orientation, a combination of extrinsic cues from the cerebrospinal fluid (CSF), intracellular pathways (e.g., Notch signalling) and their birthdate (Malatesta, Hartfuss et al. 2000, Miyata, Kawaguchi et al. 2001, Peyre and Morin 2012). Only a small fraction of RGCs give rise to PNs, the remaining forms a scaffolding for radially migrating cells by anchoring their apical processes to the ventricular surface and their basal processes to the meninges and only by the end of neurogenesis they specify into astrocytes (Franco and Müller 2013, Greig, Woodworth et al. 2013). Using asymmetric cell division, RGCs generate intermediate progenitors (IPCs) (Haubensak, Attardo et al. 2004, Miyata, Kawaguchi et al. 2004, Noctor, Martínez-Cerdeño et al. 2004) and neurons. IPCs undergo one or two rounds of symmetric and asymmetric divisions before neurogenic division.

The expanding number of dividing cells creates the subventricular zone (SVZ), where IPCs undergo neurogenic mitosis (Fietz, Kelava et al. 2010, Hansen, Lui et al. 2010, Govindan and Jabaudon 2017).

These newly generated PNs then migrate radially to populate the neocortical wall in a precise temporal order to expand the cortical thickness. During early embryonic development (around E10 in mice), the earliest differentiated cells form a primordial and transient structure known as the preplate (PP). At the same time, another cohort of transient cells, the Cajal-Retzius cells (CRs), migrate from the cortical hem and the pallial-subpallial boundary to allocate along

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the pial surface, forming the marginal zone (MZ) (Takiguchi-Hayashi, Sekiguchi et al. 2004, Bielle, Griveau et al. 2005, Yoshida, Assimacopoulos et al. 2006). CRs are responsible for Reelin secretion, a crucial protein during several steps of PNs migration (Honda, Kobayashi et al. 2011, Hirota and Nakajima 2017). First waves of PN neuroblasts elongate and anchor their leading process to the pial surface, from where it progressively shorten while the nucleus translocates towards the MZ. These early positioned PNs are eventually responsible for splitting the PP into a deeper layer, the subplate (SP), and the cortical plate (CP), where further prospective PNs allocate in an inside-out manner (i.e., the deeper layers are followed by upper layers) and progressively expand it into the cortical layers. With the emergence of the CP, PNs assume a different mode of migration towards their positioning. After leaving the VZ/SVZ, they assume a multipolar morphology in the intermediate zone (IZ), until one of their branches polarizes and becomes the leading process. The neuroblast assumes a bipolar morphology and starts to interact with RGCs. From this moment, PNs rely on RGC scaffold to migrate along it and reach their final position in the CP. In such manner, neurons of the same ontogeny progressively layer and form a column relating to their function (Gao, Postiglione et al. 2014, Beattie and Hippenmeyer 2017, Kaplan, Ramos-Laguna et al. 2017).

Neuronal subtype differentiation partly occurs at the level of progenitor cells and is facilitated by the sequential expression of different transcription factors (TFs) (Agirman, Broix et al.

2017). Late in corticogenesis, progenitors generate neurons for upper layers (e.g., Cux2) (Franco, Gil-Sanz et al. 2012, Marín and Müller 2014), while earlier on progenitors generate neurons for deep layers (e.g., Fezf2) (Chen, Schaevitz et al. 2005, Chen, Rasin et al. 2005, Molyneaux, Arlotta et al. 2005, Han, Kwan et al. 2011, McKenna, Betancourt et al. 2011, Guo, Eckler et al. 2013). Several TFs in post-mitotic cells control different subtypes of mature projection neurons, such as callosal, subcerebral or cortico-thalamic (Molyneaux, Arlotta et al.

2007, Harris and Shepherd 2015).

1.2 Inhibitory interneurons

INs make up approximately 20% of the whole cortical neuronal population. They mainly project locally and play a pivotal role in the proper functioning of cortical circuits by modulating the firing output of PNs through the synaptic release of the neurotransmitter GABA.

