SHANK3 controls maturation of social reward circuits in the VTA

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Thesis

Reference

SHANK3 controls maturation of social reward circuits in the VTA

BARISELLI, Sebastiano

Abstract

L'haploinsuffisance de SHANK3, codant la protéine d'échafaudage synaptique SHANK3, conduit à une forme hautement pénétrante des Désordres du Spectre Autistique (ASD). La manière dont l'insuffisance de SHANK3 affecte des circuits neuronaux spécifiques pour générer des symptômes liés à l'ASD demeure obscure. Nous avons utilisé ici un shRNA pour modéliser l'insuffisance de Shank3 au sein de l'Aire Tegmentale Ventrale (VTA). Nous avons identifié des changements spécifiques liés au type cellulaire, Dopaminergique (DA) et GABAergique, dans la transmission synaptique excitatrice, qui convergent pour réduire l'activité des neurones DA et générer des comportements liés à l'ASD, comprenant une détérioration de la préférence sociale. L'administration d'un modulateur allostérique positif des récepteurs métabotropiques au glutamate de type 1 (mGluR1) pendant la première semaine post-natale, a permis de restaurer la transmission excitatrice synaptique des neurones DA et les déficits liés à la préférence sociale, tandis que la stimulation optogénétique des neurones DA a été suffisante pour [...]

BARISELLI, Sebastiano. SHANK3 controls maturation of social reward circuits in the VTA. Thèse de doctorat : Univ. Genève et Lausanne, 2016, no. Neur. 168

URN : urn:nbn:ch:unige-856248

DOI : 10.13097/archive-ouverte/unige:85624

Available at:

http://archive-ouverte.unige.ch/unige:85624

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 SCIENCES

Professeur Christian Lüscher, directeur de thèse Professeure Camilla Bellone, co-directrice de thèse

TITRE DE LA THESE

SHANK3 controls maturation of social reward circuits in the VTA

THESE Présentée à la Faculté des Sciences de l’Université de Genève

pour obtenir le grade de Docteur en Neurosciences

par

Sebastiano BARISELLI

de Italie Thèse N° 168

Genève

Editeur ou imprimeur : Université de Genève 2016

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Acknowledgements

At the end of my PhD studies, I would like to thank both my thesis director Prof. Christian Lüscher and the thesis co-director Prof. Camilla Bellone. You have both been essential for my formation, especially in terms of inspiration and critical thinking. Thanks for giving me the opportunity to work in your labs.

I would like to acknowledge the members of my PhD thesis committee Prof.

Anthony Holtmaat, Prof. Alexandre Dayer and Prof. Ralf Schneggenburger. Thanks for the useful advices you gave me and for the opportunities to discuss with you about my findings.

A special thank to Prof. Camilla Bellone and Tifei Yuan, which taught me everything I know about the fascinating world of electrophysiology. I’d like to thank all the people working in the Lüscher Lab during the time I spent there for the exciting and stimulating discussions we had. In particular, Eoin C. O’Connor, for the help during the experiment setting, Meaghan Creed for the reciprocal support during our initial struggling and Nicola Platt, for your essential help with my thesis writing.

I’d like to thank Niels Ntamati and Thomas Stefanelli who offered me strong support and invaluable friendship. My thanks go also to other people I met while working at the CMU in Geneva, Ilaria, Subashika and Cristina and all the others.

I’d like to thank the people of the Bellone Group. In particular, Stamatina Tzanoulinou, thanks for your friendship. Without you I don’t think I would have been able to make it through my PhD. A thank to Christelle Glangetas and Clement Prévost-Solié, sharing my life in the lab with you it is inspirational and never boring.

Thanks to Paola Bezzi and her team, with whom I had moments of precious personal and scientific exchange.

A thank goes to my whole big family. A special one to my father Stefano, my mother Anna and to my sisters Silvia and Sara: thanks for your support and your

“vicinity”. Thanks to my amazing nices, Gaia, Irene, Emma and Claudia. You offered me moments of pure joy and fun.

Finally, my thanks go to my dearest Italian friends. Thanks for your support and the patience you have always shown me during these years.

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Abstract

Haploinsufficiency of SHANK3, encoding the synapse scaffolding protein SHANK3, leads to a highly penetrant form of Autism Spectrum Disorder (ASD). How SHANK3 insufficiency affects specific neural circuits to generate ASD-related symptoms remains elusive. Here we used shRNA to model Shank3 insufficiency in the Ventral Tegmental Area (VTA). We identified dopamine (DA) and GABA cell-type specific changes in excitatory synapse transmission that converge to reduce DA neuron activity and generate ASD-related behaviors, including impaired social preference. Administration of a positive allosteric modulator of the type 1 metabotropic glutamate receptors (mGluR1) during the first postnatal week restored DA neuron excitatory synapse transmission and rescued the social preference defects, while optogenetic DA neuron stimulation was sufficient to enhance social preference. Collectively, these data reveal the contribution of impaired VTA function to ASD-related symptoms and identify mGluR1 modulation during postnatal development as a potential treatment strategy.

Résumé

L'haploinsuffisance de SHANK3, codant la protéine d'échafaudage synaptique SHANK3, conduit à une forme hautement pénétrante des Désordres du Spectre Autistique (ASD). La manière dont l'insuffisance de SHANK3 affecte des circuits neuronaux spécifiques pour générer des symptômes liés à l'ASD demeure obscure. Nous avons utilisé ici un shRNA pour modéliser l'insuffisance de Shank3 au sein de l'Aire Tegmentale Ventrale (VTA). Nous avons identifié des changements spécifiques liés au type cellulaire, Dopaminergique (DA) et GABAergique, dans la transmission synaptique excitatrice, qui convergent pour réduire l'activité des neurones DA et générer des comportements liés à l'ASD, comprenant une détérioration de la préférence sociale. L'administration d'un modulateur allostérique positif des récepteurs métabotropiques au glutamate de type 1 (mGluR1) pendant la première semaine post-natale, a permis de restaurer la transmission excitatrice synaptique des neurones DA et les déficits liés à la préférence sociale, tandis que la stimulation optogénétique des neurones DA a été suffisante pour améliorer cette préférence. Conjointement, ces données révèlent la contribution d'une détérioration du fonctionnement de la VTA aux symptômes liés à l'ASD, et permettent d'identifier la modulation de mGluR1, durant le développement post-natal, comme potentielle stratégie de traitement.

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Contributions

Sebastiano Bariselli (S.B.) performed the viral infections, performed all the in vitro electrophysiology experiments, cell counting and contributed to the statistical analysis of the behavioral and in vivo electrophysiology experiments. Stamatina Tzanoulinou (S.T.) performed the behavioral experiments with the assistance of Clement Prevost-Soliè (C.P.S.) and Eoin C. O’Connor (E.C.O.); Christelle Glangetas (C.G.) performed (in Bordeaux, François Georges LAB) the in vivo electrophysiology experiments. Joanna Viguiè (J.V.) and Christelle Glangetas participated to the cell counting experiments. Luca Pucci (L.P.;

Lausanne, Paola Bezzi LAB) performed the Western Blot analysis. Camilla Bellone and Christian Lüscher supervised the thesis work.

