Research Doctorate in Biology
Cycle XXVIII
PhD thesis
Generation and characterization of
chemogenetic mouse models for the
study of the brainwide effects induced
by serotonergic neurotransmission
manipulation
Candidate: Andrea Giorgi
Tutor: Prof. Massimo Pasqualetti
TABLE OF CONTENTS ABSTRACT ...5 INTRODUCTION ...7 Chemogenetics ...7 History of chemogenetics ...7 DREADD receptors ...9
DREADDs for neuronal circuitry dissection ... 12
Functional MRI ... 14
The 5-HT system ... 17
5-HT discovery ... 17
5-HT-system anatomy and receptorial repertoire ... 17
Functional and molecular heterogeneity of 5-HT neurons... 20
5-HT in neuropsychiatric disorders ... 23
AIM OF THE THESIS ... 28
MATERIALS AND METHODS ... 29
Animal care ... 29
Mouse lines generation ... 29
Genotyping ... 30
Embryonic stem cell culture ... 30
Electroporation in ESCs ... 31
Southern Blot ... 31
ESCs Karyotyping ... 32
Blastocyst injection and mouse breeding ... 33
Vasectomy ... 34
Drug formulation and pharmacological treatments ... 35
Functional Magnetic Resonance Imaging (fMRI) ... 36
Immuno-histochemical analyses... 37
RESULTS ... 39
Generation of hM3Dq and hM4Di conditional knock-ins ... 39
Selective expression of hM3Dq and hM4Di in 5-HT neurons. ... 42
Ex-vivo hM3Dq-mediated phasic activation of 5-HT neurons ... 46
Ex-vivo hM4Di-mediated silencing of 5-HT neurons ... 47
Functional MRI mapping of CNO dose-response effects on wild-type animals ... 48
Chemo-fMRI mapping of brainwide effects induced by hM3Dq-mediated 5-HT phasic activation ... 51
Chemo-fMRI mapping of brainwide effects induced by hM4Di-mediated 5-HT inhibition ... 53
Brainwide c-Fos induction following hM3Dq-mediated 5-HT phasic activation ... 53
Chemo-fMRI mapping of brainwide effects induced by citalopram administration ... 55
DISCUSSION ... 60
ADDITIONAL RESEARCH... 66
hM4Di/Pet1-Cre mice for the study of synaptic plasticity at striatal inputs ... 66
hM3Dq/Pet1-Cre mice for the study of post-stroke motor recovery . 66 BIBLIOGRAPHY ... 67
Poster presentations ... 101
ABSTRACT
Serotonin (5-HT) neurotransmission affects behaviors and neuro-physiological functions via the orchestrated recruitment of distributed neural systems. Human imaging studies have employed pharmacological manipulations to probe the brainwide substrates targeted by serotonin. However, systemic drug manipulations often lack neural and receptorial specificity, and result in combined central and peripheral contributions that cannot be easily disentangled. As a result, it remains unclear whether and how serotonergic activity specifically influences regional or global functional activity. Here we combine DREADD-based chemogenetics and mouse fMRI, an approach we term “chemo-fMRI”, to causally probe the brainwide substrates endogenously modulated by phasic 5-HT neurons stimulation. To this aim, I generated of two conditional knock-in mouse models that, crossed with Pet1-Cre-transgenic mice, allowed us to remotely stimulate serotonergic transmission during fMRI scans. I show that chemogenetic stimulations of 5-HT system results in a composite pattern of activation encompassing parieto-cortical, hippocampal, and midbrain structures, as well as ventro-striatal components of the mesolimbic reward systems. Many of the activated regions also exhibit increased c-Fos immunostaining upon stimulation in freely-behaving mice, supporting a neural origin of the observed activation. Collectively, these findings identify a set of regional substrates that act as primary functional targets of endogenous serotonergic stimulation, and establish causation between phasic activation of 5-HT neurons and regional fMRI signals. They further highlight a functional cross-talk between 5-HT and mesolimbic dopaminergic, and provide a novel framework for understanding
5-HT-dependent functions and interpreting data obtained from human fMRI studies of serotonin modulating agents.
INTRODUCTION
Chemogenetics
History of chemogenetics
In 1980, the term chemogenetics was originally coined to describe the effects of natural genetic mutations on chalcone isomerase specificity for different substrates in the flowers of Dianthus caryophyllus (Forkmann and Dangelmayr, 1980). Since then, chemogenetics is every method by which proteins can be engineered in order to acquire affinity for a previously unrecognized chemical actuator (Sternson and Roth, 2014; Strobel, 1998). On this regard, in the last two decades different classes of proteins have been chemogenetically modified, including kinases and other enzymes (Bishop et al., 1998; Cohen et al., 2005; Dar et al., 2012), ligand-gated ion channels (Magnus et al., 2011; Zemelman et al., 2003) and G-protein coupled receptors (GPCRs; Alexander et al., 2009; Armbruster et al., 2007; Vardy et al., 2015). In particular, GPCR-based chemogenetic tools are numerous and include allele-specific GPCRs (Strader et al., 1991), Receptors Activated Solely by a Synthetic Ligand (RASSLs; Coward et al., 1998) and Designer Receptors Exclusively Activated by Designer Drugs (DREADDs; Armbruster et al., 2007)
GPCRs are the largest family of membrane proteins and mediate most cellular responses to hormones, neurotransmitters and drugs, as well as being responsible for vision, olfaction and taste (Rosenbaum et al., 2009; Venkatakrishnan et al., 2013). Structurally, they are characterized by an extracellular N-terminus, followed by seven transmembrane α-helices connected by three extracellular and
three intracellular loops, culminating in an intracellular C-terminus. Such domains are organized in a barrel-like tertiary structure. GPCRs activity and signaling is mostly ligand-dependent and, inside the cells, it typically relies on the consequent activation of heterotrimeric guanine-nucleotide-binding proteins (G-proteins; De Lean et al., 1980). The GPCRs C-terminus can also interact with β-arrestin, leading to the formation of alternative signaling complexes (Kroeze et al., 2015; Luttrell et al., 1999). To date, four different families of G-proteins are known: Gq, Gi, Gs and G12. Each family activates peculiar downstream
biochemical pathways. In neurons, Gq-proteins promote firing by activating the pathway of phospholipase C that, in turn, leads to inositol triphosphate-mediated calcium influx. In the same cell type, Gi-proteins activation promotes neuronal silencing mainly by inhibiting the adenylate cyclase that, in turn, leads to the opening of inward-rectifying potassium (K+) ion channels. Gs-proteins and G12-proteins
activation otherwise affect the cAMP intracellular availability and Rho GTPase activity, respectively (Syrovatkina et al., 2016)
The first chemogenetically engineered GPCR was the β-adrenergic receptor (βAR; Strader et al., 1991). The βAR primary structure was molecularly modified by replacing a single amino acid residue (D165S) in order to alter its ligand specificity, thus providing evidence about the potential for the rational design of drugs binding specific genetically engineered receptors. Several years later, RASSLs receptors were developed (Coward et al., 1998). This was the first attempt to produce tools allowing the manipulation of the broad range of G-protein-mediated physiologic responses in vivo. In fact, RASSLs were chemogenetic receptors evolved from human κ-opioids in order to lose their affinity for the endogenous peptide ligands (200-2000 fold reduction in binding dynorphin) and to gain specificity for small, no addictive and safe drugs such as spiradoline. A first evidence of their
utility in vivo was provided by the observation that activation of Ro1, a Gi-coupled RASSL, in the heart rapidly decreased heart rate by up to
80%, an expected effect of increased Gi-proteins signaling (Redfern et al., 1999). In addition, four years later, RASSLs were used to demonstrate that sweet and umami tastes are strictly dependent on T1R-receptors and that the selectivity of taste cells is determined by the nature of the receptors they express (Zhao et al., 2003). However, although in these noteworthy papers RASSLs worked as valuable tools, their ligands often showed high levels of affinity and potency also at the native receptors (Claeysen et al., 2003; Coward et al., 1998; Pauwels and Colpaert, 2000). Moreover, RASSLs had basal levels of activity leading to phenotypes also in the absence of chemical actuators (Hsiao et al., 2008; Sweger et al., 2007), thereby limiting the range of studies in which they could be used. In order to overcome RASSLs defects, DREADDs have been created (Armbruster et al., 2007; Armbruster and Roth, 2005).