Postsynaptically, GABA activates the fast chloride-permeable ionotropic GABAA receptors and the slower G-protein coupled GABAB receptors.

1.2.1 Development of cortical interneurons

IN progenitors are spatially segregated within the subpallium, which contains the ganglionic eminences (GEs), further subdivided into lateral (LGE), medial (MGE) and caudal (CGE), and

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the preoptic area (POA) (Gelman and Marin 2010). During the early stages of embryonic development (E9.5 in mice), the MGE appears as a swelling at the telo-diencephalic junction, protruding into the lateral and third ventricles (Miyoshi, Butt et al. 2007). This is then followed by a second swelling at E12, which makes up the LGE. The CGE is defined as a region posterior to the MGE and LGE, where the two regions fuse. The POA is located in the telencephalic stalk around the third ventricle (Puelles, Kuwana et al. 2000).

The different classes of neocortical INs are generated in either the MGE, CGE or POA and reach the pallium first tangentially, following different migratory routes, to then invade the forming cortex (Gelman and Marin 2010). The majority of cortical INs are derived from the MGE, while the remaining is derived from CGE and POA (Nery, Fishell et al. 2002, Butt, Fuccillo et al. 2005). The ventral part of the LGE gives rise to the striatal GABA projection neurons (caudate nucleus and putamen), while dorsal LGE gives rise to INs that migrate to the olfactory bulb, septum and the amygdala (Kohwi, Petryniak et al. 2007, Young, Fogarty et al. 2007). In addition to cortical INs, the MGE gives rise to neurons in the globus pallidus and the septum, whereas the CGE and the POA contribute to the nucleus accumbens, the bed nucleus of stria terminalis, and specific nuclei of the amygdala (Nery, Fishell et al. 2002, Nóbrega-Pereira, Gelman et al. 2010). All these subpallial structures are shaped and determined by specific spatio-temporal cascades of TFs. Although Dlx1/2 are homogeneously expressed in cells present in the VZ and SVZ, other specific TFs are induced cell- autonomously within the different subdomains and control the development and specification of distinct classes of INs (Kessaris, Magno et al. 2014, Hu, Vogt et al. 2017, Fishell and Kepecs 2020) (Fig. 1).

1.2.1.1 Medial ganglionic eminence (MGE)

The MGE gives rise to approximately 60-70% of the neocortical INs in the mouse. MGE- derived INs pattern the neocortex in an inside-out fashion, sharing their allocation with isochronically-born PNs (Miller 1985, Fairén, Cobas et al. 1986, Valcanis and Tan 2003). Both in vitro (Xu, Cobos et al. 2004, Wonders, Taylor et al. 2008) and in vivo (Butt, Fuccillo et al.

2005, Flames, Pla et al. 2007, Wonders, Taylor et al. 2008) experiments showed that the MGE gives rise to mostly Parvalbumin (PV) expressing fast spiking chandelier and basket cells and to non-fast spiking basket and Martinotti Somatostatin (SST) expressing cells (Xu, Roby et al.

2006, Miyoshi, Hjerling-Leffler et al. 2010). MGE assembly and generation of INs depend on the TF Nkx2.1, induced and maintained through Shh signalling during neurogenesis and is mainly expressed in this eminence (Xu, Wonders et al. 2005, Gulacsi and Anderson 2006, Flames, Pla et al. 2007, Fogarty, Grist et al. 2007, Liodis, Denaxa et al. 2007, Butt, Sousa et

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al. 2008, Xu, Tam et al. 2008, Zhao, Flandin et al. 2008). Nkx2.1 is maintained in post-mitotic cells, where it upregulates Lhx6. This TF, in turn, downregulate Nkx2.1 in neuroblasts, allowing them to start and engage their way out the eminence to reach and specify in the neocortex (Du, Xu et al. 2008). Expression of Lhx6 has been shown to be necessary to determine a MGE-derived INs’ fate as cortical vs. striatal (McKinsey, Lindtner et al. 2013, van den Berghe, Stappers et al. 2013, Zhou, Yang et al. 2015).