List of Abbreviations

ABA: Applied Behavioral Analysis AP: Action Potential

ASD: Autism Spectrum Disorder BNST: Bed Nucleus Stria Terminalis CNS: Central Nervous System

AMPAR: α-Amino-3-hydroxy-5-Methyl-4- isoxazolePropionic Acid Receptor

CP-AMPAR: Calcium-Permeable AMPA Receptor CI-AMPAR: Calcium-Impermeable AMPA Receptor CS: Conditioned Stimulus

DA: Dopamine

DAT: Dopamine Transporter

DIO-ChR2: Double-floxed Inverse Open reading frame- ChR2

DSM: Diagnostic and Statistical Manual for Mental Disorders

EPSC: Excitatory Postsynaptic Currents FDA: Food and Drug Administration fEPSC: field Excitatory Post-Synaptic Current GABA: Gamma Amino-Butyric Acid GAD: Glutamic Acid Decarboxylase GAT: GABA Transporter

HCN channel: Hyperpolarization-activated Cyclic Nucleotide-gated channel

HFS: High Frequency Stimulation iGluR: ionotropic Glutamate Receptor IQ: Intelligence Quotient

KR: Kainate Receptor LHb: Lateral Habenula LTD: Long Term Depression LTP: Long Term Potentiation

mEPSC: miniature Excitatory Post-Synaptic Current mGluR: metabotropic Glutamate Receptor mPFC: medial Pre-Frontal Cortex MR: Mental Retardation MSN: Medium Spiny Neuron NAc: Nucleus Accumbens

NMDAR: N-Methyl-D-aspartic Acid Receptor PAM: Positive Allosteric Modulator PFC: Pre-Frontal Cortex

PMS: Phelan-McDermid Syndrome PPN: Peduncolo Pontine Nucleus PPR: Paired Pulse Ratio PSD: Post-Synaptic Density RI: Rectification Index

RMTg: Rostro Medial Tegmental nucleus

SN: Substantia Nigra SP: Social Preference

STDP: Spike Time Dependent Plasticity STN: Subthalamic Nucleus

TH: Tyrosine Hydroxylase enzyme US: Unconditioned Stimulus vGAT: vesicular GABA Transporter vGluT2: vesicular Glutamate Transporter 2 VP: Ventral Pallidum

VS: Ventral Striatum VTA: Ventral Tegmental Area

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List of figures

Figure 1: Schematic of the excitatory post-synaptic density ... 7

Figure 2: Schematic of principal SHANK3 isoforms and their relative Knock-out mouse lines ... 17

Figure 3: Summary of electrophysiology and social behavior in transgenic lines with mutations in Shank encoding genes ... 20

Figure 4: Schematic of the main co-releasing VTA output pathways ... 25

Figure 5: AAV-shShank3 down-regulates SHANK3 in the VTA but not SN ... 45

Figure 6: AAV-shShank3 infects putative DA and putative GABA neurons ... 47

Figure 7: SHANK3 down-regulation affects excitatory transmission onto VTA DA and GABA neurons ... 50

Figure 8: Late SHANK3 down-regulation does not affect excitatory transmission and early VTA-SHANK3 does not alter NMDAR composition ... 52

Figure 9: Homer-SHANK interaction is necessary for AMPAR synaptic maturation at VTA DA neurons ... 53

Figure 10: mGluR-I pharmacological activation in vitro removes GluA2-lacking AMPARs from VTA glutamatergic synapses ... 55

Figure 11: In vivo mGluR1-PAM rescues synaptic transmission onto VTA DA but not GABA neurons ... 57

Figure 12: SHANK3 down-regulation affects VTA DA and GABA neuron activity in vivo ... 59

Figure 13: SHANK3 down-regulation impairs social preference in Vehicle treated animals ... 61

Figure 14: mGluR1-PAM treatment rescues VTA-SHANK3 social behavior ... 63

Figure 15: Synaptic and social deficits persist into adulthood and are reversed by early mGluR1-PAM treatment ... 64

Figure 16: VTA-SHANK3 mice do not show social recognition impairment ... 66

Figure 17: Optogenetic activation of VTA DA neurons augments social preference ... 68

Figure 18: VTA-SHANK3 mice exhibit stereotypies and impaired sucrose preference ... 70

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Introduction

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1. Aim of the study

The aim of the present study is to assess the role of the Ventral Tegmental Area (VTA) in social behavior, through an Autism Spectrum Disorder (ASD)-related genetic manipulation. SHANK3 is a synaptic protein, whose alterations are implicated in genetic cases of the neurodevelopmental syndromes ASDs. The protein will be down-regulated at a neonatal stage and the excitatory synaptic physiology of VTA DA and GABA neurons will be assessed in mice. In addition, the effects on neuronal activity and social motivation will be investigated to link synaptic alterations to dysfunctional VTA circuits and ASD-related behaviors. The protective effects of mGluR1 positive allosteric modulation, concomitantly with neonatal SHANK3 down- regulation, will establish a causal link between the consequences of SHANK3 down- regulation and mGluR1 activity impairment.

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2. The Autism Spectrum Disorders

According to the Diagnostic and Statistical Manual for Mental Disorders (DSM-V), Autism Spectrum Disorders (ASDs) comprise neurodevelopmental pathologies characterized by two common behavioral abnormalities: social- communication deficits and restricted and repetitive interest/behavior (5th ed.; DSM–

5; American Psychiatric Association, 2013) with a high inter-individual variability.

The Developmental Disabilities Monitoring Network Surveillance reported that, in the year 2010, 1 in 68 children were affected by ASDs with a higher prevalence in males than females (Developmental Disabilities Monitoring Network Surveillance Year 2010 Principal Investigators Centers for Disease Control and Prevention (CDC), 2014). Autism is typically diagnosed between the ages of two and three years by behavioral observations (Chen et al., 2015). However, the broad variety of symptoms and co-morbidities have posed some difficulties in early diagnosis (Matson & Nebel- Schwalm, 2007); for example, Mental Retardation (MR) is manifested in 40-55% of autistic patients. In addition, behavioral impairments such as aggression, self- injurious behavior, hyperactivity and alterations in the emotional domain (anxiety and depression) can be present. Lastly, other medical (immune system alterations, gastro- intestinal problems) or neurological (e.g seizure, sleep disturbances) symptoms appear in 10% of the autistic population (Chen et al., 2015).

Several genetic and clinical studies in humans are now aiming to elucidate the molecular deficits and the neuronal circuits underlying ASD pathology.

Clinically, the Applied Behavioral Analysis (ABA) intervention, which consists of improving behavior through reward-based reinforcement, is conducted by psychologists to treat Autism (Virués-Ortega, 2010). Despite the psychotherapy-based approach, a pharmacological treatment for the core symptoms of ASD has not yet been found and identifying the key brain regions involved in ASD would provide useful insights.

