Mapping VTA neural circuits: periaqueductal inputs and mesohippocampal outputs

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Thesis

Reference

Mapping VTA neural circuits: periaqueductal inputs and mesohippocampal outputs

NTAMATI RWAKA, Niels

Abstract

Understanding how the ventral tegmental area (VTA) shapes behaviour to avoid aversive stimuli and seek reward requires knowledge of its input and output connectivity within the network of brain structures collectively known as the reward system. In this work we employed tracing techniques and optogenetics to electrophysiologically map periaqueductal afferents to - and mesohippocampal efferents from - the VTA. We show that periaqueductal afferents are mostly excitatory and primarily supplied by the ventral subdivisions of the periaqueductal gray (PAG), implicated in opioid analgesia and threat responses, targeting both VTA dopamine and GABA neurons equally. We additionally provide the first evidence for a direct mesohippocampal projection mediated by non-dopaminergic neurons co-releasing glutamate and GABA in the dentate gyrus (DG), crucially involved in memory formation. Altogether, this work expands on the existing circuit understanding of the VTA and provides fundamental insights into how its PAG and DG connections might serve motivationally-relevant functions.

NTAMATI RWAKA, Niels. Mapping VTA neural circuits: periaqueductal inputs and mesohippocampal outputs. Thèse de doctorat : Univ. Genève et Lausanne, 2017, no. Neur.

205

DOI : 10.13097/archive-ouverte/unige:95390 URN : urn:nbn:ch:unige-953901

Available at:

http://archive-ouverte.unige.ch/unige:95390

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

UNIVERSITE DE GENEVE FACULTE DES SCIENCES

Professeur Christian Lüscher, directeur de these

MAPPING VTA NEURAL CIRCUITS:

PERIAQUEDUCTAL INPUTS AND MESOHIPPOCAMPAL OUTPUTS

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

pour obtenir le grade de Docteur en Neurosciences

par

Niels NTAMATI RWAKA

de Italie Thèse N° 205

Genève

Editeur ou imprimeur : Université de Genève 2017

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actuellement en cours de révision.

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I would like to show my warmest gratitude to all those who directly or indirectly contributed to bringing this work to fruition and supported me during these years of doctoral studies.

First, I would like to thank Prof. Christian Lüscher for the opportunity to work in his laboratory, for his motivation, and for his patience in supervising my research, which has been fundamental for my growth as a scientist. I am also grateful to Prof. Alan Carleton, Prof. Anthony Holtmaat and Prof. Manuel Mameli, for serving as my committee members and for putting time and effort in discussing my thesis work.

A special thank goes to all past and present members of the Lüscher laboratory with whom I spent these (mostly) entertaining years. In particular, I would like to thank Prof. Kelly Tan for guiding me at the beginning of this journey, and Prof. Meaghan Creed for lending me her expertise and motivational support. Also thanks to Sebastiano Barisello, Ricola Platt, Stephano Tomaselli and Petite Corre for the fun moments and the shared struggles in the lab, and big thanks to Christina the Greek, Daniel Lumprecht, Carmen Bonferroni, that tall Eastern-European with the moustache and all of the friends with whom I enjoyed many extracurricular laughs, goofs and gaffes.

I would like to thank my mother, my brother Vladimir and my sister Else for their continuous encouragement and support, and, finally, thanks to Bea, for her unconditional love and constant smiles.

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Comprendre comment l’aire tegmentale ventrale (VTA) façonne le comportement afin d’éviter les stimules aversifs et rechercher la récompense exige la connaissance de sa connectivité d’entrée et de sortie au sein du réseau des structures cérébrales connus collectivement sous le nom de "système de la récompense". Dans le présent travail, nous avons employé des techniques de traçage viraux et non viraux, ainsi que des enregistrements électrophysiologiques à l’aide de l’optogénétique, pour caractériser les connexions afférentes périaqueducales et celles efférentes mesohippocampales de la VTA.

Nous montrons ici que les premières sont fournie principalement par les subdivisions ventrales de la substance grise périaqueducale (PAG), qui sont impliquées dans l’analgésie opioïde et la réaction d’immobilisation dans les situations de menace. De plus, nous démontrons que les neurones dopaminergique et GABAergique de la VTA sont visés de la même manière par ces projections principalement excitatrices. Nous fournissons aussi la première preuve électrophysiologique d’une projection mesohippocampale gérée par des neurones non dopaminergiques de la VTA qui libère les deux neurotransmetteurs GABA et glutamate dans le gyrus denté (DG), qui est impliqué dans la formation des souvenirs. En tout, le présent travail développe la compréhension actuelle du câblage anatomique de la VTA et jette un éclairage neuf sur sa potentielle interaction avec la PAG et le DG dans les comportements motivés.

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Understanding how the ventral tegmental area (VTA) shapes behaviour to avoid aversive stimuli and seek reward requires knowledge of its input and output connectivity within the network of brain structures collectively known as the reward system. In the present work we employed viral and non-viral neuronal tracing techniques, together with optogenetically assisted electrophysiological recordings to map periaqueductal afferents to - and mesohippocampal efferents from - the VTA. We show here that periaqueductal afferents are primarily supplied by the ventral subdivisions of the periaqueductal gray (PAG), implicated in opioid-induced analgesia and freezing responses to threat stimuli, and that VTA dopamine and GABA neurons are equally targeted by these mostly excitatory projections. We additionally provide the first evidence for a direct mesohippocampal projection mediated by non-dopaminergic neurons co-releasing glutamate and GABA in the dentate gyrus (DG), which is crucially involved in memory formation. Altogether, this work expands on the existing circuit understanding of the VTA and provides fundamental insights into how its interaction with the PAG and DG might serve motivationally-relevant functions.

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List of abbreviations

5HT 5-hydroxytriptamine (serotonin) AADC L-amino acid decarboxylase ALDH1a1 aldehyde dehydrogenase 1a1 AMPA alpha-amino-hydroxy-methyl- isoxazolepropionic acid

Bic bicuculline

BNST bed nucleus of the stria terminalis

CA(1-3) cornu ammonis (1-3) ChR2 channelrhodopsin 2 CNS central nervous system

DA dopamine

DAPI diamidino-phenylindole DAT dopamine transporter

DG dentate gyrus

DIO double-floxed inverse open reading frame

DR/DRN dorsal raphe nucleus

EYFP enhanced yellow fluorescent protein

GABA gamma-aminobutyric acid GAD(65,67) glutamic acid decarboxylase (65 kDa, 67 kDa)

Glu glutamate

hsyn human synapsin

Kyn kynurenic acid

LDT/LDTg laterodorsal tegmental nucleus LHG/LHT lateral hypothalamus

LHb lateral habenula

MSN medium-sized spiny neuron

NA noradrenaline

NAc/nAcc nucleus accumbens NBQX dihydroxy-nitro-sulfamoyl- benzo[f]quinoxaline-dione

NMDA N-methyl-D-aspartate PAG periaqueductal gray (m)PFC (medial) prefrontal cortex PPR paired-pulse ratio