Studies on mutant mice, however, demonstrated that INs lacking Lhx6, although misdistributed across the layers, were still able to make it to the cortex but, more remarkably, failed to specify into PV or SST (Liodis, Denaxa et al. 2007, Zhao, Flandin et al. 2008). TFs downstream Lhx6 determining different subtypes are only starting to be elucidated (Vogt, Hunt et al. 2014, Angara, Pai et al. 2020). Nevertheless, two of them have been studied and demonstrated to be involved in PV and SST specification. The Sry-related HMG-box- containing transcription factor Sox6 is upregulated in migrating LHX6+ cells. Sox6 knockout mice failed to specify as PV, maintaining an immature electrophysiological profile instead, probably due to the lost capacity of upregulating potassium channels involved in their peculiar fast-spiking activity pattern (Azim, Jabaudon et al. 2009, Batista-Brito, Rossignol et al. 2009).

In addition, Satb1 is upregulated in an activity-dependent manner in settling LHX6+ cells and has been shown to be crucial for differentiation of SST-expressing INs (Close, Xu et al. 2012, Denaxa, Kalaitzidou et al. 2012).!

1.2.1.2 Caudal ganglionic eminence (CGE)

The CGE contributes to about 20-30% of all cortical INs (Nery, Fishell et al. 2002, Miyoshi, Hjerling-Leffler et al. 2010). CGE derived cortical INs are generated as early as E12.5 with a peak around E14.5. Unlike MGE-derived INs, they do not follow an inside-out patterning of allocation, but mostly occupy superficial layers independently of their time of birth (Miyoshi, Hjerling-Leffler et al. 2010). The TFs they express as well as the temporal and spatial location of their expression contributes to the diversity of cortical INs (Butt, Fuccillo et al. 2005).

CGE-derived INs were shown to specifically express HTR3A (Lee, Hjerling-Leffler et al. 2010, Vucurovic, Gallopin et al. 2010, Murthy, Niquille et al. 2014, Frazer, Prados et al. 2017) and to give rise to a large diversity of INs including reelin+ cells, VIP+/CR+ bipolar cells, and VIP+/CCK+ basket cells (Lee, Hjerling-Leffler et al. 2010, Miyoshi, Hjerling-Leffler et al. 2010, Murthy, Niquille et al. 2014, Prönneke, Scheuer et al. 2015). This group displays different types of morphologies and intrinsic electrophysiological profiles (Lee, Hjerling-Leffler et al.

2010).

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They display a combinatorial expression of several TFs, such as Prox1 (Rubin and Kessaris 2013, Miyoshi, Young et al. 2015, Touzot, Ruiz-Reig et al. 2016), Nr2f2 (Kanatani, Yozu et al. 2008, Cai, Zhang et al. 2013, Touzot, Ruiz-Reig et al. 2016), and Sp8 (Ma, Zhang et al.

2012) (Fig.1).

Prox1 represents the first identified TF specifically required for the embryonic and postnatal acquisition of CGE-derived cortical IN properties. Prox1 is present in nearly all striatal INs regardless of their origin. Within the cortical IN population, expression of Prox1 is confined to CGE and POA-derived cells (Rubin and Kessaris 2013, Miyoshi, Young et al. 2015).

Expression of Prox1 is selectively maintained in post-mitotic HTR3A+ IN precursors and its loss impairs the integration of these INs into superficial layers. CGE-derived INs that lack Prox1 still express Nr2f2 and Sp8 indicating independent activation of these two transcription factors (Rubin and Kessaris 2013, Miyoshi, Young et al. 2015).