DREADD receptors
Together with optogenetics, DREADDs technology is to date the mostly used tool to in vivo manipulate the activity of genetically-defined neuronal populations. DREADDs represent a family of chemogenetic GPCRs invented through direct molecular evolution of human muscarinic receptors (Armbruster et al., 2007). To generate them, the native receptors have been engineered through cyclic procedures of random mutagenesis and screening, in order to create and select receptors that had maximally lost their natural affinity to acetylcholine (Ach) and that were maximally responsive to clozapine-N-oxide (CNO), a metabolite of the antipsychotic drug clozapine (Roth et al., 1994). CNO has been chosen a priori as ligand for its excellent drug-like
properties, it is indeed a small molecule that can rapidly diffuse in the central nervous system by crossing the blood-brain barrier with a long-term residence (>60 min). In addition, it is bioavailable (Bender et al., 1994; Chang et al., 1998) and pharmacologically inert (<1µM affinity at the native muscarinic receptors; Armbruster et al., 2007). Furthermore, because its parent compound clozapine has high affinity at the native muscarinic receptors, few mutations were supposed to be required to make them highly responsive to CNO.
Basing on the interacting G-protein, three classes of DREADDs have been developed: Gq-coupled, Gi-coupled and Gs
-/β-Arrestin-coupled DREADDs. Gq-DREADDs are engineered Gq-coupled GPCRs
evolved from the human muscarinic receptor 1, 3 and 5 (hM1Dq, hM3Dq and hM5Dq respectively; Armbruster et al., 2007). Their CNO-mediated activation leads to intracellular Gq-mediated signaling
cascade, therefore they are typically used to enhance neuronal firing. Notably, hM3Dq is activated by 20-30 nM CNO, whereas it is severely unresponsive to Ach (<40.000 fold) compared to the respective, native, muscarinic receptor (hM3; Armbruster et al., 2007). Transgenic mice expressing hM3Dq receptors in CamKIIa-positive neurons showed no significant differences respect to the wild-type littermates in size, overall morphology, coat condition, body posture, and balance. In addition, no alterations have been observed in a battery of behavioral tests measuring motor coordination, anxiety, explorative attitudes, acoustic startle response, prepulse inhibition, latency to feed, spatial cognition, acquisition and reversal learning, thereby demonstrating that hM3Dq expression, in absence of CNO, does not affect behavior (Alexander et al., 2009). Similarly, DREADDs are engineered Gi-coupled GPCRs evolved from human muscarinic receptors 2 and 4 (hM2Di and hM4Di respectively). Once activated, Gi-DREADDs
neuronal activity inhibition. In vitro experiments showed that the ability of hM4Di receptors to inhibit neurons depended on the activation of the well-known GIRK-potassium (K+) channels response and was not due to the non-specific binding of CNO at the native muscarinic receptors (hM4). Importantly, hM4Di expression did not affect native receptor affinity for Ach (Armbruster et al., 2007). Recently, an axonally-targeted hM4Di receptor has been developed, showing an alternative K+ channels-independent inhibitory mechanism of action involving the repression of neurotransmitter release at synaptic level (Stachniak et al., 2014). Furthermore, as additional tool, a novel class of inhibitory DREADD has been recently generated from κ-opioid receptor (KOR). The so-called KORD (KOR-DREADD) is responsive to salvinorin B (SalB), an otherwise inert molecule, and not-responsive anymore to the endogenous ligands. Thanks to these properties, KORDs can be expressed simultaneously with excitatory Gq-DREADDs for the sequential chemogenetic activation and inhibition of neuronal populations (Vardy et al., 2015). The third class of DREADDs includes chemogenetic Gs-coupled GPCRs obtained by combining the turkey erythrocyte β adrenergic receptor and the synthetic rat M3 DREADD. The resulting Gs-DREADD (Guettier et al., 2009) has been successfully tested in vivo (Ferguson et al., 2013; Pleil et al., 2015). Finally, DREADDs exclusively acting via the β-arrestin pathways (Rq-R165L) have been developed, but they have not been tested in vivo yet (Nakajima and Wess, 2012). DREADDs are showing high translational potency since, in addition to mice, they have been already tested in rats (Ferguson et al., 2011), flies (Becnel et al., 2013) and non-human primates (Eldridge et al., 2016). Collectively, different subtypes of DREADDs have been created by genetically editing different subtypes of well-known existing GPCRs. Thereby opening the engaging possibility to efficiently and cell-specifically govern the wide range of G-protein-dependent intracellular pathways.
DREADDs for neuronal circuitry dissection
By conferring the ability to manipulate the activity of neurons, DREADD-based chemogenetics is revealing as a great tool for the in
vivo functional dissection of genetically-defined neuronal circuits,
especially in finding causal correlations with behavior, inasmuch as it allows to control neuronal activity in a non-invasive way. To this purpose, DREADDs must be selectively expressed in the neuronal subpopulation of interest. The up to date genetic biotechnologies not only enable the specific expression of a transgene in virtually every kind of genetically-identified cell, but also the spatial-temporal definition of their expression domain through several DNA recombination-based strategies (i.e., Cre-loxP, FLP-FRT, Tet-on/off systems; Sciolino et al., 2016) combined with the recently developed high-efficient viral vectors (Pina and Cunningham, 2017). Thanks to this wide set of genetic tools, to date DREADDs have been already fruitfully applied in vivo for the manipulation of serotonergic (Ray et al., 2011; Teissier et al., 2015; Urban et al., 2015), glutamatergic (Krashes et al., 2014; Zhu et al., 2014; Zhu et al., 2016) and AgRP neurons (Krashes et al., 2011) as well as striato-pallidal (Farrell et al., 2013) and POMC neurons (Koch et al., 2015). In addition, memory traces were mapped and de novo created by means of activity-dependent (c-Fos promoter) expression of hM3Dq receptor throughout the brain (Garner et al., 2012). Beside to this kind of approaches, the unprecedented opportunity to specifically manipulate neurons in a non-invasive way could be applied for studying the large-scale effects induced on the living brain. In this context, a novel method termed DREAMM (DREADD-assisted metabolic mapping) allowed to map brain activity in freely behaving animals (Michaelides and Hurd, 2015). In this thesis, in particular, I sought to exploit the peculiarity of DREADDs to map the large-scale effects induced by the modulation of
specific circuits (5-HT system) through functional magnetic resonance imaging (fMRI) technique.
Functional MRI
The brain does not store glucose, its primary energy source. However, when a brain region is biologically active, firing neurons need energy for the restoration of the ion concentration gradients following action potentials. Therefore, glucose supply is required to sustain their higher-level activity and additional glucose is provided by an increase of the local blood flow. This is the basic concept of the functional magnetic resonance imaging (fMRI), a functional neuroimaging procedure employing MRI technology to measure brain activity by detecting changes associated with cerebral blood flow (Jenkins, 2012). fMRI represented a revolutionary method in mapping brain activity. With respect to the previous methodologies such as positron emission tomography (PET) and techniques based on CBF or glucose metabolism (Kuhl et al., 1977; Raichle et al., 1976), fMRI was a non invasive tool, with reasonable contrast to noise, no requirements of radioactive compounds and relatively high spatial and temporal resolution (Jenkins, 2012). Coupling neuronal activity with a hemodynamic response is the crucial point in the application of this technique. It can be performed measuring different parameters. The blood oxygenation level dependent (BOLD) and the relative cerebral blood volume (rCBV) are the most used.