It has been hypothesized that ASD patients are unable to properly process the reward value of social interaction (Chevallier et al., 2012). One of the major neuromodulators involved in the control of the reward processing is Dopamine (DA), which is released by DA neurons located in the Ventral Tegmental Area (VTA;

Gonon, 1988). In this context, a treatment directed towards normalizing DA neuron activity or DA levels in the downstream structures could be beneficial. As mentioned,

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the FDA (Food and Drug Administration) has not yet approved a medical therapy for ASD, but it is notable that the atypical antipsychotic Risperidone, a D2 antagonist, ameliorates the social dysfunctions (McCracken et al., 2002; Nagaraj et al., 2006).

ASD is a pathology with a strong genetic component with 88% of concordance in monozygotic twins and 31% in dizygotic twins (Rosenberg et al., 2009). A seminal report described a developmental syndrome, characterized by low Intelligence Quotient (IQ) and reduced speech abilities, since named Phelan- McDermid Syndrome (Watt et al., 1985). The affected individual was carrying a long truncation in the distal region of the chromosome 22 (22q13.3; Watt et al., 1985).

Further studies revealed that, in most cases, the pathology was associated with haploinsufficiency of the SHANK3 gene, and was subsequently classified as a syndromic form of ASD (Phelan and McDermid, 2012). Other genes have been implicated in the pathogenesis of Autism such as the genes coding for the serotonin transporter (5-HTT), Reelin, Engrailed, Neuroligins, Neurexins and SHANKs (Chen et al., 2015). Importantly, SHANK3 mutations, which appear in the 0.5% of ASD cases, are one of the more common monogenic and highly penetrant causes of ASDs (Betancur & Buxbaum, 2013).

3. The ASDs and the excitatory synapse

Many genes involved in the pathogenesis of ASD, including those listed above such as Neuroligin, Neurexins and SHANKs, have been linked to synaptic development, maturation and functioning, which has led to the suggestion that ASD is a synaptic pathology, or “synaptopathy” (Zoghbi, 2003; Peça, Ting, et al., 2011; Won et al., 2013). In addition, in recent years, it has been proposed that genes found mutated in ASD cases are involved in the regulation of the metabotropic Glutamate Receptor (mGluR)-dependent synaptic plasticity (e.g. Neuroligin3; Baudouin et al., 2012) and protein translation (e.g. FMRP; Huber et al., 2002). This may suggest a unified hypothesis where genetic alterations in the mGluR complex could result in similar physiological and behavioral impairments (O'Connor et al., 2014).

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3.1 The mGluR and iGluR network

Glutamate is the main excitatory neurotransmitter of the vertebrate Central Nervous System (CNS). The structure and the molecular composition of its receptors have been elucidated and the subunits have been identified and cloned. Based on their structure and downstream effects, there are two categories of glutamatergic receptors:

ionotropic Glutamate Receptors (iGluRs), which are cation channels which open in response to glutamate allowing the flux of positively charged ions through a central pore, and mGluRs, whose effects are mediated by G-proteins. iGluRs can be further subdivided into AMPA receptors and NMDA receptors (see section 3.2 for more detail).

The location and function of iGluRs at the postsynaptic density (PSD) is controlled by a network of scaffolding and signaling proteins (Figure 1). PSD95 proteins directly interact with NMDARs (Niethammer et al., 1996) and, via another PSD protein called Stargazin, with AMPARs (Bats et al., 2007). PSD95 is linked to a protein called GKAP (Kim et al., 1997) that in turn forms a complex with the SHANK proteins.

SHANKs form a mesh-like structure with

Homer (Hayashi et al., 2009) which is able to bind IP3R (Inositol-1,4,5-Trisphosphate Receptors) and mGluRs (Tu et al., 1998). Therefore, through this network, mGluRs and iGluRs are physically linked at the PSD.

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As described in the following paragraphs, mGluR and its signaling pathway are fundamental in the maturation of iGluR transmission. It is therefore plausible that impairments occurring in the mGluR-iGluR network could affect the maturation of the synapse and play a role in the pathogenesis of ASDs.

3.2 iGluRs: basic properties

The iGluRs are further classified by their ability to be selectively activated by different agonists: the AMPA receptors, which are sensitive to the α-Amino-3- hydroxy-5-Methyl-4-isoxazolePropionic Acid, the Kainate receptors (KR), activated by the Kainate and the NMDA receptors, sensitive to the N-Methyl-D-aspartic Acid.

Structural studies have established that iGluRs are tetrameric receptors, formed by different subunits. There are four types of AMPA receptor subunit, GluA1 to GluA4 that can assemble as homomers or heteromers. So far, four Kainate receptors subunits, GluK1-GluK5, have been cloned (for review see Traynelis et al., 2010). Similarly, there are four classes of NMDAR subunit GluN1-4. All NMDARs contain two GluN1 subunits that form homo-dimers before assembling with GluN2 subunits (GluN2A- GluN2D) as di-homo-dimers (e.g. GluN1/GluN2A) or tri-heteromers (e.g.

GluN1/GluN2A/GluN2B or GluN1/GluN2B/GluN3A) (for review see Paoletti et al., 2013).

The diverse composition confers to the NMDAR different properties according to the biophysical characteristics of each subunit (for review see Traynelis et al., 2010). Subunit composition and NMDAR properties vary between synapses and change during postnatal development, defining critical periods of synaptic maturation in many brain regions (Bellone et al., 2011; Matta et al., 2011; 2013)

Briefly, each subunit of an iGluR is composed by four macro-domains: an extracellular Amino-Terminal Domain (ATD), important for the allosteric modulation, an Extracellular Binding Domain (EBD), responsible for the binding with agonists, antagonists or modulators, the Trans-Membrane Domain (TMD), that forms the channel pore and the Carboxy-Terminal intracellular Domain (CTD), involved trafficking and in the signaling cascades (for review see Traynelis et al., 2010).

3.3 GluA2-lacking AMPARs

The AMPARs are the principal mediators of fast glutamatergic signaling. As

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outlined above, AMPARs are composed of four subunits, GluA1-4. The properties of AMPARs vary greatly between those that contain GluA2 subunits and those that do not. The GluA2-lacking AMPARs, compared to GluA2-containing AMPARs, exhibit a higher single channel conductance, faster deactivation kinetics and a non-linear (rectifying) current-voltage relationship, due to the block of the pore by negatively- charged intracellular polyamines at positive membrane potentials (for review see Liu

& Zukin, 2007). Importantly, GluA2-lacking AMPARs are also Calcium Permeable (CP-AMPAR) whereas the GluA2-containing AMPARs have a positively charged Arginine residue (R) in the pore of the channel that confers selectivity for Na⁺ over Ca²⁺ and Zn²⁺(for review see Liu & Zukin, 2007).