The TF Nr2f2 (also called CoupTFII) is strongly expressed in the CGE progenitor domain but only in small proportions in LGE and MGE (Kanatani, Yozu et al. 2008, Miyoshi, Hjerling- Leffler et al. 2010). It is involved in directing INs through the caudal migration route (Kessaris, Magno et al. 2014). Sp8 and Nr2f2 are expressed in complementary gradients, with Sp8 expressed in the pallial VZ in a high to low rostrodorsal to caudoventral gradient and tend to be localised in upper layers in the mature cortex (Ma, Zhang et al. 2012).

1.2.1.3 Preoptic area (POA)

The embryonic POA is found immediately in front of the optic recess ventral to the MGE and is the source of about 10% of cortical INs (Gelman, Martini et al. 2009, Gelman, Griveau et al.

2011, Kessaris, Magno et al. 2014). The POA domain is a mosaic of different TFs, some of which shared with MGE and CGE (such as Nkx2.1, Nkx6.2, Nr2f2) and others specific to this region, such as Dbx1 and Hmx3 (Flames, Pla et al. 2007, Gelman, Martini et al. 2009, Gelman, Griveau et al. 2011) (Fig.1).

Fate mapping analyses using Dbx1-Cre mouse line showed that DBX1+ domain located in the ventral part of the POA also contributes to a population of PV+ and SST+ cortical INs (Gelman, Griveau et al. 2011, Niquille, Limoni et al. 2018). They migrate out of the POA between E11.5 and E15.5 where they preferentially populate deep layers of the neocortex. Similar to MGE derived INs, DBX1+ progenitors in the POA may also co-express Nkx2.1 and/or Nkx6.2 and Lhx6. These DBX1-expressing POA INs represent approximately another 5% of the total cortical IN population (Gelman, Griveau et al. 2011).

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Another set of cortical INs were shown to originate from HMX3+ (previously known as NKX5.1) cells located in the dorso-ventral axis of the POA. INs of the POA originating from HMX3+

cells share features with CGE-derived INs, like the expression of HTR3A and TFs such as Prox1 and Nr2f2 (and/or, in rare cases, Sp8), but only rarely the MGE related TFs Nkx2.1 or Lhx6 (Niquille, Limoni et al. 2018). Using Hmx3-Cre driver mouse line, it was demonstrated that HMX3+ INs preferentially occupy upper layers of the neocortex and express the markers Reelin and/or NPY but not VIP (Gelman and Marín 2010, Lee, Hjerling-Leffler et al. 2010, Niquille, Limoni et al. 2018). HMX3+ cells make up approximately 4% of the cortical GABAergic IN population (Gelman and Marín 2010) and exhibit the molecular, electrophysiological and morphological profile of neurogliaform cells (NGCs) (Niquille, Limoni et al. 2018).

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! Figure 1. Genetic codes controlling the development and specification of INs.

a) chronological stages of IN development. b) TFs controlling the development of INs originating from MGE, dMGE, dCGE, and POA at different stages (Kessaris, Magno et al.

2014).

1.2.2 Cortical interneuron migration

INs follow two sequential strategies of migration to disperse from their site of origin in the subpallium to reach their designated coordinates in the cortex: tangential and radial migration (Hatten 1999, Corbin, Nery et al. 2001, Marín and Rubenstein 2001, Marín and Rubenstein 2003, Métin, Baudoin et al. 2006). Tangentially migrating neurons follow two main streams parallel to the pial surface, one along the MZ and the other in the IZ/SVZ. Once they have reached the forming neocortex, INs leave the tangential migratory stream to start invading the

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CP and reach their final laminar position (Marín and Rubenstein 2001, Ang, Haydar et al.

2003, Tanaka, Nakaya et al. 2003).