The BOLD method was applied for the first time in humans (Bandettini et al., 1992; Ogawa et al., 1992) and then largely employed for pharmacological and non pharmacological fMRI studies (Jenkins, 2012). It relies on the differential magnetic properties of hemoglobin in its two functional states: oxygenated hemoglobin (Hb) and deoxygenated hemoglobin (dHb). Since Hb is diamagnetic and dHb is paramagnetic, deoxygenated blood has a magnetic susceptibility
significantly greater than fully oxygenated blood (Thulborn et al., 1982). Such property makes feasible to measure the BOLD contrast as the amount of dHb respect to Hb in the cerebral blood vessels. Changes of this parameter, termed hemodynamic response (HDR), depends by oxygen consumption (neuronal activity) and oxygen supply (blood flow and volume; Ogawa and Lee, 1990). Although HDR is coupled to neuronal activation, the underlying mechanism is still debated (Mangia et al., 2009). The rCBV-based method represents a higher sensitive technique. By using an exogenous intravascular contrast agent to increase the blood’s magnetic susceptibility, a CBV-based functional contrast is generated and measured. rCBV is a meaningful physiological component of the haemodynamic response and also a robust marker of basal metabolism (Gaisler-Salomon et al., 2009; Sheth et al., 2004), for this reason can be used for inter-group analysis of resting brain-function and evoked brain function elicited by different stimulation paradigms.
In the last two decades, pharmacological MRI (phMRI; Chen et al., 1997), indicating a branch of fMRI in which neuronal activity to be mapped is elicited using various pharmacological agents as a stimulus or as a means for modifying the response to some other non-pharmacological stimulus, has been employed in both humans and laboratory animals-based studies. Early reports of phMRI experiments were aimed to determine the brain response to amphetamine (Dodds et al., 2009), cocaine (Chen et al., 1999), apomorphine (Nguyen et al., 2000), nicotine (Stein et al., 1998), heroin (Xu et al., 2000) and 5-HT ligands (Houston et al., 2001; Scanley et al., 2001). Although, in more recent years, the effects of newly developed neuropeptides and chemical actuators have been studied (Anand et al., 2005; Bossong et al., 2012; Gozzi et al., 2008; Gozzi et al., 2005), revealing phMRI as an excellent methodology for measuring drug administration effects on the
living brain. While useful in identifying possible indirect brainwide substrates of neuromodulatory action, drug-based approaches however often lack neural and receptorial specificity, and result in combined central and peripheral contributions that cannot be easily disentangled. This aspect is especially relevant when indirect hemodynamic surrogates of brain function like fMRI are used. However, the combination of DREADD chemogenetics technology with functional MRI could overcome this issue.
The 5-HT system
5-HT discovery
Serotonin was originally isolated from the enterochromaffin cells of the gut (Erspamer and Vialli, 1937) and from the blood flow. Once termed serotonin for its role as vasoconstrictor (Rapport et al., 1948), in 1949 its structure was described as 5-hydroxytryptamine (5-HT; Rapport, 1949) and only a few years later, it was found in the brain (Twarog and Page, 1953). Given the hydrophilic nature of 5-HT, it was expected to be unable to cross the blood-brain barrier, hence its identification in the parenchymal brain tissue suggested that 5-HT could be synthetized within the central nervous system (Whitaker-Azmitia, 1999). Approximately one decade later, a cluster of 5-HT producing neurons was indeed found and visualized in the brainstem raphe nuclei (Dahlstrom and Fuxe, 1964), thereby opening to a huge number of future studies investigating morphology and functional roles of the 5-HT neurons in health and diseases.
5-HT-system anatomy and receptorial repertoire
5-HT-producing neurons localize in the brainstem and cluster in nine subregions, termed from B1 to B9 nucleus. Two main groups were classically identified depending on their derivative precursors and anatomic localization, the rostral raphe cluster (B5-B9) located anteriorly to the pontine flexure, and the caudal cluster (B1-B4), placed in the medulla (Jacobs and Azmitia, 1992; Wallace and Lauder, 1983). The caudal raphe nuclei project to the spinal cord, whereas the more rostral raphe nuclei produce a dense innervation throughout the forebrain. The rostral raphe cluster consists of the caudal linear
nucleus (CLi; B8), the median raphe nucleus (MnR; B5, B8), the lateral medial lemniscus (LML; B9) and the dorsal raphe nucleus (DR; B6, B7). The caudal cluster includes the raphe obscurus nucleus (ROb, B2), the raphe pallidus nucleus (RPa, B1, B4), the raphe magnus nucleus (R(Bang et al., 2012)Mg, B3) the lateral paragigantocellular nucleus and the intermediate reticular nuclei (IRN; B1, B3) (Jacobs and Azmitia, 1992; Wallace and Lauder, 1983). Despite their relative few number, 5-HT neurons give raise to long and highly collateralized axons that densely innervate the whole central nervous system (Bang et al., 2012; Gagnon and Parent, 2014; Jacobs and Azmitia, 1992). In particular the forebrain and the spinal cord are innervated by the rostral (B5-B9) and the caudal (B1-B4) raphe nuclei, respectively, suggesting a broad topographic organization of serotonergic projections (Bang et al., 2012). The recent optimization of viral retrograde techniques highlighted this concept, B6, B7, B8 and B9 rostral raphe nuclei have indeed distinctive and largely non-overlapping projection fields in the rostral brain (Muzerelle et al., 2016).
5-HT exerts its action through multiple receptors, expressed both pre-synaptically and post-synaptically in the nervous system as well as in non-neural tissues (Barnes and Sharp, 1999). Molecular biology techniques allowed to classify 5-HT receptors in seven distinct families (5-HT1R to 5-HT7R) depending on primary structure, downstream transduction mechanism, pharmacology and functional observations (Hannon and Hoyer, 2008). At least fifteen genes coding different 5-HT receptors exist, nevertheless post-transcriptional modifications furtherly expand to twenty the number of functional 5-HT receptors (Burns et al., 1997). In the CNS, all 5-HT receptors are present and differentially expressed, covering almost all brain structures, with particular abundance in frontal cortex, hippocampus,
amygdala, hypothalamus and striatum (Bockaert et al., 2006; Millan et al., 2008; Raymond et al., 2001). The precise localization of each 5-HT receptor is still far from complete, also because many of them show an early, as well as dynamic, expression profile (Bonnin et al., 2006; Lauder et al., 2000; Lein et al., 2007). Moreover, since selective ligands for certain receptor subtypes are not available, their anatomical distribution is mainly argued by the presence of the respective mRNAs. It remains thereby elusive the subcellular localization (Charnay and Leger, 2010; Barnes and Sharp, 1999). Similarly to other neurotransmitters such as acetylcholine, glutamate, and γ- aminobutyric acid (GABA), 5-HT primarily acts via two types of receptors: ionotropic and metabotropic. Except for the ionotropic 5-HT3Rs, which are ligand-gated ion channels (van Hooft and Yakel, 2003), all the others are G-protein-coupled receptors. Basing on the downstream intracellular signaling they induce once activated, 5-HTRs are further categorized in four groups. 5-HT1Rs act through Gi-proteins and are usually negatively coupled with adenylate cyclase thus inhibiting cAMP production; 5-HT2Rs, coupled to Gq proteins, activate phospholipase C (PLC) to increase the hydrolysis of inositol phosphates and elevate intracellular Ca2+; HT4Rs, HT6Rs, and 5-HT7Rs are positively coupled to Gs proteins; the intracellular coupling of 5- HT5Rs is still uncertain (Raymond et al., 2001). Among the metabotropic serotonin receptors, the best-documented regional and cellular localizations have been reported for 5-HT1AR, 5-HT1B/1DR and
5-HT2CR (Bockaert et al., 2006; Millan et al., 2008). 5-HT1AR is among
the most abundant and widely distributed in the brain (Pasqualetti et al., 1996) . The highest density of 5-HT1AR is found in the limbic areas,
particularly in the hippocampus and in the lateral septum, and also in cingulate and entorhinal cortex. Importantly, 5-HT1AR is localized in
cell bodies and dendrites of 5-HT neurons of the dorsal and median raphe, where it acts as autoreceptor inhibiting cell firing and 5-HT
release. Moreover, 5-HT1AR activation has been reported to induce the
secretion of S100β, a growth factor expressed by astrocytes with beneficial effects on serotonergic neuronal system development (Whitaker-Azmitia, 2001). 5-HT1B and 5-HT1D are present on 5-HT
terminals as well as dopaminergic, GABAergic and glutamatergic terminals, where they seem to inhibit neurotransmitter release (Starke et al., 1989). 5-HT1BR mRNAs was identified in rat basal ganglia
(substantia nigra, globus pallidus, vental pallium) and in many other brain regions (Bruinvels et al., 1994), whereas 5-HT1DR mRNA is
present in the nucleus accumbens, caudate putamen and locus coeruleus (Hamblin et al., 1992). 5-HT1BR and 5-HT1DR are also
present in the cortex (Bonnin et al., 2006) and in raphe nuclei of both human and rodents, likely acting as autoreceptors. In addition, 5-HT2CR expression has been detected in choroid plexus, cortex,
nucleus accumbens, amygdala, hippocampus, caudate nucleus and substantia nigra (Abramowski et al., 1995; Pasqualetti et al., 1999). All studies suggest that the activation of 5- HT2CR inhibits dopaminergic
firing (Gobert et al., 2000; Ji et al., 2006). 5-HT2C receptors are also
expressed in the arcuate nucleus of the hypothalamus.