In general, AMPARs in the adult CNS contain the GluA2 subunit, except for interneurons in the spinal cord, amygdala and hippocampus where GluA2-lacking AMPARs mediate synaptic transmission (Gu et al., 1996; Mahanty & Sah, 1998;

Lamsa et al., 2007; Szabo et al., 2012). GluA2-lacking AMPARs are also inserted in vivo, at thalamic synapses onto principal neurons in the lateral amygdala after the acquisition of fear-conditioning and their removal is required for fear-extinction (Clem & Huganir, 2010). CP-AMPARs can also be inserted at the synapse in pathological conditions. For example, GluA2-lacking AMPARs are present at glutamatergic synapses onto VTA DA neurons after a single cocaine injection (Bellone & Lüscher, 2006), at medial Prefrontal Cortex (mPFC) synapses onto D1 Medium Spiny Neurons (MSNs) of the Nucleus Accumbens (NAc) after 30 days of withdrawal from cocaine self-administration (Pascoli et al., 2014) or after ischemic insult in hippocampal neurons in vitro (Liu et al., 2006).

In addition, during the expression of Long Term Potentiation (LTP) at CA1 hippocampal synapses, it has been proposed that there is a transient incorporation of CP-AMPARs required for LTP consolidation (Plant et al., 2006; but see Adesnik &

Nicoll, 2007).

The consequence of CP-AMPARs expression is a change in the rules for plasticity induction, which results in anti-Hebbian forms of AMPA-LTP. For example, in hippocampal interneurons (Lamsa et al., 2007) and in VTA DA neurons of cocaine injected mice (Mameli et al., 2011), CP-AMPARs are permissive for a form of synaptic potentiation that relies on neuronal hyperpolarization.

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3.4 Group-I mGluRs: a focus on synaptic transmission control

The mGluRs are members of the GPCR (G-Protein Coupled Receptor) family, which are encoded by 8 different genes and give rise, in total, to at least 8 splice variants (for review see Niswender & Conn, 2010). According to their different effectors, the mGluRs are subdivided in 3 groups: I, II and III. mGluR1 and mGluR5 are group I mGluRs, which are coupled to Gq proteins and activate the Phospholipase-C/inositol 1,4,5-trisphosphate/diacyl-glycerol pathway. The group-II and III mGluRs inhibit the Adenyl Cyclase enzyme leading to a reduction in cAMP (cyclic Adenosine Mono-Phosphate) cellular levels (for review see Hermans &

Challiss, 2001).

In the VTA DA neurons the group-I mGluRs control the strength and quality of synaptic transmission. At excitatory synapses of young (early adolescence) rat VTA DA neurons, pharmacological activation of group I mGluRs (5 mins, 20 µM 3,5- Dihydroxyphenylglycine, DHPG) induces two coexisting forms of synaptic depression: the first is rapid and endocannabinoid-dependent and the second is long- lasting and depends on the removal of GluA2-lacking AMPARs (Bellone & Lüscher, 2005). In mature mouse VTA DA neurons, brief bath application of DHPG or Ro- 677476 (10 mins, mGluR1 positive allosteric modulator) does not induce LTD (Bellone & Lüscher, 2006). However, a form of DHPG-induced LTD, which relies on the synthesis and insertion of GluA2 subunits, is expressed when the animals have been exposed to cocaine (Bellone & Lüscher, 2006; Mameli et al., 2007).

In the genetic landscape of ASD, it should be noted that alterations in proteins involved in the mGluR-signaling pathway, such as FMRP (Fragile Mental Retardation Protein), hemartin (TSC1) and tuberin (TSC2), lead to monogenic ASDs known as Fragile X Syndrome and Tuberous Sclerosis (Peça, Ting, et al., 2011). The pathogenic mechanisms have been identified in alterations of the mGluR-dependent plasticity (Huber et al., 2002; Bateup et al., 2011; Tsai et al., 2012), due to a dysregulation in the AKT/mTOR (or Protein Kinase B/mammalian Target Of Rapamycin) pathway.

3.5 AMPA transmission maturation

The composition of AMPAR changes during the first post-natal weeks and it is

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2013). The rearrangement of iGluR composition takes place within a critical temporal window in an activity-dependent manner. In recent years, efforts have been made to elucidate the molecular mechanisms underlying the subunits involved, the major molecular players and the forms of plasticity that accompany this process.

The earliest time point at which CP-AMPARs have been detected is Embryonic day 17 and 18 (E17-E18) in the rat when GluA2-lacking AMPARs are expressed in motor neurons and contribute to the rise in synaptic Ca²⁺ (Metzger et al., 2000).

GluA2-lacking AMPARs have been detected in dissected rat cochleae in Inner Hair Cell (IHC) before Post-natal day 10 (P10) with the Cobaltum (Co²⁺) uptake assay in vitro. These Ca²⁺ permeable and Joro Spider Toxin-sensitive (a blocker of GluA2- lacking AMPAR) AMPARs are abundant before hearing onset, suggesting that their presence is predominant in the absence of sensory stimulation (Eybalin et al., 2004).

In the neocortex, CP-AMPARs are present immediately after birth and they are replaced by GluA2-containing AMPARs during neural network formation. By measuring the current-voltage relationship (I/V) and the polyamine sensitivity, CP- AMPARs have been detected in layer 5 pyramidal neurons of neonatal but not juvenile (after P16) rats (Kumar et al., 2002). The CP-AMPARs are also expressed at different developmental time points at layer 4 (before P7-P8) and layer 2/3 (before P12-P14; Brill & Huguenard, 2008). Therefore, the maturation of synaptic transmission does not follow the neocortical layer formation and further studies are required to investigate how neuronal network activity may influence the subunit switch.

CP-AMPARs are also expressed at forming Mossy Fiber (MF)-CA3 synapses.

They were detected during the first three postnatal weeks through I/V curve measurement and Philantotoxin (GluA2-lacking AMPAR blocker) sensitivity.

Interestingly, when the PDZ-domain containing protein Interacting with C Kinase 1 (PICK1) was absent, CP-AMPARs were not expressed. In addition, a form of Voltage-gated Ca²⁺ channel-dependent Long Term Depression (LTD) accompanied the removal of GluA2-lacking AMPARs and was occluded in PICK1 KO mice (Ho et al., 2007), providing a key molecular player involved in the control of CP-AMPAR synaptic incorporation.

Lastly, CP-AMPARs are expressed at excitatory synapses onto VTA DA neurons within the first 2 weeks after birth in mice. Importantly, in vivo and in vitro studies have shown that the mGluR1 and its effector, Phospholipase C (PLC), are

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necessary for the AMPAR subunit exchange (Bellone et al., 2011).