PPTg pedunculopontine tegmental nucleus

PSC postsynaptic current

PTX picrotoxin

RabG rabies envelope glycoprotein RMTg rostromedial tegmental nucleus RVG RabG-deficient rabies virus RVM rostral ventromedial medulla

SN substantia nigra

TH tyrosine hydroxylase VGAT vesicular GABA transporter VGLUT(1-3) vesicular glutamate transporter (1-3)

VP ventral pallidum

VTA ventral tegmental area

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This principle is not restricted to normative ethics but can also be understood as a basic tenet of biology and, more specifically, ethology, since all voluntary behaviors are driven by the motivation to avoid aversive behavioral states and seek reward. In mammalian brains, the fine balance between these motivated behaviors is carefully orchestrated by the activity of an assembly of interconnected neural structures collectively known as the reward system. Over the decades, researchers took growing interest in this system and set out to achieve a clear picture of its anatomical and physiological architecture. This is of paramount importance, as an understanding of the biology underlying motivated behaviors in physiology and pathology requires knowledge of the neural pathways connecting the core of the reward system - the ventral tegmental area (VTA) - to its various upstream and downstream partners.

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2 The following chapters will thus provide an overview of the VTA, its subpopulations, and the neuronal pathways wiring it to other reward system regions, with a focus on the hippocampus and the periaqueductal gray (PAG).

1 A historical overview of the VTA

The concept of a reward system - a collection of brain structures appointed to the encoding and expression of the pleasure (liking) and the incentive salience (wanting) associated with rewarding stimuli - took form over sixty years ago, after the seminal findings of psychologists James Olds and Peter Milner (Olds and Milner, 1954). Their investigation on the rewarding properties of intracranial electrical self-stimulation identified the septal area as a region capable to induce a strong and compulsive reinforcing behavior. In subsequent years, these findings were corroborated by a series of human studies conducted by a team of researchers lead by psychiatrist Robert G. Heath (Bishop et al., 1963; Moan and Heath, 1972). These clinical studies, while ethically controversial now, were important in supporting the results obtained in animal studies and confirming the involvement of the septal area in rewarding responses (Baumeister, 2000). Further investigation into the identity of the neurotransmitters released in this region would have soon established dopamine (DA) as the key molecule involved in reward-related neural signaling, and the ventral tegmental area (VTA) as its principal source (Miliaressis and Cardo, 1973; Mogenson et al., 1979; Vives et al., 1983; Wise and Bozarth, 1984).

The identification of DA in the central nervous system (CNS), however, could not happen until the development of the Falck-Hillarp fluorescence method (Falck et al., 1962). This sensitive histochemical technique allowed, for the first time, to visualize monoamines like DA, noradrenaline (NA) or serotonin (also known as 5-hydroxytriptamine, 5HT) in the CNS.

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3 This soon lead to the identification of the catecholaminergic (NA and DA) cell groups A1 through A16, and the indolaminergic (5HT) cell groups B1 through B9 (Björklund and Dunnett, 2007; Dahlstroem and Fuxe, 1964; Schofield and Dixson, 1982). While the differentiation between NA (A1-A7) and DA cell groups (A8-A16, Fig. 1) initially required particular chemical manipulations of the brain samples in order to enhance the different fluorimetric properties of these molecules (Björklund et al., 1968; Lindvall et al.), further understanding of the catalytic steps involved in catecholamine synthesis together with the development of immunostaining protocols allowed for a simpler and more reliable distinction between NA- and DA-releasing neurons, as will be detailed in the following section.

FIGURE 1 | Dopaminergic cell nuclei. Schematic illustrating the distribution of the main DA cell groups within the CNS, along with their main projections. The VTA corresponds to the A10 cell group. (Modified from Björklund and Dunnett, 2007)

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4 2 VTA neuronal populations

The VTA, a collection of neurons situated in the midbrain, medial to the substantia nigra (SN) and just dorsal and lateral to the interpeduncular nucleus (Brown, 1943; Tsai, 1925), was initially characterized as a DA-releasing nucleus, due to its anatomical overlapping with the largest dopaminergic nucleus of the midbrain – the A10 cell group. However, further analysis unveiled the heterogeneity of the neuronal populations inhabiting this region, identifying significant number of neurons releasing the inhibitory neurotransmitter gamma- amino butyric acid (GABA) (Margolis et al., 2012; Olson and Nestler, 2007) as well as a smaller amount of neurons releasing the excitatory neurotransmitter L-glutamate (Yamaguchi et al., 2007). In more recent years, more sophisticated genetic, electrophysiological and pharmacological approaches allowed for a better elucidation of the complexity of the VTA, identifying several neuronal populations presenting mixed DA- glutamate, DA-GABA and GABA- glutamate phenotypes.

The aim of the following sections will thus to explore more deeply the cellular composition of the VTA, with a focus on their neurochemical identity and known function.

2.1 Dopamine-releasing neurons

The principal cellular subpopulation of the VTA, constituting over 60% of all the neurons in this region, is represented by DA-releasing neurons (Margolis et al., 2006a; Swanson, 1982).

The synthesis of DA, as that of all neurotransmitters of the catecholamine group, starts from the L-amino acid tyrosine. Through the activity of the enzymes tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC, also known as DOPA decarboxylase), tyrosine is first converted into L-dihydrophenylalanine (L-DOPA) and finally into DA (Kuhar et al.,

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5 1999). Cytoplasmic DA is the transported inside synaptic vesicles by the vesicular monoamine transporter 2 (VMAT2) and, following synaptic release, it is reuptaken from the extrasynaptic space through the presynaptic membrane DA transporter (DAT) (Eiden et al., 2004; Torres et al., 2003). Any and all of these proteins, as well as the transmitter intermediates themselves, could therefore be selected as molecular markers for DA neurons. It should be noted, however, that TH is the rate-limiting enzyme involved in the synthesis of all catecholamines, and would therefore not be suitable by itself for the distinction of DA neurons from NA neurons. These neurons follow the identical enzymatic steps for the synthesis of DA but also express an additional enzyme, DA beta-hydroxylase (DBH), that catalyzes the conversion of DA into NA inside presynaptic vesicles of noradrenergic axonal terminals (Kuhar et al., 1999). Prototypical DA neurons would therefore be identified by the expression of TH, DAT, VMAT2 and the exclusion of DBH. In practice, however, the DBH exclusion is always implicit fact when studying VTA, which has long been established as a dopaminergic nucleus.