Functional and molecular heterogeneity of 5-HT neurons
Adult 5-HT neurons appear as a heterogeneous class of cells. This heterogeneity mirrors the non uniform embryonic development of 5-HT neuronal precursors. 5-HT neurons are indeed generated starting from the embryonic day 9.5 (E9.5) and 12.5 (Briscoe et al., 1999) in the mouse ventral hindbrain, when it is still subdivided in seven compartments, known as rhombomeres (r1 to r7; Lumsden and Krumlauf, 1996). Notably, the final anatomical localization of 5-HT neurons is linked to their rhombomeric origin. In particular, B6 and B7
entirely derive from r1, B8, B9 and B5 from r1, r2 and r3, and B1, B2, B3 and the others caudal nuclei from r5-r7 (Jensen et al., 2008). During mouse neurodevelopment, r1-derived neurons are the first to be specified, while only a day later the others appear. The region of the neural tube in which the 5-HT neurons precursors arise is dorso-ventrally delimited by a gradient of the Sonic hedgehog morphogenetic factor (Shh). But, within this area, the specification of the rostral nuclei requires the additional expression of the fibroblast growth factor 8 (FGF8), whereas FGF4 for the caudal ones (Ye et al., 1998). The differentiation of serotonergic neurons is finely regulated by distinct transcription factors. Nkx2.2, Nkx6.1, and Ascl1/ Mash1 contribute to the generation of 5-HT precursors, whereas for the subtype differentiation Gata2, Insm1, Lmx1b and Pet1 are required (Kiyasova et al., 2011). The transcriptional cascades regulating 5-HT neurons development converge on the activation of Pet1, a key transcription factor. In the mouse brain, Pet1 became detectable in 5-HT precursors starting form E12, just before the expression of serotonergic terminal differentiation markers (Tph2, SERT), and its expression is maintained in adult 5-HT neurons too (Hendricks et al., 1999; Kiyasova and Gaspar, 2011). At these stages, Pet1 is important for serotonergic neuron terminal differentiation (Hendricks et al., 2003), however, in mice devoid of Pet1, 30% of 5-HT neurons properly develops in adult 5-HT-releasing neurons. Despite this aspect, Pet1 is considered as a specific marker of developing and mature 5-HT neurons, and is commonly used to give 5-HT specificity to various experimental approaches, such as conditional knock-outs generation, intersectional fate mapping as well as optogenetic and chemogenetic targeting.
Accordingly with their multiple developmental origin, adult 5-HT neurons differ each other in electrophysiological properties, transcriptomic profile and innervation pathways (Fernandez et al.,
2015; Gaspar and Lillesaar, 2012; Muzerelle et al., 2016; Okaty et al., 2015; Tork, 1990). In addition, 5-HT neurons with diverse neurotransmitter repertoires have been identified (Gaspar and Lillesaar, 2012). Among them, it is remarkable the identification of 5-HT neurons expressing the vesicular glutamate transporter 3, whose presence suggests the ability to additionally release glutamate (Hioki et al., 2010; Sos et al., 2016).
The electrophysiology of 5-HT neurons is likewise variegated. They are generally characterized by slow, rhythmic activity in spontaneously active cells, broad spikes and large after hyperpolarization potential (Beck et al., 2004). However, region-specific differences in the excitability properties have been recently observed (Crawford et al., 2010). In fact, correlation studies showed that anatomical localization and electrophysiological properties of 5-HT neurons identify three forebrain-projecting 5-HT populations, termed cluster 1, cluster 2-1 and cluster 2-2. 5-HT neurons belonging to the cluster 1 are characterized by larger time constant and high input resistance, an example of this neurons type are those innervating the amygdala. Conversely, the majority of neurons projecting to the hippocampus and the prefrontal cortex belong to the cluster 2-2, which is characterized by low capacitance and fast time constant (Fernandez et al., 2015). Finally, 5-HT neurons belonging to the cluster 2-1 have lower excitability and lower level of spike frequency adaptation.
From the molecular point of view, diverse transcriptomic landscapes have been demonstrated to differentially characterize 5-HT neurons depending from their rhombomeric origin and their final localization within the raphe nuclei (Okaty et al., 2015), suggesting the existence of function-specific 5-HT sub-populations. In support of this thesis, a role in controlling the respiratory dynamics was demonstrated for two subgroups of 5-HT neurons (Egr2- and Tachyinin-expressing
neurons; Brust et al., 2014; Hennessy et al., 2017), a role in the regulation of sensorimotor gating has been attributed to the r2-derived 5HT neurons of the MRN (Okaty et al., 2015) and a role in regulating aggressive behavior has been associated to the D1- and D2-expressing 5-HT neurons (Niederkofler et al., 2016).
Electrophysiological, anatomical and molecular variety of 5-HT neurons mirrors the wide range of physiological processes, behaviors and neuronal circuits affected by 5-HT. In this regard, 5-HT was found actively involved in the regulation of blood circulation, thermoregulation and respiration (Alenina et al., 2009; Audero et al., 2008; Brust et al., 2014; Hendricks et al., 2003; Hickner et al., 2014; Hodges et al., 2009; Ray et al., 2011) as well as pain perception (Savelieva et al., 2008) and peristalsis regulation of the gastrointestinal tract (Kim and Camilleri, 2000). Furthermore, it is clear the influence of 5-HT on circadian rhythms and sleep-awake cycle (Alenina et al., 2009; Whitney and Shemery, 2016). 5-HT likewise affects behavior and cognitive processes, such as learning and memory (Fernandez et al., 2015), food intake (Lam et al., 2010; Wu et al., 2012), sexual behavior (Hull et al., 2004; Liu et al., 2011; Zhang et al., 2013), emotional behavior (Teissier et al., 2015), reward processing (Li et al., 2016; Liu et al., 2014; Miyazaki et al., 2014; Teissier et al., 2015), visual-related synaptic plasticity (Maya Vetencourt et al., 2008) as well as in development (Bonnin et al, 2007 - A transient placental source of serotonin for the fetal forebrain; Migliarini et al, 2013).