This study was the first demonstration that the removal of CP-AMPARs is temporally paralleled by the partial exchange of synaptic GluN2B with GluN2A subunit of NMDARs. In particular, in the first postnatal week, compared to juvenile age, NMDAR transmission was more sensitive to the GluN2B-containing NMDAR blocker ifenprodil and less sensitive to the GluN2A blocker Zn²⁺. In addition, interfering with mGluR1 and PLC functionality blocked the subunit switch for both AMPAR and NMDAR, proving that maturation of both types of iGluR share two molecular effectors (Bellone et al., 2011). Future studies are needed to further characterize the molecular cascades involved in the maturation processes of both AMPARs and NMDARs.

4. Preclinical Models of ASDs: SHANK-family members

Human studies have indicated that many genes implicated in ASD cases encode for synaptic proteins, suggesting that altered synaptic physiology could be a common substrate for ASD (Peça, Ting, et al., 2011). With increasing evidence from human studies on the genetic nature of ASDs, together with the development of techniques for the generation of transgenic mouse lines, genetic models of ASDs have been developed, including SHANK knock out mice (Figure 2 and 3).

4.1 SHANK-family proteins

The SHANK proteins were first described as proteins with a SH3 and an ANKirin domain (Naisbitt et al., 1999). Three genes (SHANK1, SHANK2 and SHANK3) with multiple internal promoters give rise to a vast array of isoforms with five conserved domains (for review see Jiang & Ehlers, 2013). Considering the existence of several protein isoforms, which contain a combination of domains, different variants could be expressed at specific synapses where they may exert different roles.

Shank expression is post-natally regulated. From the Allen Developing Mouse Brain Atlas, it emerges that the mRNA expression of the Shank3 gene peaks at the first postnatal week and reduces during the second/third week, suggesting its involvement in synaptic maturation. However, an in situ hybridization study reported that the mRNAs of the SHANK proteins follow different spatial and temporal patterns

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of expression: SHANK3 increases after P16, while SHANK1 and SHANK2 are already highly expressed at birth (Böckers et al., 2004). Spatially, SHANK1 expression is excluded from the cerebellum granular cell layer and the Caudate and Putamen Nuclei; SHANK3 is enriched in the granular cell layer of the cerebellum and in the striatum but it is excluded from the Purkinje cells; SHANK2 is not detectable in the hypothalamus and in the granular cell layer (Böckers et al., 2004).

Recently, in vitro and in vivo studies have tried to elucidate the roles of SHANK protein isoforms at the molecular, synaptic and behavioral level.

4.2 SHANK1

The SHANK1 gene is located, in humans, on chromosome 19 and microdeletions are associated with ASD cases (Sato et al., 2012). Particularly, a genetic screening detected a hemizygous elimination of exon 1 to exon 20 and a de novo deletion between Synaptotagmin3 and SHANK1 locus (Sato et al., 2012).

The SHANK1 protein is involved in postsynaptic spine formation, in fact its over-expression in vitro leads to spine enlargement and recruitment of the postsynaptic partners GKAP, PSD95 and IP3R (Sala et al., 2001). Co-transfection of SHANK1 and Homer induces IP3R recruitment and enhances the mGluR responses through increased Ca²⁺ release from the intracellular stores (Sala et al., 2005).

The Shank1 KO mouse shows a reduced number of spines in hippocampal CA1 neurons, a reduction in miniature Excitatory Post-Synaptic Current (mEPSC) frequency and no changes in mEPSC amplitude, pointing to a pre-synaptic alteration.

In the same study no alterations in hippocampal synaptic plasticity were detected (Hung et al., 2008). From a molecular point of view, the Post-Synaptic Density (PSD) of forebrain protein extracts showed down-regulation of Homer and SAPAP/GKAP, but no change in NMDAR subunit expression in Shank1 KO mice (Hung et al., 2008). Behaviorally, Shank1 KO mice do not show any social or self-grooming abnormality (Silverman et al., 2011), but alterations in ultrasonic vocalizations have been found, suggesting impairments in social communication (Wöhr et al., 2011).

4.3 SHANK2

The SHANK2 gene is located on the long arm of chromosome 11 and a microarray genome-wide screening has linked Shank2 PDZ domain deletions to ASD.

Two de novo deletions of 120 Kilo-bases (loss of exon 6 and 7) and 69 Kilo-bases

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(loss of exon 7), which result in frameshift mutation and Shank2 haploinsufficiency, were found in affected individuals. In the same study, the authors found 8 further putative mutations, de novo or inherited associated with ASD (Berkel et al., 2010).

The synaptic alterations caused by these mutations have been evaluated in vitro. In particular, the insertion of a stop codon, R426X, led to the disruption of the Proline-Rich and the C-terminal domain. The over-expression of SHANK2 R426X in neonatal or adolescent animals reduced the amplitude of mEPSC, in hippocampal and layer 2/3 neurons, and altered the surface expression of AMPAR. Animals over- expressing SHANK2 R426X, in the neocortex and hippocampus from P0, had cognitive deficits (Berkel et al., 2012).

SHANK2 has been found to be involved in the regulation of mGluR- dependent Ca²⁺ signal, through PLC-β3. Particularly, PLC-β3, which is part of the mGluR signalling pathway, interacts with the SHANK2-PDZ domain, Homer1b and IP3R (Hwang et al., 2005). The exposure to a C-terminal dominant negative peptide for PLC-β3 blocked the co-immunoprecipitation of SHANK2 and attenuated the cellular Ca²⁺-rise induced by the mGluR agonist ACPD (1-Amino-1,3- dicarboxycyclopentane; Hwang et al., 2005).

Considering the clinical and molecular relevance of SHANK2, two different transgenic lines have been developed (Figure 3). In the ProSAP1/Shank2^-/-mouse model, increased NMDAR and GluA2 subunit expression was found in striatal and hippocampal synaptosomal preparations (Schmeisser et al., 2012). The basal hippocampal CA1 synaptic activity was altered (reduction in field Excitatory Post- Synaptic Potentials, fEPSC, mEPSC frequency and increased NMDA/AMPA ratio) and High-Frequency Stimulation-induced LTP (HFS-LTP) was enhanced. The ProSAP1/Shank2^-/-mice showed stereotypies, altered reciprocal social interaction and reduced ultrasonic vocalization (Schmeisser et al., 2012).

In the Shank2^-/- model, a whole brain synaptosomal fractionation revealed increased GluN1 expression together with reduced phosphorylation of CamKII and Extracellular-signal Regulated Kinases 1 and 2 (ERK1/2) (Won et al., 2012). In contrast to the observations conducted in the ProSAP1/Shank2^-/- model, in the Shank2^-/- a reduction in NMDA/AMPA ratio and LTP have been reported together with an impaired LTD (Won et al., 2012) and no alterations have been observed in fEPSP and mEPSC frequency or amplitude. The synaptic alteration discrepancies are

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and 7, but rather technical differences during the recordings. From a behavioral perspective, similar social alterations were found (Won et al., 2012), strengthening the validity of Shank2 KO models as recapitulating behavioral symptoms of ASDs.