In the past decades, the Cre/loxP DNA recombination system (Hamilton and Abremski, 1984; Voziyanov et al., 1999) has been widely exploited in the generation of conditional transgenic mouse lines and has proven paramount in the study of genetically-identified cellular populations (Nagy, 2000; Rossant and McMahon, 1999). Two such transgenic lines that have been extensively used by researchers in order to selectively target midbrain DA neurons are the TH-Cre and the DAT-Cre mouse lines, in which the selective Cre/loxP recombination is restricted in the cells expressing the TH or DAT genes, respectively (Gelman et al., 2003; Zhuang et al., 2005). Therefore, it should be expected that either mouse line provide a reliable method to identify VTA DA neurons. However, an extensive

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6 analysis performed by Li and colleagues in 2013 suggested a more complex expression pattern of the aforementioned DA markers (Li et al., 2013). The authors employed immunohistochemistry, in situ hybridization and quantitative reverse transcription polymerase chain reaction (qRT-PCR), to visualize and quantify these markers across the A10 region. Their study revealed that, virtually all TH-immunolabelled neurons have the ability to synthesize DA - as indicated by the co-expression of the AADC mRNA, but have a differential capacity to store and reuptake the neurotransmitter, with medial neurons expressing lower to no levels of VMAT2 and DAT compared to lateral neurons. A similar finding had previously been described also in the mouse VTA, where medial DA neurons display lower DAT expression levels compared to more lateral DA subpopulations (Lammel et al., 2008).

This VTA DA molecular heterogeneity is also paralleled by an equally varied electrophysiological profile. Classically, DA neurons have been identified by the presence of a large hyperpolarization-activated current (Ih), large action potential width, low firing frequency (<10Hz) and sensitivity to dopamine D2 receptors (D2Rs) (Chiodo, 1988; Ungless and Grace, 2012) (Fig. 2). However, the reliability of these electrophysiological markers, which were generally used to distinguish DA neurons from surrounding GABA neurons, has often been debated, especially in light of their variation across the medio-lateral axis of the VTA (Margolis et al., 2006a). For instance, Lammel and colleagues (Lammel et al., 2008) have shown that different subgroups of VTA DA neurons, often characterized by a distinct projection profile - e.g. projecting to the medial prefrontal cortex (mPFC) vs. the lateral shell of the nucleus accumbens (NAc), display a markedly smaller or absent Ih, and fire action potentials with a larger width and at a higher average frequency in medial compared to lateral DA neurons.

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7 Due to the molecular and electrophysiological diversity of VTA DA neurons, each single method employed for their identification will necessarily come with its own shortcomings, and the best possible outcome will only be obtained by a combination of these approaches.

FIGURE 2 | Classical electrophysiological markers for DA and non-DA neurons. Based on the initial characterization in the SN, DA (left) and non-DA neurons (right) where distinguished based on their action potential width (top), spontaneous firing rate (middle) and presence of an Ih current (or lack thereof, bottom). (Modified from Lacey et al., 1989)

For instance, the uneven expression of DAT across the mediolateral axis of the VTA (Lammel et al., 2008; Li et al., 2013) might, at least in principle, result in an incomplete and more lateral-biased selection of VTA DA neurons with DAT-Cre mice. Similarly, the use of the TH- Cre line is insufficient to confirm a dopaminergic identity, as the TH gene could be active and be expressed at the mRNA level but lead to no detectable levels of TH protein in some neurons (Yamaguchi et al., 2015). This is particularly important when investigating the VTA

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8 subpopulations that express markers for multiple neurochemical identities and therefore cannot be defined as dopaminergic (Stamatakis et al., 2013), as will be discussed later.

2.2 GABA-releasing neurons

The second major neuronal grouping of the VTA consists of neurons synthetizing and releasing GABA (Margolis et al., 2012; Olson and Nestler, 2007), which account for 35% of the total VTA neuronal population (Nair-Roberts et al., 2008; Taylor et al., 2014). These neurons are scattered throughout the VTA, but are more concentrated in a rostrally and dorsolaterally, as well as caudally, where GABA neurons form ovoid clusters flanking the interpeduncular nucleus (Jhou et al., 2009a; Olson and Nestler, 2007). This caudal GABA cluster has recently been described as a separate structure - the rostromedial tegmental nucleus (RMTg)(Jhou et al., 2009b; Lavezzi and Zahm, 2011), which densely projecting to VTA DA neurons, whereas some researchers have labelled it the tail of the VTA, emphasizing its contiguity and the lack of a clear boundary with the VTA (Bourdy and Barrot, 2012;

Perrotti et al., 2005).

GABA is the principal inhibitory neurotransmitter in the adult brain and is mainly produced from the amino acid glutamate in the presence of either of the two known glutamic acid decarboxylase (GAD) isoforms – GAD1 and GAD2 (Schousboe and Waagepetersen, 2007).

These two enzymes are encoded by separate genes and have a different molecular weight of 67 kDa and 65 kDa, respectively, hence their alternative names GAD67 and GAD65. Since the demonstration of their selective expression in GABA-synthetizing neurons, the two GADs have long been employed as a reliable marker of GABAergic neurons regardless of the isoform identity (Ribak et al., 1976; Saito et al., 1974). Nonetheless, there is the possibility that the GAD65 and the GAD67 neuronal populations might not be totally overlapping, as

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9 evidence exists of their differential expression regulation and preferential subcellular localization - GAD65 being enriched in synaptic terminals, while GAD67 is more uniformly distributed (Martin and Rimvall, 1993). Another marker employed for the identification of GABA-releasing neurons is the vesicular GABA transporter (VGAT), which is responsible for the neurotransmitter packaging in the presynaptic boutons (Chaudhry et al., 1998; McIntire et al., 1997). Consequently, these markers have been used to visualize GABA neurons through in situ hybridization, immunohistochemistry or, with the advent recombinant DNA technology, through the generation of transgenic mouse lines expressing fluorescent reporters or the Cre recombinase under their promoters’ regulation (López-Bendito et al., 2004; Tamamaki et al., 2003; Tan et al., 2012; Wang et al., 2009; van Zessen et al., 2012).

While the GAD or VGAT expression is sufficient for the identification of GABA neurons, the use of these markers can still incur some level of false negatives, as researchers have shown GABA can be obtained by an alternative synthetic pathways that doesn’t require the GADs.

One such pathway relies on the metabolism of putrescine, an intracellular polyamine, and subsequent synthesis of GABA by aldehyde dehydrogenase 1a1 (ALDH1a1) (Sequerra et al., 2007; Shelp et al., 2012; Yamasaki et al., 1999). However, while this alternative pathway has been described in the developing mammalian brain, in the adult brain it has only been characterized in a subgroup of mesostriatal neurons releasing both DA and GABA, and has yet to be demonstrated in “pure” GABA neurons (Kim et al., 2015).

2.3 Glutamate-releasing neurons

The third neuronal subgroup known to populate the VTA consists of neurons releasing glutamate, the major CNS excitatory neurotransmitter. Glutamate is an amino acid that can be synthetized from the amino acid glutamine by the enzyme glutaminase, which is

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10 expressed in several neuronal and glial cells in the brain (Hogstad et al., 1988; Kaneko and Mizuno, 1988; Kaneko et al., 1990; Weiler et al., 1979).

The first suggestion of a glutamatergic connectivity originating from the midbrain came from a study of long-range mesocortical projections, in which electrical stimulation of the VTA gave rise to fast excitatory responses in the rat frontal cortex (Mercuri et al., 1985).