5-HT in neuropsychiatric disorders
Growing evidences have been strengthened the ideas that alterations in the normal central serotonergic signaling are involved in the patho-physiology of several neuropsychiatric disorders such as
schizophrenia, depression, anxiety and autism (Lucki, 1998; Mann et al., 1990). Supporting this hypothesis, selective re-uptake inhibitors (SSRIs) are the most efficient antidepressants and currently represent the first line treatment for depression, obsessive-compulsive disorders and anorexia nervosa. In addition, several lines of evidence suggest that 5-HT may play a pivotal role in the etiology of behaviour and pathology relevant to autism spectrum disorders (ASD; Moy et al., 2009; Pardo and Eberhart, 2007). The most consistent findings derive from neuroimaging, genetic and pharmacological intervention showing that young autistic children lack the developmental peak in whole brain 5-HT synthesis capacity normally present in control infants (Chugani et al., 1999). Moreover, genetic studies in autistic populations have identified abnormalities in several 5-HT-related genes including Tph2 (Coon et al., 2005), SERT, MAOA and 5-HT receptors 2A and 7
(Polleux and Lauder, 2004). Further evidences supporting 5-HT as a neurobiological factor in ASD comes from pharmacological interventions as selective serotonin reuptake inhibitor fluoxetine causes improvements in social behaviour while decreasing aggressive and stereotyped behaviours in children with autism (Hollander et al., 2003). Conversely, tryptophan depletion results in decreased brain 5-HT and exacerbated symptoms in patients with ASD, increasing various stereotyped autistic behaviours (McDougle et al., 1996). There is finally growing evidence that chronic, endogenous 5-HT deficiency is implicated in depression vulnerability (Levinson, 2006). The hypothesis originally derived from the clinical observation that drugs enhancing 5-HT neurotransmission by inhibiting SERT or MAOA displayed antidepressant activity (Coppen and Wood, 1978). Moreover, common functional polymorphisms of the SERT gene promoter have been associated to elevated trait anxiety, and increased vulnerability to affective disorders (Levinson, 2006). In mice it has been demonstrated that alteration of serotonergic signalling through 5-HT1AR inactivation
during early postnatal development prompt to anxiety-like behaviour in the adult (Gross et al., 2002). More recently, Jacobsen and collaborators have demonstrated that the R439H mouse analogue of the R441H human polymorphism identified in the Tph2 gene, a mouse model of 5-HT deficiency, displayed depression-like alterations in 5-HT biomarkers such as cerebrospinal fluid 5-HIAA and fenfluoramine-induced plasma prolactin (Jacobsen et al., 2012). All together these observations strongly support the hypothesis of the involvement of 5-HT neurotransmission deficiency in the origin of neurodevelopmental and affective disorders (Ansorge et al., 2008). The idea that 5-HT activity contributes to the etiology of schizophrenia evolved from the observation that lysergic-acid diethylamide (LSD), a drug structurally similar to 5-HT, is hallucinogenic (Pieri et al., 1978) and it has been suggested to produce its psychotomimetic effects through the stimulation of 5-HT2Rs (Sanders-Bush et al., 1988). The LSD psychosis has been found to be a close model for the reality distortion syndrome in schizophrenia (Slade, 1976). Moreover, changes in 5-HT receptor and SERT gene expression, altered 5-HT levels and behavioural studies support the hypothesis that dysfunctions in 5-HT system may alter synaptic plasticity that is sufficient to cause developmental changes leading to schizophrenia (Sodhi and Sanders-Bush, 2004). Impairment to the normal 5-HT neurotransmission could be a contributory factor, if not a primary cause, also in some neuropsychiatric disorders leading to behavioural impairment, such as eating disorders, addiction and stress related disorders (Gaspar et al., 2003; Sodhi and Sanders-Bush, 2004). For example, antidepressants such as SSRI have been shown to promote neurogenesis in the adult hippocampus requiring several weeks to integrate into the neural circuits, which could account for the delay that is necessary before antidepressants start being effective (Santarelli et al., 2003). Analysis performed to shed light on the biological functions of Tph2 gene has
permitted the identification of genetic variants or different expression levels of this gene involved in the pathogenesis of various psychiatric disorders including depression, schizophrenia and aggression. Zhang and co-workers have also identified in the human Tph2 gene a functional SNP (G1463A), which replaces the highly conserved Arg441 with His (namely R441H, or Arg441His). This substitution results in 80% reduction in the enzymatic activity when expressed in cell culture system. Moreover, the authors found that the mutant 1463A allele was more abundant in subjects affected by unipolar major depression, suggesting that deficiency of brain 5-HT synthesis, due for example to a functional SNP mutation in Tph2 gene, may be an important risk factor for certain neuropsychiatric disorders, such as unipolar major depression (Zhang et al., 2005). To investigate the effects of R441H Tph2 mutation in vivo, Beaulieu and colleagues have generated a knockin mouse line in which the human R441H Tph2 allele is engineered at the equivalent R391H amino acid residue of the mouse Tph2 gene. The results showed that R439H mutation recapitulates the changes reported in the human genetic variant, leading to a 80% reduction in the 5-HT levels in the brain and induced the activation of GSK3β, a signalling molecule modulated by many psychiatric therapeutic agents (Beaulieu et al., 2008). Moreover, R439H Tph2 genetic variation is sufficient to induce behavioural abnormalities, which are alleviated by pharmacological inactivation of GSK3β. These studies strongly support a role for 5-HT in neuropsychiatric disorders, and identify GSK3β as a key factor in the pathway through which 5-HT deficiency may lead to abnormal behaviours, and a possible target for therapeutic strategies. In the last years, applying SNP haplotype and linkage disequilibrium studies, it has been reported an association between polymorphic variants of the human Tph2 gene and the pathogenesis of many behavioural disturbances: affective disorders, aggressive behaviour (Kulikov et al., 2005), autism (Coon et al., 2005),
attention-deficit/hyperactivity disorder (ADHD; Walitza et al., 2005), obsessive-compulsive disorders (ODC; Mossner et al., 2006), major depression (Zill et al., 2004a) and suicide behaviour (Zill et al., 2004b).
AIM OF THE THESIS
5-HT neurons exert archetypical modulatory effects via the concerted recruitment of multiple differentially distributed pre-and post-synaptic receptors subtypes (Nichols and Nichols, 2008). Recent imaging studies have probed the brain regions modulated by 5-HT receptor ligands in humans (Anderson et al., 2008) and animals (Schwarz et al., 2007). The mapped effects however only provide an indirect view of the substrates targeted by 5-HT-based neuromodulation, and are plausibly contaminated by peripheral vasoactive effects mediated by 5-HT receptor populations located on vascular endothelial walls (Martin, 1994). In this thesis, I sought to map the brainwide targets of 5-HT-producing neurons through the combined use of mouse cerebral-blood volume based fMRI (Squillace et al., 2014) and 5-HT neuron-specific DREADD chemogenetics. An approach we term chemo-fMRI aiming to circumvent these issues by causally linking central 5-HT stimulation to regional functional responses. To this scope we generated and characterize two DREADD-based mouse lines for the conditional expression of the excitatory hM3Dq and the inhibitory hM4Di DREADD in 5-HT neurons.
MATERIALS AND METHODS
Animal care
All research involving animals were carried out in accordance with the European directive 86/609/EEC governing animal welfare and protection, acknowledged by the Italian Legislative Decree no. 116, 27 January 1992. Animal research protocols were also reviewed and consented to by a local animal care committee. Male C57BL/6J mice (12-20 weeks) were used throughout the study unless otherwise specified. hM3Dq/Pet1-Cre, littermate controls DIO-hM3Dq and littermate controls wild-type mice rederived on a C57BL/6J genetic background, were obtained by generating the conditional knock-in hM3Dq mouse line and by maintaining and crossing it with the previously generated Pet1210-Cre mice.
Mouse lines generation
The hM3Dq and hM4Di sequences in-frame-fused with the mCherry reporter and flanked by two couples of Lox sites (LoxP and Lox2722; DIO-hM3Dq and DIO-hM4Di, Double-floxed Inverse ORF) were isolated by EcoRI digestion from hSyn-DIO-hM3Dq and pAAV-hSyn-DIO-hM4Di, respectively. They were inversely cloned downstream to the CAG promoter in the pCX-CAG-eGFP plasmid, thus replacing the eGFP coding sequence (CDS). The CAG-DIO-hM3Dq fragment and the CAG-DIO-hM4Di were extracted from the pCX-CAG-hM3Dq and the pCX-CAG-hM4Di, respectively, by a SpeI/XbaI double digestion and cloned into the unique XbaI site placed between the right (RA) and the left (LA) ROSA26 homology arms included in the pROSA26-1Sor plasmid. Pgk-Neo/Kana cassette
(NEO) and Pgk-Difteric Toxin A cassette (DTa) were used for positive and negative selection, respectively. The PvuI-linearized pROSA26-1Sor-LA-CAG-DIO-hM3Dq-NEO-RA-DTa and pROSA26-1Sor -LA-CAG-DIO-hM4Di-NEO-RA-DTa were independently electroporated in E14Tg2a.4 embryonic stem cells (ESCs). Positive recombinants identified by southern blot were microinjected in the host C57BL\6J blastocysts, which were injected in utero of pseudopregnant CD1 females (3.5 dpc) to give rise to chimeras (n=29). The hM3Dq conditional knock-in and the hM4Di conditional knock-in mice did not show evident differences in respect to their wild-type littermates.