During tangential migration, all cortical interneurons, regardless of their origin, engage in either the MZ or SVZ/IZ stream. Although how INs choose to adopt one migratory stream over the other is not fully clear, studies have shown that cells in different route of migration express different surface receptors (Antypa, Faux et al. 2011). This distinction was independently of the subpallial origin of the migrating cells, suggesting that differential expression of ligands guides migrating neurons into different migratory streams in their journey to the neocortex. In addition, the embryonic vascular networks provides support and guidance cues to regulate the spatially distinct streams of tangentially migrating INs. Mouse embryonic slice culture preparation demonstrated that tangential migration is guided by endothelial cells of the periventricular vascular network that are distinct from the pial network (Won, Lin et al. 2013).

Unlike migrating PNs, INs do not rely on a physical support to reach the pallium and allocate (Denaxa, Kyriakopoulou et al. 2005). Instead, INs navigate towards their destination by relying on environmental cues, to which a response is achieved through up- and down-regulation of receptors on their surface. Growth cones present at the tip of each leading process are the steering machinery of a migrating IN, allowing consequent nucleokinesis and somal translocation (Martini, Valiente et al. 2009, Valiente and Marín 2010, Yanagida, Miyoshi et al.

2012).

Expression and precise coordination of chemoattractive and chemorepulsive cues within the developing brain are necessary to allow INs reach their final destination in the cortex or to subcortical areas (Marín, Yaron et al. 2001, Polleux, Whitford et al. 2002, Marín and Rubenstein 2003, Wichterle, Alvarez-Dolado et al. 2003, Métin, Baudoin et al. 2006, Pasterkamp 2012). Although several examples for IN-environment interaction have been described, the best known remains the differential expression of Neuropilin 2 receptor (NRP2) on a subset of MGE- and POA-derived INs as soon as they leave their site of origin. NRP2+

INs engage in tangential migration to the dorsal pallium, avoiding the striatal primordium enriched in Semaphorin (SEMA) 3A and 3F. Conversely, those INs not expressing NRP2 invade and populate the striatum and amygdala (Marín, Yaron et al. 2001, Kanatani, Honda et al. 2015). In addition to guidance cues, neurotransmitters such as glutamate, GABA, glycine, dopamine and 5HT also promote interneuron migration and facilitate their path to the neocortex (Nguyen, Rigo et al. 2001, Soria and Valdeolmillos 2002, Heng, Moonen et al.

2007). As an example, 5HT in IN migration is discussed below in the dedicated section.

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1.2.3 Cortical plate invasion and final allocation

INs avoid the CP of the developing cortex while journeying through the migratory streams.

The chemokine CXCL12 was shown to serve as a chemoattractant for maintaining tangentially migrating interneurons expressing CXCR4/7 (Stumm, Zhou et al. 2003, Li, Adesnik et al. 2008, López-Bendito, Sánchez-Alcañiz et al. 2008, Hernández-Miranda, Parnavelas et al. 2010, Wang, Li et al. 2011). The cell-autonomous down-regulation of these receptors make INs lose responsiveness to CXCL12 and leave the tangential stream. Recently, it has been shown that, at least for MGE-derived INs, this phenomenon coincides with upregulation of the ErbB4 receptor, which convey an attractive response to Neuregulin3, secreted by PNs in the CP (Bartolini, Sánchez-Alcañiz et al. 2017). Previous work in our lab demonstrated that functional HTR3A is upregulated in INs during CP invasion (Murthy, Niquille et al. 2014). This coincides with the emergence of serotonergic fibers in the developing cortex, strongly suggesting a role for this neurotransmitter in inducing CGE-derived INs to leave the tangential migration and invade the CP (Gaspar, Cases et al. 2003).

MGE-derived INs establish in the neocortex in an inside-out manner, sharing lamination with PNs born at the same time. Indeed several studies have shown that PNs constitute an attractive source for these partnering INs and more likely being responsible for their final allocation (Pla, Borrell et al. 2006, Lodato, Rouaux et al. 2011, Bartolini, Sánchez-Alcañiz et al. 2017). HTR3A+ INs, however, do not follow the same allocation pattern, but they rather occupy superficial layers independently of their time of birth (Métin, Baudoin et al. 2006, Miyoshi, Hjerling-Leffler et al. 2010). A similar mechanism of attraction exerted by PNs has been also proposed for HTR3A+ INs allocation, but more comprehensive molecular studies are still missing (Wester, Mahadevan et al. 2019). Recently, our lab has shown a novel interplay based on SEMA3A-PLXNA4 signalling, where MGE-derived INs would instruct HTR3A+ INs to allocate into superficial layers (Limoni, Niquille et al. 2020) . In addition to these cell-extrinsic mechanisms, cell-autonomous mechanisms have also been reported.