Subsequently, visual identification of these neurons usually relied on their expression of vesicular glutamate transporters (VGLUTs), of which three isoforms are known to exist in the mammalian brain – VGLUT1, 2 and 3 (Shigeri et al., 2004). While VGLUT3 has been observed in multiple neuronal types, including serotonergic, cholinergic and GABAergic neurons, as well as in glia (Fremeau et al., 2002; Gras et al., 2002; Schäfer et al., 2002), VGLUT1 and VGLUT2 have been more consistently used as reliable markers restricted to glutamatergic neurons (Bellocchio et al., 1998, 2000; Fremeau Jr. et al., 2001; Takamori et al., 2001). An mRNA expression analysis performed by Kawano and colleagues showed that VGLUT2 was widely expressed in discrete cell groups of the ventral midbrain, while VGLUT1 and VGLUT3 showed little or no expression (Kawano et al., 2006). Consistently, VGLUT-2 positive neurons have been described in the VTA by different groups and estimated to account for a small proportion of the total population (2-5%) yet highly enriched in more rostral and medial segments of the VTA (Hnasko et al., 2012; Nair-Roberts et al., 2008). The existence of this small population of glutamatergic neurons has thus been demonstrated to be conserved across mammals, being present in rodents, nonhuman primates and humans (Root et al., 2016; Yamaguchi et al., 2007, 2015) and confirmed to establish functional, asymmetric synaptic contacts with electrophysiology and electron microscopy (Wang et al., 2009).

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11 2.4 Mixed-phenotype neurons

In 1954, John C. Eccles proposed that, from a single neuron, “the same chemical transmitter is released from all the synaptic terminals”, in describing what he called Dale’s principle (Eccles et al., 1954). While this assertion came from a time when the only two neurotransmitters known were acetylcholine and noradrenaline, the interpretation of this principle was progressively challenged as new transmitters and peptides where discovered to be released in the CNS, especially when intended to imply the release of a single transmitters from all of a neuron’s synapses (Nicoll and Malenka, 1998). This concept is particularly relevant in the VTA, where co-release of multiple chemicals from the same neurons has been extensively described. Neurotransmitter-neuropeptide co-expression has been observed by several groups in the VTA, demonstrating the presence of brain-derived neurotrophic factor, neurotrophin-3, corticotropin-releasing hormone, neurotensin and cholecystokinin in subsets of VTA DA and GABA neurons (Grieder et al., 2014; Jayaraman et al., 1990; Olson and Nestler, 2007; Seroogy et al., 1988, 1994). Similarly, co-release of two neurotransmitters (or a neurotransmitter and a neuromodulator, in the case of DA), has been reported to occur through distinct possible mechanisms in several VTA subpopulations (Fig. 3).

Early evidence of DA and glutamate co-release came from in vitro studies showing how dissociated VTA DA neurons establish glutamatergic synapses and express VGLUT2 in culture (Dal Bo et al., 2004; Sulzer et al., 1998). Later in 2004, Chuhma and colleagues also suggested DA neurons could mediate fast glutamatergic signaling in an ex vivo slice preparation encompassing the VTA-NAc pathway, with a stimulation protocol that, however, lacked genetic specificity and relied on the known pharmacological modulation of

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12 VTA DA neurons by D2 agonists (Chuhma et al., 2004).The co-expression of TH and VGLUT2 in single neurons was subsequently demonstrated, in rodents as well as in primates, by several studies investigating the mRNA and protein expression patterns of dopaminergic and glutamatergic markers (Kawano et al., 2006; Root et al., 2016; Yamaguchi et al., 2015).

While representing a small fraction of all VTA DA or glutamate neurons, their density is particularly high around the midline nuclei of the VTA (Yamaguchi et al., 2011). However, while these mixed-phenotype, TH- and VGLUT2-positive neurons also express AADC, which makes them potentially able to synthetize DA, it appears only a fraction of these mixed- phenotype neurons is able to sustain its synaptic release and recycle, as some lack VMAT or DAT (Li et al., 2013). Nonetheless, with the help of optogenetics, several groups have since provided voltammetric and electrophysiological confirmation that co-release of the two transmitters, from separate microdomains of the same VTA-NAc axons, does indeed occur (Stuber et al., 2010; Tecuapetla et al., 2010; Zhang et al., 2015).

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13 FIGURE 3 | Mechanisms of neurotransmitter co-release. Co-transmission from the same neuron can occur through several mechanisms, including the packaging in the same vesicle through the same (a) or distinct (b) vesicular transporters. Alternatively, a heterovesicular storage can lead to the release of neurotransmitters from separate vesicle pools in the same presynaptic bouton (c) or in distinct release sites (d). Co-transmission from the same presynaptic neuron could have different effects if the postsynaptic neurons do not express receptors for all of the co-released neurotransmitters (e).

(Tritsch et al., 2016)

Similarly, the co-release of GABA from DA terminals has been demonstrated to occur in the striatum and the NAc (Kim et al., 2015; Tritsch et al., 2012, 2014). Originally, Tritsch and colleagues proposed that co-release from these neurons does not rely on GABA synthesis through any of the GAD enzymes nor it depends on VGAT for vesicle filling in the axonal terminals. In alternative to the conventional GABA synthesis and accumulation mechanism, they showed that these neurons rely on uptake from the extrasynaptic space and on VMAT- mediated vesicle loading to sustain the co-release of GABA (Tritsch et al., 2012, 2014). Later,

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14 Kim and colleagues expanded on these findings demonstrating that these DA/GABA co- releasing neurons, while lacking the canonical GABA synthetizing machinery, are still capable of endogenous GABA synthesis and rely on the alternative ALDH1a1-dependent pathway for its production (Kim et al., 2015).

More recently, GABA and glutamate co-releasing neurons have also been described in VTA neurons projecting to the lateral habenula (LHb) (Root et al., 2014a). In this study, Root and colleagues characterized a subpopulation of TH-negative neurons that express VGLUT2, GAD and VGAT and make symmetric and asymmetric synaptic contacts within a single axon terminal onto postsynaptic LHb dendritic spines or dendrites expressing GABAA receptors adjacent to glutamatergic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Electrophysiological recordings ex vivo and in vivo confirmed these neurons’ ability to elicit dual fast exhitatory/inhibitory responses onto LHb neurons, suggesting a possible role of these VTA projections in controlling LHb activity in a finely- tuned and temporally-specific fashion. In the present thesis, we will see how GABA/glutamate co-release could be a feature shared by other VTA projection neurons.

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15 3 Afferent connectivity of the VTA

From the initial discovery of the A10 dopaminergic cell group populating the VTA, anatomical investigation on the input connectivity of the VTA gradually evolved from lesion studies and tract tracers to, more recently, genetically-encoded viral tracers and transgenic mice (Oades and Halliday, 1987; Yetnikoff et al., 2014). With these techniques, researchers have identified over the years a massively convergent system of afferents originating from the cortex, the basal forebrain, the hypothalamus and the brainstem (Carr and Sesack, 2000;

Fadel and Deutch, 2002; Omelchenko and Sesack, 2005, 2010; Omelchenko et al., 2009;

Phillipson, 1979; Wallace et al., 1992; Watabe-Uchida et al., 2012) (Fig. 4). Here will be provided an overview of the main glutamatergic, GABAergic and neuromodulatory VTA afferents, and the chapter will conclude with an introduction of the PAG, whose connection to the VTA will be further characterized in this thesis.