Genotyping
Mice were routinely genotyped by PCR DNA amplification with specific oligonucleotides as primers: 5’-GAGGGGAGTGTTGCAATACC-3’ as forward, and 5’-AGTCTAACTCGCGACACTGTA-3’ as reverse, for the wild-type ROSA26 allele; the alternative reverse 5’-GTCCCTATTGGCGTTACTATG-3’ for the hM3Dq and the DIO-hM4Di alleles; GTCATCTCCTTTGTCCTTTGG-3’ as forward and GGAGCTGGGTTTCCAGCTC-3’ as reverse for the hM3Dq CDS,
5’-AGCAGTGACCTTTGGCACAG-3’ as forward and
5’-GTCCTTATCAGCCACGGGG-3’ as reverse for the hM4Di CDS
Embryonic stem cell culture
The feeder-independent ES cell line E14Tg (BayGenomics) derived from the 129/Ola strain of mice was used. The ES cell line was maintained on gelatinized tissue culture dishes in ES cell medium containing the leukaemia inhibitory factor (LIF) according to BayGenomics protocols available online http://baygenomics.ucsf.edu.
Electroporation in ESCs
The pROSA26-1Sor-LA-CAG-DIO-hM3Dq-NEO-RA-DTa and the pROSA26-1Sor-LA-CAG-DIO-hM4Di-NEO-RA-DTa targeting vectors were purified on a Qiagen column kit and digested with PvuI. The linearized DNA was purified with phenol/chloroform/isoamyl alcohol, precipitated with ethanol and dissolved in sterile water at a concentration of 1 mg/ml. The day of the electroporation, cells were 80% confluents and the medium was changed 2 hours before the cells are harvested. The ES cells were trypsinized, resuspended at a concentration of 2 x 107 cells/ 0.7 ml of PBS and electroporated with 30 μg of linearized targeting vector, by using a Bio-Rad Gene Pulser unit using the Time Constant Protocol at a setting of 0.2 msec and 800 volts. After pulsing, the results screen indicated a capacitance of 10 microF and infinite resistance. Electroporated ES cells were recovered in the cuvette for 20 minutes at RT and then transferred in 40 ml of ES cell medium. 10 ml of ES cell medium with about 5 × 106 cells were plated into 10 cm diameter tissue-culture petri dishes. The following day the medium was replaced with medium containing 12.5 g/ml active geneticin (G418). The medium was changed daily. Approximately seven days after electroporation cells that have integrated the targeting vector form colonies about 1 mm in size and were picked. Recombinant resistant colonies were picked and replicated in 96-well plates. Clones contained in one replica-plate were stocked at -80°C while clones contained in the second set of plates were used for Southern blot analysis.
Southern Blot
Once picked, the ES recombinant clones were expanded according to BayGenomics online protocols. Total genomic DNA was extracted
from each of the ES cell clones with phenol-chloroform and ethanol precipitation, digested with specific restriction enzymes and separated according to size by gel-electrophoresis, lifted to a nylon filter and hybridized with a proper radiolabeled probe. In particular, to screen for CAG-DIO-hM3Dq and CAG-DIO-hM4Di targeted integration in ROSA26 locus, ES cells DNA was digested with HindIII and a probe external to the left homology arm was used to screen for the 5’ correct recombination.
ESCs Karyotyping
1-2 x106 ES cells are seeded in a 60 mm gelatinized plate and culture o/n. Then, 0.06 μg/ml of colcemid is added to culture medium for 2-3 hours and is washed with 2 ml of PBS. Cells are collected in a 15ml tube and are then incubated in 1 ml of trypsin/EDTA for 5 minutes, which is inactivated adding 4 ml of medium. Cells are resuspended carefully by pipetting up and down to obtain a single cell suspension and then pelleted at 1000 rpm for 5 minutes and resuspended again. 5 ml of hypotonic solution (0.56% KCl in water) is added and cells are incubated 6 minutes a RT to swell the cells. Cells are pelleted, the hypotonic solution is removed and cells are resuspended very carefully. 5 ml of cold fixative solution (3 methanol:1 glacial acetic acid) is added pipetting several times to avoid formation of clumps and incubated 20 minutes a RT. Cells are pelleted again, resuspended in 5 ml of fresh fixative and incubated 5 minutes a RT twice. Then slides are prepared: slides are washed in acetone and then in water and air-dried. After dipping in ice-cold 40% methanol slides are pulled out and the excess of liquid is drain off without shaking. Hold the slides at 45° angle 1-3 drops of cell suspension are dropped from about 40-50 cm height with a 1 ml pipetman. Then slides are dried o/n and the next day
are placed 5 minutes in 5N HCl, rinsed for 5 minutes by constant flow of distilled H2O, then stained with Giemsa dye for 40 minutes. The chromosome can be also visualized with DAPI staining. Chromosomes were then manually counted to exclude aneuploidy
Blastocyst injection and mouse breeding
ES cell lines were thawed and passed for 6 days in ES cell medium in the absence of G418. The day of injection, the medium was changed several hours before harvesting the cells. A confluent 25 cm2 flask was trypsinized for 3-4 minutes and diluted into 9 ml of cold ES cell medium without LIF, pelleted and resuspended in 0.8 ml of ES cell medium (without LIF) in a sterile 1.5 ml screw-top microcentrifuge tube. Before they were added to the injection chamber, cells were kept on ice (for up to several hours) to prevent clumping. Blastocysts were flushed from pregnant C57BL/6 females and collected into a CO2-independent medium containing 10% FBS. Blastocysts were expanded for 1-2 hours in ES cell medium in a 37°C / 6% CO2 incubator, transferred to a hanging drop chamber, and cooled to 4°C. ES cells were added to the hanging drops and the blastocysts were injected with enough cells (20 or more) to fill the blastocoele. Injected blastocysts were then transferred to pseudopregnant recipient females (10-15 blastocysts/uterine horn). Typically, injection of 10 blastocysts will yield an average of twice male chimeras with germline mosaicism. Of note, the 129/Ola cells carry the recessive pinkeye (p) and chinchilla (cch) mutations; strong chimeras exhibit patches of cream coloured fur and can have pink eyes.
Vasectomy
Vasectomized males are required to generate pseudopregnant recipients females. Vasectomy is performed on CD1 males mice at 6-8 weeks of age. 10- 14 days after vasectomy, the animals were tested to verify their sterility. One or two fertile female mice were placed with the vasectomized male and were checked for plugs the following morning. Females with plug were sacrificed and their oviducts were flushed 24 hours later. The eggs should be at one cell stage or unfertilized because whether they are at two cells stage, the vasectomy is not performed correctly.
Patch clamp
Brains were sectioned at 0.07 (mm/s) to obtain 300 μm coronal slices of the DRN. In a recording chamber slices were submerged in normal, oxygenated aCSF (28-30° C, 2mL/min flow rate) for at least 30 minutes before performing whole-cell patch clamp experiments. Borosilicate electrodes with a pipette filled with internal solution (135 mM KCl-gluconate, 5 mM NaCl, 2 mM MgCl2, 10 mM HEPES, 0.6 mM EGTA, 4 mM ATP and 0.4 mM GTP, pH= 7.35, 290 mOsmol) were used to patch cells in the DRN. Signals were acquired using a Multiclamp 700B amplifier and analyzed with Clampfit 10.3 software (Molecular Devices, Sunnyvale, CA, USA). The effects of CNO were determined in current clamp mode. After 5 minutes of stable baseline, CNO (10 μM) was bath applied for 10 minutes while recording changes in membrane potential. For excitability experiments, the current threshold (rheobase) necessary to induce cell firing were determined in current clamp mode using a current ramp protocol from 0 to 100 pA. Next, a 10 pA current step protocol from 0 to 200 pA was applied, from
which V-I plots were determined (i.e., the number of action potentials vs. current).