4.4 SHANK3

The Shank3 gene is composed of 22 exons and it encodes a 1730 amino acid protein. The gene contains several internal promoters, resulting in many splicing variants and a vast array of proteins (Jiang & Ehlers, 2013). The existence of this transcriptional framework complicates the generation of a total KO for Shank3 but many animal models lacking different isoforms are available allowing the investigation of the specific function of each SHANK3 domain (Figure 2).

SHANK3 over-expression in cerebellar aspiny neurons was able to recruit glutamatergic receptors, inducing the formation of functional dendritic spines and increasing mEPSC amplitude and frequency (Roussignol et al., 2005). On the contrary, the down-regulation of SHANK3 reduced the spine number of hippocampal neurons suggesting a bi-directional control of spine formation and excitatory receptors recruitment. Moreover, SHANK3 down-regulation in hippocampal neuronal cultures was found to reduce mGluR5 expression (Verpelli et al., 2011), establishing a link between SHANK3 expression and mGluR functionality. SHANK3 down- regulation reduced the frequency of mEPSCs and impaired DHPG-induced LTD but mGluR5 positive allosteric modulation (with CDPPB, 3-Cyano-N-1,3-diphenyl-1H- pyrazol-5-yl benzamide) rescued these alterations.

Different animal models have been generated to study the functions of SHANK3 from a molecular, physiological and behavioral perspective (Figure 3). The animal models that lack the Ankirin (ANK)-repeated domain (and the containing isoforms) are the exon4-9B (Bozdagi et al., 2010), the exon 4-9J (Wang et al., 2011), the Shank3a KO (which lacks the exon4-7; Peça, Feliciano, et al., 2011) and the exonΔ9 (Lee et al., 2015). Considering the genomic position of these exons, it is conceivable that, in these KOs, the isoforms whose transcription starts downstream are preserved, leading to alterations caused by the lack of only the ANK-containing isoforms. From a molecular point of view, the expression of synaptic proteins has been accurately analyzed in the exon 4-9J model. In particular, GluA1, GluN2A, Homer1b/c and SAPAP expression is reduced in the forebrain (Wang et al., 2011).

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Similarly, in the exon4-9B^+/- a reduction in GluA1 expression was found (Bozdagi et al., 2010). From a physiological point of view, excitatory transmission, assessed in the CA1 region of the hippocampus, was found to be altered both pre-synaptically (increased mEPSC frequency and reduced Paired Pulse Ratio, PPR) and post- synaptically (reduced mEPSC amplitude and fEPSP), together with an impaired LTP (Bozdagi et al., 2010). Accordingly, a reduction in the fEPSP was present in the striatum of Shank3a KO (Peça, Feliciano, et al., 2011). In addition, the exonΔ9 KO model showed a reduction in the fEPSP at Schaffer Collateral-CA1 synapses without any obvious alteration in mEPSC amplitude, frequency or PPR (Lee et al., 2015). In contrast, in the exon4-9J no differences in excitatory hippocampal basal activity were found but a decrease in the HFS-induced LTP was revealed (Wang et al., 2011).

Interestingly, in the exonΔ9 KO, an increase and a reduction of the miniature Inhibitory Post-Synaptic Current (mIPSC) frequency were found in CA1 and layer 2/3 of the mPFC, respectively (Lee et al., 2015). Although this work showed a role for SHANK3 for inhibitory transmission, these differences could be due to a non-cell autonomous effect. In fact, since SHANK3 is likely expressed at excitatory PSD of inhibitory interneurons, it could influence their output activity.

Behaviorally, the exon4-9J and the exon4-9B have reduced social interaction (Bozdagi et al., 2010; Wang et al., 2011). In the Shank3a KO, social preference was unaltered but social recognition was impaired (Peça, Feliciano, et al., 2011). In contrast with what was reported in the previous models, the exonΔ9 KO model displayed normal social preference and social novelty recognition (Lee et al., 2015).

Alterations in other behavioral domains have been assessed in these KO animal models: stereotypies, such as self-grooming or novel-environment induced rearing were respectively reported for the exon4-9B, exon4-9J and exonΔ9 KO (Bozdagi et al., 2010; Wang et al., 2011; Lee et al., 2015), as well as communication deficits in exon4-9B and exon4-9J KO mice (Bozdagi et al., 2010; Wang et al., 2011). It should be noted that, even if the aforementioned KO models have mutations in the ANK- repeated domain, the physiological and behavioral discrepancies could be due to the different knock-out strategies used: the exon4-9 mice are lacking a larger portion of the ANK-domain compared to the Shank3a (exon4-7) or the more downstream mutated mouse model exonΔ9. Although in all cases the exon removal is impacting the N-terminal region of the protein, a residue of different isoforms could be present,

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The Shank3b KO was generated by targeting the PDZ-domain of SHANK3, (Peça, Feliciano, et al., 2011) (Figure 2). The KO has reduced expression of PSD93, Homer1b/c, SAPAP3, GluA2, GluN2A and GluN2B. While in the CA1 region no major changes in glutamatergic transmission have been found, in the striatum the fPSP, frequency and amplitude of mEPSC were reduced. From a behavioral perspective, the mice showed impaired social preference and recognition together with a reduction in social exploration (Peça, Feliciano, et al., 2011). These mice also displayed high self-grooming levels accompanied with anxiety-like traits, reminiscent of the stereotypies present in ASD patients.

Recently, in an attempt to generate a mouse model lacking all the major isoforms of SHANK3, a more downstream deletion (exon21) has been made in the mouse genome generating the Shank3^ΔC/ΔC model (Kouser et al., 2013). In contrast with the other Shank3 KO, hippocampal PSD fractionation failed to show any major change in the iGluR subunit expression. Importantly, in opposition to what was reported by the in vitro experiments (Verpelli et al. 2005), the Shank3^ΔC/ΔCmouse model showed a strongly up-regulated hippocampal expression of the mGluR5. From a synaptic point

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of view, the hippocampal CA1 synapses exhibited reduced NMDA/AMPA, a diminution in mEPSC frequency and fEPSP. In addition, the HSF-induced LTP was impaired. From a behavioral point of view, the Shank3^ΔC/ΔC mice had a mild impairment in social recognition and, during adulthood, increased self-grooming (Kouser et al., 2013).

In order to mimic the ASD pathogenesis a mouse model lacking the high molecular weight isoforms of SHANK3, Shank3^G/G has been generated by inserting a Guanine nucleotide in the exon21, causing a frameshift mutation found in two autistic brothers (Speed et al., 2015). The targeted exon21 codes for the Homer-binding site that indirectly interacts with mGluRs and possibly controls their function. Similarly to the Shank3^ΔC/ΔC model, synaptosomal preparation from the hippocampus reported a lack of the longest SHANK3 isoforms. However, even if the loss of SHANK3 appears to be similar, the increased mGluR5 expression found in the Shank3^ΔC/ΔCwas absent.