FIGURE 4 | Known VTA afferents. Illustration of the confirmed monosynaptic inputs to VTA DA (a) and GABA (b) neurons. Yellow circles: dopaminergic neurons; red circles: glutamatergic neurons;

blue circles: GABA neurons; blue triangle: GABAergic medium-sized spiny neuron. (Modified from Morales and Margolis, 2017).

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16 3.1 Excitatory inputs

The principal excitatory affererents of the VTA arise from the PFC (Sesack and Pickel, 1992) and the lateral hypothalamus, which, in addition to glutamate, also releases the peptides orexin and alpha-melanocyte-stimulating hormone (Fadel and Deutch, 2002; Semba and Fibiger, 1992). The amygdala was also shown to modulate VTA DA neurons, yet not within the VTA itself. As observed by Howland and colleagues, stimulation of the basolateral (BLA), but not the central nucleus of the amygdala (CeA), is able to elicit DA release from VTA terminals in the PFC and the NAc (Howland et al., 2002). This effect can be blocked by the local infusion of AMPA receptor antagonists in the PFC or NAc but not directly in the VTA, suggesting an axo-axonic interaction with DA fibers.

The bed nucleus of the stria terminalis (BNST), whose glutamatergic neurons are activated by noxious stimulim was recently shown to innervate the VTA (Jennings et al., 2013).

Optogenetic activation of these fibers demonstrated that they elicit aversive and anxiogenic behaviors through a direct excitation of VTA GABA neurons. Additionally, VTA GABA neurons (as well as GABAergic neurons in the tail of the VTA/RMTg) receive strong excitatory inputs from the LHb that have been shown to signal behavioral aversion (Faget et al., 2016; Jhou et al., 2009a; Lecca et al., 2012). At the same time, LHb also sends direct projections to VTA DA neurons, targeting preferentially a medially-located subset of mPFC-projecting neurons that has been proposed to be activated by aversive stimuli (Lammel et al., 2011, 2012)

Several lines of evidence indicate that the excitatory inputs regulating VTA DA activity are subject to experience-dependent plasticity. For instance, rewarding experiences (e.g.

cocaine administration) have been extensively investigated in their ability to induce synaptic

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17 changes leading to a long lasting increased efficacy of glutamatergic transmission (Bellone and Lüscher, 2006; Mameli et al., 2009; Ungless et al., 2001). Intriguingly, whereas psychoactive substances with no addictive liability (e.g. fluoxetine) fail to induce the same synaptic adaptations, aversive experiences such as glucocorticoid-dependent stress, food restriction or formalin-induced inflammatory pain are also able to enhance the excitatory drive onto VTA DA (Branch et al., 2013; Daftary et al., 2009; Lammel et al., 2011; Saal et al., 2003). As will be discussed later, however, these two types of reward- and aversion-induced plasticity tend to occur on segregated populations of VTA DA neurons that differ in their electrophysiological properties and projection targets (Lammel et al., 2011).

3.2 Inhibitory inputs

Another major input to the VTA is provided by GABAergic neurons of the ventral pallidum (VP) and the NAc (Faget et al., 2016; Geisler and Zahm, 2005; Groenewegen et al., 1993;

Kalivas et al., 1993). The accumbal fibers, in particular, have been shown to arise from medium-sized spiny neurons expressing the D1 DA receptor (D1-MSNs), as well as the peptides substance P and dynorphin (Fallon et al., 1985; Lu et al., 1998). Recently, an anatomical study that employed rabies virus for neurotransmitter-defined retrograde monosynaptic quantification of VTA inputs, concluded that NAc afferents target preferentially DA neurons, compared to GABA or glutamate (Faget et al., 2016) (Fig. 5). This is however in contrast with earlier electrophysiological demonstration that NAc D1-MSNs elicit inhibitory postsynaptic responses only in VTA GABA neurons (Bocklisch et al., 2013; Xia et al., 2011), indicating how anatomical evidence of a high fiber convergence does not necessarily reflect a strong functional connection in terms of synaptic currents.

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18 An additional source of inhibition is provided by the BNST, which, aside from the excitatory projections described in the previous section, also sends a direct inhibitory input onto VTA GABA neurons (Jennings et al., 2013). This pathway, whose activity is normally suppressed following noxious stimulation, produces rewarding and anxiolytic effects upon selective optogenetic activation. Finally, inhibitory inputs are also provided by GABA neurons in the tail of the VTA/RMTg as well as local VTA GABA themselves, that inhibit VTA DA neurons leading to a strong behavioral aversion (Jhou et al., 2009b; Tan et al., 2012).

FIGURE 5 | Rabies-assisted retrograde tracing. EnvA-pseudotyped, RabG (RG)-deficient rabies virus is used to selectively infect neurons expressing TVA and RG (starter cells), from which input neurons are trans-synaptically labeled. (Modified from Watabe-Uchida et al., 2012)

3.3 Other brainstem inputs

In the brainstem, the peduncolopontine tegmental nucleus (PPTg) and the laterodorsal tegmental nucleus (LDT) are an additional source VTA afferents, providing a significant number of glutamatergic, GABAergic and cholinergic inputs (Cornwall et al., 1990; Oakman et al., 1995; Semba and Fibiger, 1992). PPTg neurons, which receive a large input from the amygdala and project several midbrain structures, have been proposed to relay

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19 somatosensory signals to the VTA, as their pharmacologic inactivation was shown to suppress the conditioned sensory responses of VTA DA neurons associated with rewards (Pan and Hyland, 2005; Semba and Fibiger, 1992). LDT neurons, receiving a strong input from the PFC and projecting primarily to the VTA, is thought to regulate the burst firing of VTA DA neurons (Lodge and Grace, 2006). The LDT-VTA pathway has been shown to be segregate depending on the neurotransmitter identity and the projection-target of VTA neurons. For instance, excitatory and inhibitory afferents from the LDT make synaptic contacts with both PFC-projecting DA and GABA neurons, whereas NAc-projecting DA and GABA neurons receive exclusively cholinergic or inhibitory inputs from the LDT (Omelchenko and Sesack, 2005).

Among the serotonergic raphe nuclei clustering along the brainstem midline, the dorsal raphe nucleus (DR) has long been known to provide an input to both TH-positive and TH- negative neurons in the VTA (Hervé et al., 1987). Alongside 5HT, the DR also contains neurons releasing DA, GABA and glutamate, the latter of which constitute the majority of the DR-VTA projections and mediate reward behaviors by exciting VTA DA neurons in a 5HT- independent but glutamate-dependent fashion (Charara and Parent, 1998; Fremeau et al., 2002; Hioki et al., 2010; McDevitt et al., 2014). Surrounding the DR, the PAG also sends projections to VTA neurons (Faget et al., 2016; Omelchenko and Sesack, 2010), but information on their neurochemical identity, subregional distribution or their electrophysiological function is still lacking.