Drug formulation and pharmacological treatments
All drugs were administered intravenously as this route of administration maximizes the rate of fMRI signal change leading to sharp fMRI response that can be more easily discriminated by linear alterations in baseline due to scanner or physiological drifts (Mandeville et al., 2014). Clozapine-n-Oxide (Sigma Aldrich) was dissolved in saline solution at a concentration of 0,125 μg/ml. The CNO dose employed for chemo-fMRI mapping (0,5 mg/kg i.v., volume 10 mL/kg) was selected out of dose-response experiments (0,5-1-2 mg/kg i.v.) described in the result section. The dose chosen was the maximum non-effective dose in control studies in wild-type mice. Citalopram (Sigma Aldrich) was dissolved in saline solution at a concentration of 3 or 1.5 ml/kg for 10 mg/kg and 5 mg/kg dosing, respectively. The citalopram doses tested were previously shown to be behaviourally effective in C57Bl6/J mice (Browne and Fletcher, 2016).
In pilot c-Fos mapping studies in which we employed fMRI dose regimen of CNO, the prolonged restriction required for intravenous administration of CNO resulted in broad unspecific FOS increases under baseline conditions. We therefore opted for the administration of a dose of 2 mg/kg to account for the reduced maximum plasma concentration associated with intraperitoneal administration with respect to intravenous administration. The dose employed is in line with the amounts of CNO tested by other investigators in DREADD-based studies (Roth, 2016).
Functional Magnetic Resonance Imaging (fMRI)
Animal preparation for functional magnetic resonance imaging has been previously described in great detail (Liska et al., 2017; Sforazzini et al., 2014a). The protocol utilized is optimized for physiological stability and permits monitoring of peripheral parameters critical to the success of pharmacological fMRI (phMRI), such as peripheral blood pressure (Ferrari et al., 2012) and arterial blood gases. Briefly, mice were anaesthetized with isoflurane (5% induction), intubated and artificially ventilated (2.5% surgery). The left femoral artery was cannulated for continuous blood pressure monitoring and blood sampling. Surgical sites were infiltrated with a non brain-penetrant local anesthetic (Ferrari et al., 2010). At the end of surgery, the animal was placed in supine position onto a water-heated custom cradle and isoflurane was discontinued and replaced by halothane (0.7%), an anaesthetic that preserves cerebral blood flow auto-regulation and neurovascular coupling (Gozzi et al., 2007). Functional data acquisition started 30 minutes after isoflurane cessation. Ventilation parameters were adjusted to maintain arterial paCO2 levels < 40 mmHg (Pepelko and Dixon, 1975) and paO2 levels > 90 mmHg, values which correspond to the 98% of hemoglobin saturation (Table I).
fMRI data were acquired as previously described (Galbusera et al., 2017; Squillace et al., 2014) on a 7T Pharmascan (Bruker, Ettlingen, Germany) by using a 72-mm birdcage resonator and a 4 channel anatomical shaped Bruker mouse brain coil, placed dorsally to the animal head. Co-centered anatomical and fMRI images were acquired using a Rapid Acquisition Relaxation-Enhanced and a Fast Low-Angle Shot MRI sequence (TReff = 288 ms, TEeff = 3.1 ms, α=30°; 180 x 180 x 600 µm resolution, dt = 60 s, Nr = 60 corresponding to 60 min total acquisition time). Images were sensitized to reflect alterations in relative cerebral blood volume (rCBV) by previous administration of 5
µl/g of blood-pool contrast agent (Molday Ion, Biopal, Worcester, MA, USA). Twenty-five minutes later each subject received an intravenous administration of vehicle or drug. fMRI responses were mapped and quantified as previously described (Errico et al., 2015). Briefly, fMRI time series were spatially normalized to a common reference space and signal intensity changes were converted into fractional rCBV changes. rCBV time series before and after drug or vehicle injections were extracted and analysed. Voxel-wise group statistics was performed using FEAT Version 5.63, with 0.5 mm spatial smoothing and using a boxcar input function that captured the main alteration in rCBV signal observed upon the pharmacological treatment.
The composition of experimental groups was as follows:
Study 1: – CNO dose-response in C57Bl6J mice: intravenous treatment with CNO (0,5 mg/kg n=6; 1 mg/kg n=4; 2 mg/kg n=5) or vehicle (n=6).
Study 2: Chemo-fMRI of 5-HT neurons: intravenous treatment with CNO in hM3Dq/Pet1-Cre (n = 20), or control DIO/hM3Dq (n = 14).
Study 3: fMRI of citalopram in C57Bl6J mice: intravenous injection of citalopram (5 mg/kg n=8; 10 mg/kg n=7) or vehicle (n=10).
Immuno-histochemical analyses
Ninety minutes after CNO administration, mice were deeply anesthetized with avertin 1.25 % and perfused transcardially with 4% paraformaldehyde. Brains were dissected, post-fixed overnight in 4% paraformaldehyde at 4°C and sectioned in slices 50 µM thick by a Leica Microsystems vibratome. Free-floating sections were incubated overnight at RT with goat anti-cFos (SC-52-G, Santa Cruz
Biotechnology, 1:1000) in a 5% horse inactivated serum solution. Sections were then incubated overnight at 4°C with donkey anti-goat (Alexa Fluor 488, Invitrogen, 1:500). For mCherry and GFP immunodetection, free floating sections were incubated at 4°C overnight with rabbit anti-RFP (ab62341, Abcam, 1:500) and chicken anti-GFP (Ab13970, Abcam, 1:1000). Sections were then incubated overnight at 4°C with goat anti-rabbit (Rhodamine Red, Invitrogen, 1:500) and goat anti-chicken (Alexa Fluor 488, Invitrogen, 1:500). Cell counts for c-Fos quantification and mCherry/eGFP co-localization analysis were performed using FIJI-ImageJ.