Notably, the upregulation of potassium-chloride co-transporter KCC2 would decrease INs motility through activation of GABA receptors, reduce membrane potential and increase intracellular calcium level (Bortone and Polleux 2009). This hypothesis tested in vitro, however, failed to be fully reproduced in vivo, leaving the question still a matter of debate (Zechel, Nakagawa et al. 2016).

1.3 Cortical interneuron classification

Cortical INs constitute a heterogeneous population. The classical way of classification involves their morphological, physiological and molecular characteristics (DeFelipe 1993, Cauli,

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Audinat et al. 1997, Gupta, Wang et al. 2000, Kawaguchi and Kondo 2002, Ascoli, Alonso- Nanclares et al. 2008, DeFelipe, López-Cruz et al. 2013). The emergence and development of RNA-sequencing techniques at single cell resolution, as well as the improvement of paired electrophysiological techniques coupled with morphological reconstructions, allowed rapid and deeper knowledge of their diversity (Jiang, Shen et al. 2015, Zeisel, Muñoz-Manchado et al. 2015, Fuzik, Zeisel et al. 2016, Tasic, Menon et al. 2016, Tasic, Yao et al. 2018). The need for a community effort to have a unified taxonomy and nomenclature that combines and converges different classification criteria has been recently suggested (Yuste, Hawrylycz et al. 2020).

Although over 60 subtypes have been identified at transcriptomic level (Tasic, Yao et al. 2018) (Fig.2), three canonical non-overlapping classes have been primarily described in the neocortex, based on the main markers they express (Rudy, Fishell et al. 2011, Wamsley and Fishell 2017, Fishell and Kepecs 2020) and are considered to broadly cover their diversity (Fig.3A).

PV-expressing INs make up about 40% of the total neocortical INs and are found throughout layers 2 to 6 (Tasic, Yao et al. 2018) (Fig.2A; Fig.3B). Morphologically, they are chandelier cells, which make contact with the initial axon segment of PNs, and basket cells, which inhibit PNs on the soma and dendrites (Jiang, Shen et al. 2015, Fishell and Kepecs 2020) (Fig.3).

The latter, in addition, have been shown to mediate gamma oscillations (30-80Hz), enabling integration of multiple sensory inputs (Pouille and Scanziani 2001, Cardin, Carlén et al. 2009).

All PV+ INs are characterized by peculiar non-adapting fast-spiking electrophysiological properties (Jiang, Shen et al. 2015).

A second group constituting approximately 30% of INs includes cells expressing SST.

Although, at transcriptomic level, over 20 different subtypes have been identified (Tasic, Yao et al. 2018) (Fig.2A), morphologically and electrophysiologically, they can be reduced to three main groups (Fig.3A). Martinotti cells, the most numerous of this subtype, are characterized by an intricate axonal arborisation extending to layer 1 (Lim, Pakan et al. 2018), forming a feedforward-feedback inhibition loop onto PNs (Jiang, Shen et al. 2015). Other SST+ INs have a bitufted morphology, but being a heterogeneous group of cells themselves, their characteristic features are still to be determined (Jiang, Shen et al. 2015). At last, small group of SST+ cells co-expresses nNOS1. They are only located in layer 6 and extend a long axon towards the white matter to project long range (Jaglin, Hjerling-Leffler et al. 2012, Magno, Oliveira et al. 2012, Wamsley and Fishell 2017, Tasic, Yao et al. 2018). Electrophysiologically,

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nNOS1+ INs show fast-spiking and adapting properties and are inhibited by monoaminergic afferents (Kilduff, Cauli et al. 2011, Tricoire and Vitalis 2012).