3.4 Periaqueductal gray

The PAG is a dense and heterogeneous collection of neurons that surround the midbrain aqueduct, a canal connecting the third and fourth cerebral ventricles. This structure has

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20 been implicated in the modulation of a plethora of biological functions, including aggression, maternal behavior, cardiovascular and respiratory function (Dampney et al., 2013; Lee and Gammie, 2010; Sukikara et al., 2010). Most of the studies on the PAG, however, have focused on its role in mediating analgesia and fear responses (Carrive, 1993;

Heinricher et al., 2009). Early evidence of the PAG’s involvement in pain modulation came from experiments performed in rats and cats in which intracranial electrical stimulation was shown to strongly reduce or totally abolish responsiveness to noxious stimuli in a manner similar to morphine-induced analgesia (Liebeskind et al., 1973; Mayer and Liebeskind, 1974).

Since then, researchers have characterized the PAG as being a major site of action for opiate analgesics like morphine, as well as housing several endogenous opioid-expressing neurons (Simantov et al., 1977; Yaksh et al., 1976). Consequently, PAG stimulation leads to the release of endogenous opioids in the rostral ventromedial medulla (RVM), an area that, like the PAG, contains subpopulations of pain-responsive (ON and OFF cells) and non-responsive (neutral cells) (Heinricher et al., 1987; Morgan et al., 2008). In turn, the RVM sends serotonergic and non-serotonergic projections down to trigeminal and spinal interneurons in order to suppress the transmission of nociceptive signals (Fields, 2000; Mason, 1999).

Another striking observation was that electrical stimulation of the PAG, particularly its dorsal segment, can also produce aversive reactions and lead to the development of conditioned fear responses (Di Scala et al., 1987). Similarly, pharmacological block of GABAergic transmission in the PAG causes behavioral activation and elicits flight behaviors, suggesting their tonic suppression in normal conditions (Di Scala et al., 1984). These studies, together with the observation that the behavioral display of fear (i.e. freezing) disappears or is strongly reduced following PAG lesions, strongly implicate this structure’s crucial role in

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21 the expression of defensive responses (LeDoux et al., 1988). The existence of these diverse modulatory functions of the PAG is explained by its longitudinal columnar organization into dorsomedial (dmPAG), dorsolateral (dlPAG), lateral (lPAG) and ventrolateral (vlPAG) segments. The most striking differences are observed with the vlPAG, which is involved in opioid analgesia, hyporeactivity, hypotension and bradycardia, in contrast to the dlPAG (often merged with the lPAG into a dl/lPAG column), which mediates active defensive behavior, hypertension, tachycardia and fear-induced, non-opioid analgesia (Bandler and Shipley, 1994) (Fig. 6).

FIGURE 6 | Functional differences between PAG columns. Illustration of the lPAG and vlPAG, and the behavioral functions associated with each column, along the rostro-caudal axis of the PAG.

(Bandler and Shipley, 1994)

A recent study by Tovote and colleagues (Tovote et al., 2016) confirmed this functional segregation by characterizing a vlPAG inhibitory circuit and its modulation by two competing flight- or freezing-promoting pathways. For instance, CeA-mediated disinhibition of vlPAG

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22 VGLUT2-output neurons is sufficient to elicit a freezing response, whereas the alternative activation of dl/lPAG excitatory neurons stimulates vlPAG local GABA interneurons, thereby suppressing vlPAG glutamatergic output (and consequent freezing) in order to promote the flight response. It is therefore tempting to imagine a similar subregional segregation could also be displayed by PAG projections to neuronal circuits subserving distinct affective and behavioral responses, such as the reward and aversion circuits in the VTA.

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23 4 Efferent connectivity of the VTA

From the initial intracranial electrical stimulation experiments over 50 years ago, to the anatomical tract-tracing investigations and, finally, to the more recent cell-specific optogenetic-assisted circuit mapping, researchers have identified a number of brain structures targeted by projections. This final chapter will thus describe the major VTA efferent pathways, with a focus on the hippocampus and the current evidence supporting its innervation by VTA DA and non-DA fibers (Fig. 7).

FIGURE 7 | Major VTA inputs and outputs. Efferents are color-coded to illustrate DA percentage in each projection. Direct VTA connections where DA contribution was not known are shown in black.

Other connections are shown in gray. (Fields et al., 2007)

4.1 Dopaminergic projections

In 1982, a systematic study of VTA projections using retrograde tracers coupled with immunodetection of TH was the first to show that all VTA efferents are only partially dopaminergic, the proportion of which varies depending on the projection target (Swanson, 1982). For instance, the mesolimbic pathway, which consists of VTA efferents to the NAc,

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24 with over 80% of its fibers being TH-positive represents the most DA-rich VTA projection. In comparison, the mPFC-projecting mesocortical pathway and the BLA-projecting mesoamygdaloid pathways contain significantly smaller proportions of TH-positive fibers – around 30% and 50%, respectively (Fig. 6). Different studies employing paired retrograde tracer injections have reported low levels of collateralization among these VTA efferents, suggesting the existence of minimally overlapping, projection-specific subsets of VTA neurons (Fallon et al., 1984; Swanson, 1982). More recently, Lammel and colleagues (Lammel et al., 2008) proposed the existence of two classes of molecularly and electrophysiologically distinct midbrain DA neurons in the lateral and medial VTA. The former consists of neurons with more classical markers of DA neurons, e.g. expression of DAT, prominent Ih current, low maximal firing frequency (10 Hz) and low basal AMPA to NMDA (N-methyl-D-aspartate) ratio (A/N) - a proxy measure of the relative expression of AMPA and NMDA receptors which, if changed, can be indicative of excitatory synaptic plasticity. This group, which consists of the DA neurons projecting to the NAc lateral shell and localize preferentially in the lateral VTA, adjacent to the physiologically similar DA neurons of the SN pars compacta. In contrast, the medial VTA houses DA neurons that project to the NAc medial shell, NAc core, BLA and mPFC. Compared to lateral shell- projecting DA neurons, these medial neuronal subgroups display relatively lower levels of DAT protein, smaller or absent Ih, higher maximal firing (20Hz) and higher A/N at basal conditions (Lammel et al., 2008). Neurons projecting to mPFC, in particular, also lack the dopamine D2 receptor and GIRK2 channels, but express kappa opioid receptors instead (Margolis et al., 2006b). Moreover, depending on their projection targets, VTA DA neurons can display different reward- or aversion-induced plasticity, further suggesting their function in fundamentally distinct behaviors. Cocaine administration, for example, increases A/N in

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25 both NAc shell-projecting DA subgroups, whereas is observed in more medial mesocortical DA neurons. Instead, these mPFC-projecting neurons show an augmented A/N following an aversive stimulus (intraplantar formalin injection), similarly to DA neurons projecting to the NAc lateral shell, but not medial shell (Lammel et al., 2011).