These two models show similar synaptic alterations at hippocampal synapses, such as a reduced NMDA/AMPA ratio, fEPSP and mEPSC frequency. In this model, the HFS-induced LTP was normal but the DHPG-induced LTD was altered, consistent with a possible alteration in the mGluR pathway (Speed et al., 2015). From a behavioral point of view, the mice did not show any social alteration or stereotyped behavior (Speed et al., 2015).

In conclusion, the Shank KO animal models provide an efficient tool to study the genetic alterations underlying the ASDs. Although some discrepancies are present in the above models, they may help to understand the pathological mechanisms underlying the genetic forms of Autism. Due to the technical limitations of generating a complete KO, particularly an absence of specific isoforms from all the neuronal types and compensatory mechanisms, understanding the consequences of the SHANK absence from a specific neuronal circuit is challenging. These technical difficulties may hinder the elucidation of the causal mechanisms that lead to the pathology, thus delaying the development of rationally-designed ASD therapies.

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Transgenic Line Target protein

Excitatory Post-synaptic protein level

Synaptic Physiology Behavior

Shank1^-/- Hung et al., 2008 Silverman et al., 2011 Wöhr et al., 2011

Shank1 Forebrain (PSD)

ê pan-Shank ê Homer ê SAPAP

= GluN1

= GluN2B

Hippocampus (CA1) ê mEPSC frequency

= mEPSC amplitude ê fEPSP

= AMPAR/NMDAR

= LTP (HFS)

= LTD (LFS)

= self-grooming

= social interaction (dyadic)

= social preference (three- chamber)

= social recognition (three- chamber)

ê ultrasonic vocalization

ProSAP1/Shank2^-/- Schmeisser et al., 2012

Shank2e*

Shank2a*

Shank2b*

Hippocampus synaptosome é GluN1

é GluN2B ê Shank1

Striatum synaptosome é GluN1

é GluN2A é GluA2 é Shank3 é PSD95

Hippocampus (CA1) ê fEPSP

ê mEPSC frequency

= mEPSC amplitude é NMDAR/AMPAR é LTP (HFS)

é self-grooming

ê social interaction (dyadic) ê ultrasonic vocalization

Shank2^-/-

Won et al., 2012 Shank2e*

Shank2a*

Shank2b*

Whole brain synaptosome é GluN1

= GluN2A

= GluA2

= mGluR1

= mGluR5 ê pCamKII ê pERK1/2 ê p38 ê pGluA1

Hippocampus (CA1)

= fEPSP

ê NMDAR/AMPAR ê LTP (HFS, TBS) ê LTD (LFS)

= DHPG-LTD

= GluN2B-EPSC

= PPR

= mEPSC frequency

= mEPSC amplitude

é self-grooming ê social interaction ê social preference ê ultrasonic vocalization

Shank3 exon4-9B^+/- Bozdagi et al., 2010

Shank3a*

Shank3b*

Immunohistochemistry ê GluA1

Hippocampus (CA1) ê fEPSP (AMPA) ê mEPSC amplitude é mEPSC frequency ê PPR

ê LTP (HFS; TBS;

TBP)

ê social interaction (dyadic test)

ê ultrasonic vocalization

Shank3 exon4-9J^-/-

Wang et al., 2011 Shank3a*

Shank3b* Forebrain PSD ê Homer1b/c ê SAPAP Forebrain PSD ê GluA1 ê GluN2A

= fPSP

= mEPSC amplitude

= mEPSC frequency ê LTP (HFS)

= PPR

ê social preference (three- chamber)

ê social interaction (dyadic test)

é self-grooming ê ultrasonic vocalization Shank3 exonΔ9

Lee et al., 2015

Shank3a^# Shank3b^#

Hippocampus (CA1) ê fEPSP

= mEPSC amplitude

= mEPSC frequency

= PPR

é mIPSC frequency Prefrontal Cortex (layer 2/3) ê mIPSC frequency

= social preference

= social novelty é rearing

Shank3a KO Peca et al., 2011

Shank3a*

Shank3b*

Striatum ê fEPSP

= social preference ê social recognition (three- chamber)

Shank3b KO Peca et al., 2011

Shank3a*

Shank3b*

Shank3c*

Shank3d*

Striatum (PSD) ê SAPAP3 ê Homer1b/c ê PSD93 ê GluA2 ê GluN2A ê GluN2B

Striatum ê fPSP

ê mEPSC frequency ê mEPSC amplitude

= PPR

= fPSP

= PPR

= mEPSC frequency

= mEPSC amplitude

ê social preference (three- chamber)

ê social recognition (three- chamber)

ê social interaction (dyadic)

Shank3^ΔC/ΔC Kouser et al., 2013

Shank3a^# Shank3c^# Shank3d^#

Hippocampus (PSD) é mGluR5

Hippocampus (CA1) êNMDA/AMPA êfEPSP

ê social recognition (three- chamber)

é self-grooming (in adulthood)

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Shank3e^# Shank3f^#

ê mEPSC frequency ê LTP (HFS) Shank3^G/G

Speed et al., 2015 Shank3a^#

Shank3c^# Shank3d^# Shank3e^# Shank3f^#

Hippocampus (PSD)

= mGluR5

Hippocampus (CA1) êNMDA/AMPA ê fEPSP

ê mEPSC frequency = LTP (HFS) êLTD (DHPG)

= social behavior

= self-grooming

Figure 3. Summary of electrophysiology and social behavior in transgenic lines with mutations in Shank encoding genes.

Adapted from (O'Connor et al., 2014)

Table abbreviations:

*= Shank isoforms as reported in Jiang and Ehlers (2012)

#= non-classified in Jiang and Ehlers (2012) but inferred from exon targeting PSD: Post-synaptic Density

SPM: Synaptic Membrane

HFS: High-frequency stimulation (NMDA-dependent)

In Hung et al., 2008; Schmeisser et al, 2012; Won et al., 2012: 100 Hz for 1 sec. In Bozdagi et al. 2010: 4 trains at 100Hz for 1 sec, separated by a 5 min interval. In Wang et al., 2011: 2 trains at 100 Hz for 1 sec, separated by a 15 sec interval.

LFS: Low Frequency Stimulation (NMDA and mGluR-dependent)

In Hung et al., 2008; Won et al., 2012; Bozdagi et al., 2010; Huber et al., 2002; Sperow et al, 2012; 1 Hz for 15 min TBS: Theta-Burst Stimulation (NMDA-dependent)

In Won et al., 2012: 10 bursts of 4 pulses at 100 Hz, separated by a 200 msec interval, repeated 4 times every 10 sec. In Bozdagi et al., 2010: 15 bursts of 4 pulses at 100 Hz separated by a 200 msec interval

TBP: Theta-Burst pairing

In Bozdagi et al., 2010: 2 trains, separated by 20 sec, of 5 bursts at 5 Hz each containing 5 pulses at 100 Hz PP-LFS: Paired-Pulse Low Frequency Stimulation (mGluR-dependent)

In Bozdagi et al. 2010: Paired-pulse stimulation (50 msec interval) at 1 Hz for 20min. In Schmeisser et al. 2012; Huber et al., 2002; Bateup et al., 2011: Paired-Pulse stimulation (50 msec interval) at 1 Hz for 15 min

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5. ASD and Dopamine: a dysfunctional reward system?