! Figure 2. Molecular diversity of interneurons.

A-B) Diagrams showing IN molecular diversity with SST, PV, Lamp5, Sncg and VIP defining non-overlapping subclasses in L1-4 (blue), and L5-6C-D) Dendrograms depicting layer distribution of MGE-derived SST and PV INs (C) and CGE derived VIP, Lamp5, Sncg, and Serpinf1 INs (Tasic, Yao et al. 2018).

Finally, the last 30% of remaining cortical INs are characterized by the expression of the HTR3A (Fig.3) (Batista-Brito, Rossignol et al. 2009, Lee, Hjerling-Leffler et al. 2010, Vucurovic, Gallopin et al. 2010, Rudy, Fishell et al. 2011, Murthy, Niquille et al. 2014). This group is composed of 30 different subtypes, expressing VIP, Reelin, Lamp5, Serpinf1 and Sncg as main neuromarkers (Fig.2B) (Wamsley and Fishell 2017, Tasic, Yao et al. 2018, Fishell and Kepecs 2020). Fate-mapping experiments demonstrated that these INs are exclusively derived from the CGE and POA (Nery, Fishell et al. 2002, Lee, Hjerling-Leffler et al. 2010, Vucurovic, Gallopin et al. 2010, Murthy, Niquille et al. 2014, Niquille, Limoni et al. 2018). Given

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the high relevance of these INs to my research work, they will be extensively described in the following section.

! Figure 3. Diversity and classification of neocortical INs

A) Morphologically distinct INs organised according to their expression of PV, SST, and Htr3a.

A small fraction of PV+ cells also express SST. B) A schema depicting the laminar distribution of neocortical INs from layers 1-6 extending to the white matter (wm) and corpuscalosum (cc).

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!

1.3.1 Family of HTR3A+ interneurons

HTR3A-expressing INs cover the population of neuronal cells born in the CGE and in the non- NKX2.1-expressing POA. Although they can be found across the whole neocortical thickness, HTR3A+ INs are preferentially located in supragranular layers, complementary to MGE- derived ones (i.e., PV+ and SST+), with whom they do not overlap (Murthy, Niquille et al.

2014, Tremblay, Lee et al. 2016). They have been shown to be modulated by serotonergic and cholinergic fibers, as well as respond to thalamocortical afferents innervating layer 1 (Lee, Hjerling-Leffler et al. 2010, De Marco García, Karayannis et al. 2011, Murthy, Niquille et al.

2014, De Marco García, Priya et al. 2015, Niquille, Limoni et al. 2018). Although still a lot remains to be understood about these INs, their peculiar position and characteristics may endow them with a pivotal role in the top-down regulation of cortical processing (Larkum 2013, Tremblay, Lee et al. 2016). HTR3A+ INs consist of two major subgroups with distinct synapse connectivity: VIP+ and Reelin+ INs. In this section, HTR3A INs will be described according to their expression of VIP or Reelin and their allocation in layer 1.

1.3.1.1 VIP interneuron subpopulation

VIP+ INs are prevalently enriched in layers 2 and 3 of the neocortex, where they represent about 40% of the HTR3A+ population (Lee, Hjerling-Leffler et al. 2010, Rudy, Fishell et al.

2011, Wamsley and Fishell 2017). VIP+ INs in L5 appear morphologically distinct from those in upper layers and comparatively little is known about them (Prönneke, Scheuer et al. 2015, Naka and Adesnik 2016).

VIP+ INs co-express other markers such as the choline acetyltransferase (ChAT), cholecystokinin (CCK; ~10-30%) and CR (~50-70%) (Tremblay, Lee et al. 2016, Wamsley and Fishell 2017) and these two subpopulations are largely non-overlapping (Fig.3). Most VIP INs have a vertically oriented bipolar-like dendritic arborisation, while the remaining exhibit multipolar morphology (Bayraktar, Welker et al. 2000, Cauli, Zhou et al. 2014, Jiang, Shen et al. 2015, Pronneke, Scheuer et al. 2015, Tremblay, Lee et al. 2016).