4.2 Non-dopaminergic projections

Aside from the function as local inhibitory interneurons mediating onditioned aversion and disrupting consummatory behavior (Tan et al., 2012; van Zessen et al., 2012), VTA GABA neurons have also been known to send long range projections to the mPFC and the NAc (Margolis et al., 2006b; Van Bockstaele and Pickel, 1995). The latter projection has been recently characterized and shown to selectively innervate NAc cholinergic interneurons (CINs) (Brown et al., 2012). In their study, Brown and colleagues demonstrated that VTA GABA terminals can be optogenetically activated to force a pause in CINs’ tonic activity and, when this pause is induced during the learning of a stimulus-outcome association, there is an enhancement in the discrimination between motivationally relevant conditioned stimuli and neutral stimuli.

Similarly, glutamate neurons locally excite DA and non-DA neurons within the VTA (Dobi et al., 2010) but also provide long-range projections to many of the same regions targeted by VTA DA and GABA inputs, including the mPFC, amygdala, NAc, VP and LHb (Hnasko et al., 2012). Whereas optogenetic stimulation of VTA glutamate neurons was shown to drive rewarding behaviors that were blocked by the intra-VTA infusion of glutamatergic antagonists (Wang et al., 2015), which suggests a net local activation of DA neurons over GABA neurons, glutamatergic efferents can instead produce aversive responses through the

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26 direct excitation of LHb neurons (Root et al., 2014b) or through the indirect, PV interneuron- mediated inhibition of NAc MSNs (Qi et al., 2016).

4.3 Combinatorial projections

The various VTA subpopulations expressing and co-releasing a combination of neurotransmitters that have been described so far appear to be mostly restricted to the mesolimbic and mesohabenular pathways. For example, both VTA DA/glutamate (Stuber et al., 2010) and DA/GABA co-releasing neurons (Tritsch et al., 2014) have been shown to synaptically target NAc MSNs, but only the latter group also innervates the dorsal striatum.

In contrast, VTA neurons co-expressing VGLUT2 and GAD have been estimated to account for close to 90% of all the LHb-projecting fibers originating from the VTA (Root et al., 2014a), which strongly suggests that the aversive responses produced by activating the VTA-LHb VGLUT2-positive pathway (Root et al., 2014b) is also accompanied by the release of GABA.

However, understanding of the behavioral significance of this and other types of neurotransmitter co-release from the VTA is still lacking.

4.4 Mesohippocampal pathway

Another VTA efferent pathway that has received comparatively little attention over the years is the mesohippocampal pathway, consisting of DA and non-DA projections spreading throughout the hippocampal formation (Amaral and Cowan, 1980; Scatton et al., 1980). This medial temporal lobe structure has a crucial role in the formation of episodic memories and is critical for spatial navigation (Leutgeb et al., 2005). The hippocampal formation consists of the dentate gyrus (DG), the cornu ammonis (CA) regions (CA3, CA2 and CA1) and the subiculum. At the circuit level, these regions are sequentially connected, the DG being the main input region and the subiculum the major output of the hippocampal formation (David

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27 and Pierre, 2006). Retrograde-tracing the mesohippocampal pathway with Fluoro-gold revealed that VTA neurons send projections to virtually all of the dorsal and ventral hippocampal regions (Gasbarri et al., 1994). Furthermore, immunodetection of TH in the VTA indicated that only 6-18% of these mesohippocampal neurons express the dopaminergic marker (Gasbarri et al., 1994; Swanson, 1982) (Fig. 8). Despite this minor dopaminergic contribution, the overwhelming majority of the investigations on this pathway have so far focused on the role of VTA DA signaling, owing in part to the abundance of dopamine receptors expressed throughout the hippocampus, suggestive of an important role in its functional modulation (Ciliax et al., 2000; Defagot et al., 1997).

FIGURE 8 | Retrograde identification of mesohippocampal neurons. Fluoro-Gold was injected at different locations along the septo-temporal axis of the hippocampus to retrogradely label and quantify the total number of neurons (FG), or the number of DA neurons (FG + TH), projecting to each hippocampal region. Levels 36, 42 and 44 refer to the plate number on the Paxinos Mouse Brain Atlas. R3-R65 are the identification number of the injected animals. (Gasbarri et al., 1994)

The possibility to selectively manipulate VTA DA terminals in the hippocampus in order to confirm the presence of functional synapses and causally determine their physiological role has only come about since the recent development of optogenetic tools and reliable transgenic mouse lines. Rosen and colleagues, for instance, demonstrated how changes in DA firing patterns can differentially influence the transmission efficacy between CA3 and CA1 (Rosen et al., 2015). This excitatory transmission was shown to be depressed by low,

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28 tonic DA activity, and enhanced by higher, phasic DA activity, suggesting a method by which transition between motivational states could affect memory encoding. Interestingly, McNamara and colleagues have shown that a similar tonic to phasic transition in DA activity can be observed when a mouse experiences novelty (e.g. exposure to a new environment), and when a similar phasic DA activity is induced in the CA1 during spatial learning of a maze task, it can promote the persistence of spatial memories (McNamara et al., 2014).

A recent anatomical study revisiting the mesohippocampal innervation of the rat DG found that most TH fibers in the region do not originate from VTA DA neurons but are provided by noradrenergic locus coeruleus neurons (Ermine et al., 2016). Their finding was supported by the observation that most DG-retrogradely labelled neurons in the VTA are non- dopaminergic, which is consistent with Gasbarri and colleagues’ estimation of a small (16%) proportion of TH-positive cells among DG-projecting neurons in the VTA (Gasbarri et al., 1994). These results suggest the intriguing, yet untested hypothesis that VTA GABA and glutamate neurons might provide a substantial contribution to the innervation of the hippocampus.

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29 5 Aims of the study

The aim of the present work is to further the understanding of the reward circuitry by providing an anatomical and electrophysiological description of two poorly characterized input and output connections of the ventral tegmental area (VTA).

Evidence exists that the VTA receives synaptic innervation from the PAG, yet whether this pathway is physiologically functional has not been tested, and the identity of the neurons involved in the connection remains unknown. Thus we want to test whether VTA input neurons are localized in specific dorsal or ventral PAG columns, and whether VTA-projecting PAG neurons (GABA- or glutamatergic) show any projection-specificity towards particular VTA subpopulations.

Secondly, we will focus on the VTA projection to the hippocampus, which has been described by a number of studies, and the presence of a DA innervation, especially in the CA1, is well established. However, despite reports that DA neurons are minor contributors to the mesohippocampal pathway, innervation of the hippocampus by non-DA afferents from the VTA is still lacking empirical support. We will therefore test the hypothesis that non-DA neurons of the VTA project to the hippocampus and consequently characterize the functional properties of such synaptic connection.

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31

GABA neurons

The following chapters will illustrate a series of experiments, done in collaboration with Dr.