RESULTS
Generation of hM3Dq and hM4Di conditional knock-ins
DREADD receptors permit the modulation of neuronal activity by trans-acting on the wide range of GPCRs-dependent intracellular pathways (Armbruster et al., 2007), thereby opening the possibility to functionally characterize neurons in a non-invasive fashion. In order to produce tools allowing the induction of Gq- or Gi-coupled DREADD expression in genetically-defined neuronal populations, we generated two conditional knock-in mouse lines harboring a double floxed inverse open reading frame (DIO) of either the hM3Dq or the hM4Di DREADD genes, respectively. In both lines, the transcriptional activation of
DREADDs will be achieved upon Cre-mediated somatic
recombination, inducing the expression of hM3Dq or hM4Di for the spatio-temporal enhancement or inhibition of neuronal firing, respectively. To this aim, we took advantage of a homologous recombination-based gene targeting strategy including the insertion of the hM3Dq and the hM4Di genes within the Gt(ROSA26)1Sor genomic locus (ROSA26). ROSA26 is localized on mouse chromosome six and it encodes a ubiquitous, dispensable, non-coding nuclear RNA whose function is still uncertain. We chose ROSA26 because it has been largely used as target locus for exogenous DNA integration via homologous recombination (Friedrich and Soriano, 1991; Irion et al., 2007). Both hM3Dq and hM4Di targeting vectors were similarly designed and built in parallel (see materials and methods section). Within the two ROSA26 homology arms (HAs), we cloned the chicken β-actin (CAG) promoter, the double floxed (LoxP and Lox2722) inverse copy of hM3Dq (DIO-hM3Dq) and hM4Di (DIO-hM4Di) genes respectively, and the neomycin expression cassette (NEO). Notably,
hM3Dq and hM4Di genes were in frame fused with mCherry, a red fluorescent reporter (Fig. 1A). The
pROSA26-CAG-DIO-hM3Dq/mCherry and the pROSA26-CAG-DIO-hM4Di/mCherry
targeting vectors were designed in order to avoid constitutive expression of DREADDs, whose transcription will occur only after Cre-mediated stable inversion of the DIO-constructs (LoxP-Lox2722 system). In order to insert hM3Dq and hM4Di donor vectors in ROSA26, PvuI-linearized targeting vectors were independently electroporated in E14Tg2a.4 embryonic stem cells (ESCs). At this step, three scenarios could occur: cells that successfully integrate the construct at the targeting locus via homologous recombination (positive recombinants), cells that randomly integrated the construct and cells that did not integrate the exogenous DNA. In order to maximize the rate of positive recombinants, we provided targeting vectors with both positive (NEO) and negative (difteric toxin A expression cassette, DTa) selection markers. In particular, NEO encodes neomycin that confers resistance to the antibiotic G418, whereas DTa encodes the lethal difteric toxin A. Since we cloned NEO inside and DTA outside the HAs, only ESCs that integrated the donor vector via homologous recombination should survive, thereby evolving to colonies. To this purpose, after the electroporation G418 was maintained for seven days in the medium. Among the resistant ESC colonies, positive recombinants were identified by southern blot analysis (Fig. 1B). I found 7 out of 96 positive clones for the hM3Dq-elettroporated ESCs and 32 out of 192 for the hM4Di-hM3Dq-elettroporated ones. Karyotype analyses have been thus performed in order to discard positive recombinants that had accumulated macroscopic mutations affecting the number and/or the appearance of chromosomes throughout all procedure passages. For the generation of mice harboring the hM3Dq or the hM4Di recombinant alleles, we microinjected the selected ESC clones in C57BL/6 host blastocysts
(3.5 dpc). A few hours later, we injected the blastocysts in utero of pseudopregnant CD1 females (3.5 dpc) that continued the pregnancy until the birth of chimeras (n=29). The presence of DREADD genes in the chimeric mice was molecularly assessed by PCR DNA amplification (Fig. 1d). By backcrossing chimeras with C57BL/6 wild-type mice, we obtained the germline transmission of the recombinant alleles for the 60% out of the total number of chimeras. In this way, we generated the hM3Dq and hM4Di conditional knock-in (cKI) founders, in which we further validated the presence of the recombinant hM3Dq and hM4Di alleles within the ROSA26 genomic locus via PCR (Fig. 1C) by means of specifically designed primers (see material and methods section). Such mouse lines have been brought on the C57BL/6 genetic background through consecutive backcrosses (F6) with C57BL/6 wild-type mice. Notably, no evident differences have been observed between DIO-hM3Dq, DIO-hM4Di mice and their respective wild-type littermates.
Fig 1. Generation of hM3Dq and hM4Di conditional knock-in mouse lines. Targeted integration of the hM3Dq and hM4Di constructs in the ROSA26genomic locus. (A) From the top to the bottom, the wild-type structure of the ROSA26 mouse genomic locus; the hM3Dq targeting vector, green (LoxP) and blue (Lox2722) arrowheads indicate the location of Lox sites, yellow circles are used for FRT sites; the transcriptionally inactive DIO-hM3Dq allele, in which the hM3Dq-mCherry CDS is inverted in respect to the CAG promoter; the transcriptionally active hM3Dq allele, in which the hM3Dq-mCherry has been reverted upon a Cre-mediated somatic recombination, the hM4Di targeting vector; the transcriptionally inactive DIO-hM4Di allele, in which the hM4Di-mCherry CDS is inverted in respect to the CAG promoter; the transcriptionally active hM4Di allele, in which the hM4Di-mCherry has been reverted upon a Cre-mediated somatic recombination (B) Southern blot analysis on genomic DNA extracted from eight embryonic stem cells clones after HindIII digestion (hM3Dq allele=6.7 kb; wild-type allele=4.4 kb; hM4Di allele=11.1 kb). (C) PCR amplification of ROSA26 locus on DNA extracted from a wild-type (3), two mutants (1 and 4) and two heterozygous (2 and 5) hM3Dq and hM4Di mice.
Selective expression of hM3Dq and hM4Di in 5-HT neurons.
5-HT neurons are a widely distributed population whose exact influence on brain circuits and behavior is still controversial. In order to produce tools allowing the in vivo modulation of serotonergic neurotransmission, we crossed both hM3Dq+/- and hM4Di+/- mouse lines with the previously generated Pet1210-Cre mouse line, in which
the transgenic expression of Cre-recombinase is driven by the promoter of the Pet1 gene, a well known marker of both mature and developing 5-HT neurons (Pelosi et al., 2014). The resulting hM3Dq/Pet1-Cre and hM4Di/Pet1-Cre trans-heterozygous mice, upon CNO administration, will enable serotonergic neurons firing activation and firing inhibition, respectively. To confirm hM3Dq expression in 5-HT neurons we performed a double immunofluorescence analysis on the triple heterozygous (Fig. 2D) hM3Dq/Pet1-Cre/Tph2GFP+/- mice (Fig. 2A-C), in which the GFP reporter was under the control of the
endogenous promoter of the 5-HT marker gene Tph2 (Migliarini et al., 2013). We found mCherry expression in 5-HT dorsal raphe neurons as well as in median and caudal raphe nuclei We found also that mCherry colocalized with GFP in the 95% out of the total 5-HT neurons (n=3 mice, 2909±119.64 counted cells, 2658.33±119 mCherry+ GFP+ cells, 124.33±12.42 mCherry- GFP+ cells; Fig. 2E), raising the possibility to can in vivo enhance the activity of nearly all 5-HT neurons. Similarly, to confirm hM4Di expression in 5-HT neurons we performed a double immunofluorescence analysis on the triple heterozygous (Fig. 3D) hM4Di/Pet1-Cre/Tph2::eGFP mice (Fig. 3A-C). We found mCherry expression in 5-HT dorsal raphe neurons as well as in median and caudal raphe nuclei.
Fig 2. Selective hM3Dq expression in 5-HT neurons and patch clamp recordings of CNO treated hM3Dq-Pet1-Cre 5-HT neurons. (A-C) from the top of the left panel, confocal images showing (A) mCherry expression, (B) GFP expression and (C) merged channels in dorsal raphe nucleus. (D) Schematic genotype representation of hM3Dq/Pet1-Cre/ Tph2GFP+/- triple trans-heterozygous mice, green (LoxP) and blue (Lox2722) arrowheads indicate the location of Lox sites after Cre-mediated recombination. (E) Quantification of hM3Dq-expressing 5-HT neurons: mCherry (in-frame-fused with hM3Dq) is present in the 95% of the total GFP-positive (Tph2-positive) cells (n=3 mice 2909±119.64 counted cells, 2658.33±119 mCherry+ GFP+ cells, 124.33±12.42 mCherry- GFP+ cells). (F) Electrophysiological properties
identifying 5-HT neuron. (G) From the left to the right, mean effect, image of the recorded cell and single-cell firing tracking of CNO treated hM3Dq/Pet1-Cre mCherry+ neuron. (H) From the left to the right, mean effect, image of the recorded cell and single-cell firing tracking of CNO treated hM3Dq/Pet1-Cre mCherry- neuron. (I) From the left to the right, mean effect, image of the recorded cell and single-cell firing tracking of CNO treated wild-type 5-HT neuron. The purple line indicates CNO administration time window.
Fig 3. Selective hM4Di expression in 5-HT neurons and patch clamp recordings of CNO treated hM4Di-Pet1-Cre 5-HT neurons. (A-F) from the top of the left panel, confocal images showing (A) mCherry expression, (B) GFP expression and (C) merged channels in dorsal raphe nucleus. (D) Schematic genotype representation of hM4Di/Pet1-Cre/Tph2GFP+/- triple heterozygous mice, green (LoxP) and blue (Lox2722) arrowheads indicate the location of Lox sites after Cre-mediated recombination. (E) Electrophysiological properties identifying 5-HT neuron. (L) From the left to the right, mean effect, image of the recorded cell and single-cell firing tracking of CNO treated HM4DI/Pet1-Cre mCherry+ neuron. (M) From the left to the right, mean effect, image of the recorded cell and single-cell firing tracking of CNO