Bipolar VIP+ INs have narrow dendritic trees that cross several layers in either direction, thus conferring them translaminar inputs in several layers restricted to one column. Layer 2/3 VIP+

INs send their dendrites through layer 1 (L1) reaching the pial surface. There, they receive inputs from various intracortical and subcortical projections coming into L1 (Tremblay, Lee et al. 2016). Subtle morphological differences can be appreciated in VIP+ INs with vertically oriented dendrites: bitufted, single tufted, bipolar/tripolar. However, it is not clear that these

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differences in dendritic morphology are physiologically significant as they tend to sample similar intracolumnar and translaminar sectors. Therefore, the term ‘bipolar’ is used to denote all VIP+ INs with vertically oriented dendritic arbor (Tremblay, Lee et al. 2016).

Layer 2/3 VIP+ INs dendrites are found in supragranular layers and extend their axons in both vertical directions, where it also reaches layers 4 and 5/6 (Porter, Cauli et al. 1998, Bayraktar, Welker et al. 2000, Jiang, Shen et al. 2015, Pronneke, Scheuer et al. 2015, Wamsley and Fishell 2017). Bipolar VIP+ INs in deeper layers, however, follow different axonal and dendritic arborisation. These cells have dendrites extending through the supra- and infragranular layers, but their axons are restricted to layers 5/6 (Jiang, Shen et al. 2015, Prönneke, Scheuer et al. 2015).

VIP+ INs with multipolar morphology include those co-expressing CCK and those that are found in L6 with an intralaminar axon spanning laterally (Prönneke, Scheuer et al. 2015, Tremblay, Lee et al. 2016). Unlike bipolar cells, VIP+ INs with multipolar morphology have local axonal arbor (Tremblay, Lee et al. 2016). In the BLA, VIP+ INs preferentially innervate distal dendrites, although they also form synapses on the soma of PNs and a subset of calbindin expressing INs (Babaev, Piletti Chatain et al. 2018).

Electrophysiologically, VIP-expressing INs often display an ‘irregular-spiking’ (IS) pattern.

These IS neurons have repetitive firing pattern with irregular spike frequency and monotonic amplitude during larger depolarisations (Cauli, Audinat et al. 1997, Porter, Cauli et al. 1998, Férézou, Cauli et al. 2002, Galarreta, Erdélyi et al. 2004, Lee, Hjerling-Leffler et al. 2010, Miyoshi, Hjerling-Leffler et al. 2010). This firing pattern could be a consequence of their high input resistance, making them particularly sensitive to excitatory inputs (Lee, Hjerling-Leffler et al. 2010, Tremblay, Lee et al. 2016). In addition to IS VIP+ INs, those that exhibit bursting and strongly adapting electrophysiological properties have been described (Lee, Hjerling- Leffler et al. 2010, Prönneke, Scheuer et al. 2015), although the extent to which these differences in firing pattern contribute to a distinct subpopulation and their functional relevance is still unclear (Tremblay, Lee et al. 2016).

In addition to showing nicotinic ACh responses, VIP+ INs are strongly depolarised by HTR3A agonists (Lee, Hjerling-Leffler et al. 2010), suggesting that neuromodulatory activity from the raphe and basal forebrain neurons could rapidly activate these INs (Tremblay, Lee et al. 2016).

1.3.1.2 Interneurons in layer 1

The neocortical L1 is unique as it is the main target for top-down regulation of cortical and subcortical inputs. L1 contains a dense network of excitatory and inhibitory axons, dendrites

Role of serotonin 3a receptor in early life stress and amygdala-medial prefrontal cortex networks during fear processing

Thesis

Reference