Meaghan Creed, seeking to provide an anatomical and electrophysiological organization of PAG inputs to the VTA. All tissue processing, confocal imaging, and electrophysiological experiments were performed and analysed by the author.

1 Abstract

Neurons in the periaqueductal gray (PAG) are known to modulate threat responses, as well as to mediate the anti-nociceptive and rewarding effects of opioid drugs. Similarly, an extensive body of research has implicated the ventral tegmental area (VTA) in the regulation of both aversive and reinforcing behaviors. Here we study the interconnection between these two midbrain structures by describing the anatomical and electrophysiological organization of the VTA-projecting PAG neurons. Using rabies-mediated, cell type-specific retrograde tracing we observed that VTA DA- and GABA-retrogradely labeled input neurons are similarly distributed withing the PAG, most of them being concentrated in its posterior and ventrolateral segments. When optogenetically stimulated, we observed that the PAG- to-VTA pathway is predominantly excitatory and targets similar proportions of Ih-expressing VTA DA and GABA neurons. Taken together, these results contribute to a better

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32 understanding of the reward circuitry and provide relevant insights into the interplay between PAG and VTA in the regulation of reward and aversion.

2 Introduction

The periaqueductal gray (PAG) is a neurochemically and functionally heterogeneous midbrain structure that is critical for the endogenous modulation of nociception and for the expression of defensive behaviors (Behbehani, 1995). These functions have been shown to be mediated by neurons anatomically segregated in the longitudinal columns corresponding to the dorsolateral (dl), lateral (l) and ventrolateral (vl) subdivisions of the PAG (Bandler and Shipley, 1994).

Anatomical tracing studies have described ascending and descending projections from the PAG to a variety of brain structures (Vianna and Brandão, 2003). Among these projection targets is the ventral tegmental area (VTA), a major component of the brain reward system (Geisler and Zahm, 2005). While it is primarily known for its role in reward prediction, and positive reinforcement (Fields et al., 2007; Schultz et al., 1997), the VTA has also been implicated in the modulation of nociception and in the expression of fear and aversive responses (Kender et al., 2008; Li et al., 2016; Pezze and Feldon, 2004; Tan et al., 2012). The engagement of VTA dopamine- (DA) and gamma-aminobutyric acid (GABA)-releasing neurons with PAG afferents through both symmetric and asymmetric synaptic contacts has been demonstrated with rabies-assisted retrograde tracing and ultrastructural immunoelectron microscopic analyses (Faget et al., 2016; Omelchenko and Sesack, 2010).

It is not yet known, however, whether the input neurons providing these VTA afferents spatially segregate within specific PAG columns, potentially associating the PAG-to-VTA pathway with specific anti-nociceptive or defensive functions. Moreover, evidence for an

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33 electrophysiologically functional connection, and knowledge of its excitatory or inhibitory effect onto VTA DA and GABA neurons is still lacking. To this end, the present study will describe the rostrocaudal distribution of VTA-projecting neurons across the PAG columns, and will test whether these neurons exhibit a preferential excitatory or inhibitory effect on DA and GABA neurons of the VTA.

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34 3 Materials and methods

3.1 Animals

Experiments were performed on DAT-Cre (Zhuang et al., 2005) and GAD65-Cre mice (Kätzel et al., 2011) of both sexes. All animal procedures were performed in accordance with the authors' university animal care committee's regulations.

3.2 Injection procedures

All stereotaxic intracranial injections were performed under isoflurane anesthesia (2-5%, Attane) using glass capillary pipettes connected to a microinjection pump (Narishige) at a rate of ~100 nl min^-1. The coordinates used were (from bregma, in mm): AP -3.4, ML

±0.5, DV -4.3 for VTA injections; AP -4.0, ML ±0.3, DV -2.6 for PAG injections. For retrograde tracing experiments, 300 nl of a 1:1 mixture of AAV8-CAG-DIO-RG and AAV5-EF1a-DIO-TVA- mCherry was injected unilaterally in the VTA, followed 2 weeks later by the injection of 200 nl of RVG-EnvA-EGFP at the same coordinates. For patch patch clamp experiments, animals were bilaterally injected with AAV5-EF1a-DIO-mCherry in the VTA and with AAV2- hsyn-ChR2-EYFP in the PAG (all viruses from the UNC Vector Core Facility).

3.3 Electrophysiology on acute slices

Coronal VTA slices (180 m) were prepared using a vibratome in ice-cold cutting solution containing (in mM): NaCl 87, NaHCO3 25, KCl 2.5, MgCl2 7, NaH2PO4 1.25, CaCl2 0.5, glucose 25, and sucrose 75. Slices were incubated in the same solution for 20 min at 31°C before being transferred to regular room-temperature artificial cerebrospinal fluid (aCSF), containing (in mM): NaCl 119, NaHCO3 26.2, KCl 2.5, MgCl2 1.3, NaH2PO4 1, CaCl2 2.5, and

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35 glucose 11. After at least one hour for recovery, slices were transferred to the recording chamber, superfused with aCSF at 2 ml/min. All solutions were constantly bubbled with 95%

O2 and 5% CO2. Postsynaptic currents were evoked by stimulating ChR2 with brief (4 ms) blue light pulses using a 470nm LED mounted on the microscope and powered by an LED driver under computer control. Kynurenic acid (kyn, 4 mM, Sigma) and picrotoxin (PTX,M, Sigma) were added to the aCSF in order to block glutamate- and GABA- mediated currents, respectively. All representative traces were made from averaging at least 20 consecutive sweeps. Neurons were visually identified using an IR CCD camera mounted on an Olympus BX45 microscope. Borosilicate glass pipettes at a resistance range of 2-4 M were used for recording. The internal solution used contained (in mM): K- gluconate 30, KCl 100, MgCl2 4, creatine phosphate 10, Na2ATP 3.4, Na3GTP 0.1, EGTA 1.1, and HEPES 5. The calculated reversal potential with this internal solution for Cl^- was -5mV and cells were voltage-clamped at -70 mV. Currents were amplified, filtered at 2 kHz, digitized at 10 kHz, and saved on a hard disk. The liquid junction potential was small (-4 mV) and traces were therefore not corrected. Access resistance was monitored by a hyperpolarizing step of -4 mV at the onset of every sweep, and the experiment was discarded if the access resistance changed by more than 20%.

3.4 Histological procedures and imaging

Mice were deeply anesthetized with pentobarbital (300 mg/kg i.p.) and transcardially perfused with 0.1M phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA, Sigma). Brains were removed, post-fixed in 4% PFA for 24 h at 4°C, and cut in 50 m sections on a vibratome. For tyrosine hydroxylase (TH) immunohistochemistry, brain sections were rinsed in PBS (0.1 M) and incubated for 1 h at room temperature in a blocking

Mapping VTA neural circuits: periaqueductal inputs and mesohippocampal outputs

Thesis

Reference

Mapping VTA neural circuits: periaqueductal inputs and mesohippocampal outputs

MAPPING VTA NEURAL CIRCUITS:

Niels NTAMATI RWAKA

List of abbreviations

Table of Contents

GABA neurons