The social motivation hypothesis for ASD has proposed that autistic patients show impairments in the social motivation domain that subsequently lead to alterations in social cognition (Chevallier et al., 2012). Social motivation has been defined as the “psychological dispositions and biological mechanisms biasing an individual to preferentially orient to the social world (social orienting), to seek and take pleasure in social interaction (social reward) and to work to foster and maintain social bonds (social maintaining)” (Chevallier et al., 2012). In this regard, autistic patients are unable to orient their attention toward social stimuli (social orienting;

Osterling et al., 2002), to engage in social interaction/helping behaviors (Liebal et al., 2008), to appropriately respond to social reward versus monetary rewards (social reward; Demurie et al., 2011) and to engage in ingratiating behaviors (social maintenance; Hobson & Lee, 1998).

Considering the role of DA activity in reward processing (Schultz et al., 1997;

Stuber et al., 2008), an intriguing hypothesis is that the lack of social skills is a consequence of alterations in the mesolimbic DA system. Along these lines, a functional Magnetic Resonance Imaging study has found a reduced activation of the Ventral Striatum (VS) after reward presentation in ASD patients compared to non- affected individuals, with a marked reduction in responsiveness to social reward (Scott-Van Zeeland et al., 2010). A possibility is that the VS alterations could be secondary to impairments in midbrain function, but a direct proof of VTA dysfunction in humans is still lacking. Interestingly, rare DAT variants in humans are linked to ASD (Bowton et al., 2014) and D2R functional impairments have been observed in a mouse model of altered sociability (Squillace et al., 2014). Therefore, Autism-related genetic manipulations restricted to the VTA could be relevant to establish a causal link between DA system dysfunction and behavioral impairments.

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6. The Ventral Tegmental Area: a source of Dopamine for the brain

In the midbrain there are three dopaminergic nuclei known as the Retrorubal Field (corresponding to the Broadmann Area, A8), the Substantia Nigra (SN, A9) and the Ventral Tegmental Area (VTA, A10; German & Manaye, 1993). The midbrain DA neurons send projections toward the forebrain regions through three ascendant pathways: the “mesostriatal” pathway, originating from the SN and VTA to the Dorsal and Ventral Striatum (Nucleus Caudatus, Putamen and Accumbens), the

“mesolimbic” pathway, toward the Amygdala, the Septum and the Olfactory Tubercle and the “mesocortical” pathway, which projects to the Prefrontal, Perirhinal and Cingulate Cortices (for review see Björklund & Dunnett, 2007).

The VTA is a nucleus composed of different neuronal populations with distinct electrophysiological properties, including most prominently DA neurons and GABA neurons. In recent years, it has been demonstrated that VTA neurons can co-release DA and glutamate (Stuber et al., 2010) or DA and GABA (Tritsch et al., 2014), thus increasing the complexity of the system. In the following paragraphs, special attention will be dedicated to the DA neurons of the VTA in terms of electrophysiological and connectivity features.

6.1 VTA neurons: neuronal subpopulations and basic physiological properties A study based on immunohistochemistry and in situ hybridization of the rat VTA revealed that 55-65% of neurons express the Tyrosine Hydroxylase enzyme (TH⁺, critical for DA synthesis, approximately 40.000 cells), 30% are positive for the Glutamic Acid Decarboxylase mRNA (GAD⁺, necessary for GABA synthesis, approximately 20.000 cells) and a small percentage (2-3%) expresses the vesicular Glutamate Transporter 2 (vGluT2, for re-uptake and packaging of glutamate), suggesting the existence of intermingled neuronal subclasses (German & Manaye, 1993; Nair-Roberts et al., 2008). The electrophysiological properties of VTA DA and GABA neurons have been characterized both in vitro and in vivo. Typically, a VTA DA neuron displays a low pace-making activity in the range of 2-10 Hz, with bursting episodes composed by 2 to 10 spikes with an inter-spike interval of 80 to 160 msec (for review see Ungless & Grace, 2012). Juxtacellular labeling of TH combined with

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in vivo recordings demonstrated that the presence of a bi- or tri-phasic action potential, with a half-width duration equal to or longer than 1.1 msec, can reliably identify a DA neuron. Two other characteristics of DA neurons are the spike inhibition upon application of DA or a DA agonist (e.g. apomorphine or quinpirole) in vivo and the presence of the hyperpolarization-induced K⁺ current (Ih current) mediated by HCN channels (Hyperpolarization-activated Cyclic Nucleotide-gated channels) in vitro (Ungless & Grace, 2012). However, the medially-located DA neurons have small or absent Ih (Lammel et al., 2014).

During in vivo recordings, the identification of GABAergic neurons of the VTA relies on their rapid and non-bursting pattern of spikes and short-duration action potential (Steffensen et al., 1998; Tan et al., 2012; Bocklisch et al., 2013).

The segregation between GABA-releasing and DA-releasing VTA neurons has been recently questioned. In fact, ChR2 stimulation of a “unique” population of TH⁺ neurons (in TH-Cre:DIO-ChR2, Double-floxed Inverse Open reading frame-ChR2), characterized by small Ih current, quinpirole (D2 agonist) insensitivity and high-firing rate, was able to elicit light-evoked IPSCs and reduce the activity of Lateral Habenula (LHb) neurons (Stamatakis et al., 2013). This population was characterized by lower levels of D2R and DAT mRNAs compared to the TH⁺NAc projecting neurons (Stamatakis et al., 2013).

In addition, DA neurons are able to form inhibitory synaptic contacts with NAc Medium Spiny Neurons (MSNs); in fact, ChR2 expressed in DAT-Cre VTA neurons is able to optically evoke IPSCs sensitive to the GABAA blocker Picrotoxin (Tritsch et al., 2014). However, no expression of GAD enzyme was detected in DAT-Cre (Tritsch et al., 2014) suggesting no overlap between TH⁺-GAD⁺ (Figure 4, in purple;

Stamatakis et al., 2013) and DAT⁺-GAD^- neurons (Figure 4, in blue; Tritsch et al., 2014). The differential expression of the GAD enzyme points to a segregation between the GABA-releasing DA neurons projecting to the NAc or the LHb.

However, the co-release hypothesis for VTA DA neurons has been challenged by the fact that the Cre recombinase in TH-Cre is expressed in non-DA cells and in nuclei adjacent of the VTA, implying that some TH::ChR2 expressing fibers may originate from GAD-expressing GABAergic neurons (Lammel et al., 2015; Stuber et al., 2015).

The third neuronal population in the VTA expresses vGluT2. This subclass can be further divided in 2 subpopulations: one that expresses vGluT2-only (Yamaguchi

SHANK3 controls maturation of social reward circuits in the VTA

Thesis

Reference