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Cannabinoid receptor function in the medial prefrontal cortex

den Boon, F.S.

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Citation for published version (APA):den Boon, F. S. (2013). Cannabinoid receptor function in the medial prefrontal cortex.

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CANNABINOID RECEPTOR FUNCTION

IN THE MEDIAL PREFRONTAL CORTEX

This research was financially supported by Top Institute Pharma,

grant T5-107-1

Cover Silke Kuilman

Print CPI Wöhrmann Print Service

Cannabinoid receptor function

in the medial prefrontal cortex

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus

prof. dr. D.C. van den Boom

ten overstaan van een door het college voor promoties

ingestelde commissie,

in het openbaar te verdedigen in de Agnietenkapel

op woensdag 19 juni 2013, te 10.00 uur

door

Femke Susanne den Boon

geboren te Amsterdam

Promotiecommissie

Promotor: Prof. dr. W.J. Wadman

Copromotor: Dr. T.R. Werkman

Prof. dr. C.G. Kruse

Overige leden: Prof. dr. L. de Haan

Dr. H.J. Krugers

Prof. dr. P.J. Lucassen

Prof. dr. E.M.A. Aronica

Prof. dr. F.H. Lopes da Silva

Prof. dr. H.D. Mansvelder

Prof. dr. B.H.C. Westerink

Faculteit der Natuurwetenschappen, Wiskunde en Informatica

The leafless tree looked like a brain The birds within were all the thoughts and desires within me Hoppin’ around from branch to branch Or snug in their nests listenin’ in

- Bill Callahan -

Table of contents

Chapter 1 9 General introduction Chapter 2 31 Cannabinoid receptor activation reverses short-term synaptic plasticity in the rat medial prefrontal cortex Chapter 3 45 Excitability of prefrontal cortical pyramidal neurons is modulated by activation of intracellular type-2 cannabinoid receptors Chapter 4 63 Endocannabinoids produced upon action potential firing evoke a Cl- current via type-2 cannabinoid receptors in the medial prefrontal cortex Chapter 5 81 Modulation of the balance between excitation and inhibition in the rat medial prefrontal cortex by type-1 cannabinoid receptors Chapter 6 103 General discussion Addendum 121 References Nederlandse samenvatting List of publications Curriculum Vitae Dankwoord

Chapter 1

General introduction


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The endocannabinoid system The term ‘endocannabinoid (eCB) system’ refers to a group of endogenous modulatory lipids called endocannabinoids (eCBs) that play a role in various physiological processes, both in the brain and in the periphery (Kano et al. 2009). In addition to the endogenous ligands, the eCB system contains at least two G protein-coupled receptors (GPCRs), cannabinoid type-1 receptors (CB1Rs) and cannabinoid type-2 receptors (CB2Rs), enzymes responsible for the synthesis and degradation of eCBs and eCB transporter proteins. The eCB system is present throughout the central nervous system (CNS) and the periphery and is involved in many functions of the CNS, including decision-making, working memory and other executive functions (Pattij et al. 2008).

Compounds interacting with the eCB system are found in nature. The most widely known cannabinoids are found in the plant Cannabis sativa. Cannabis, obtained from this plant, is one of the most widely used drugs of abuse worldwide. The major psychoactive component of cannabis, Δ9-tetrahydrocannabinol (Δ9-THC), was discovered in 1964 (Gaoni and Mechoulam 1964). Δ9-THC increases dopamine release in the striatum in animals and humans (Tanda and Goldberg 2003; Gardner 2005; Bossong et al. 2009). This characteristic of Δ9-THC is associated with the abuse liability of cannabis (Maldonado and Rodríguez de Fonseca 2002; Maldonado et al. 2011).

Since the discovery of Δ9-THC, other cannabinoids have been isolated from Cannabis sativa and synthetic cannabinoids have been discovered (Mechoulam and Hanus 2000; Pertwee et al. 2010). Although cannabis is classified as an illegal substance in most countries, cannabis is now officially prescribed to relieve symptoms of several diseases. For example, cannabis and Δ9-THC are used as antiemetic in patients with cancer and acquired immune deficiency syndrome (AIDS) and for the treatment of chronic pain in patients with cancer and spasticity in multiple sclerosis. Cannabis use in adolescents is associated with an increased risk of schizophrenia and chronic cannabis use with a reduction in cognitive control and neuropsychological decline (Arseneault et al. 2002; Harding et al. 2012; Meier et al. 2012). Medicinal use of cannabis is increasing, while our current understanding of the eCB system in the CNS is far from complete. eCB research aims to discover the role of the eCB system in the regulation of brain functions that are involved in psychopathological syndromes, such as addiction and cognitive disorders.

The work in this thesis was performed as a part of a TIPharma research consortium with the title ‘The neurophysiological role of the eCB system in support of smoking cessation, fighting addiction and treating cognitive decline’. The goal, as defined by this research consortium, is to elucidate the role of the eCB system in the development of psychological disorders. The consortium consists of several research groups, each with their own research techniques and specific research questions. The experimental work presented here relies on the use of electrophysiological and biochemical techniques and aims to investigate the functioning of the eCB system at the cellular and local network level. The targeted brain area is the medial prefrontal cortex (mPFC), a brain region involved in higher cognitive functions, such as working memory, planning complex behaviour, decision making and impulse control

Chapter 1

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(Miller and Cohen 2001; Koechlin et al. 2003). The mPFC has been implicated in various psychopathological and cognitive disorders and has traditionally received attention from researchers in the addiction field. Furthermore, although it is known that the eCB system is present in the mPFC, a detailed description of the functioning of this system in the mPFC is lacking. Here, our main research questions concern the role of CB receptors in neuronal communication, at the level of the local cortical network and individual neurons. The eCB system and neuropathologies Recent years have seen an increase in research into the involvement of the eCB system in a growing number of neuropathologies, such as multiple sclerosis, Huntington’s, Parkinson’s and Alzheimers’ diseases, schizophrenia, autism and epilepsy. Some research has focused on the potential role of cannabinoid ligands as therapeutics, whereas others have investigated harmful effects of cannabinoids (Arseneault et al. 2002; Scotter et al. 2010; Harding et al. 2012; Meier et al. 2012). A recent report shows that persistent cannabis use, particularly among adolescent-onset cannabis users, was associated with neuropsychological decline across several domains of functioning. Among adolescent-onset users, cessation of cannabis use did not fully restore performance on a broad range of neuropsychological tests (Meier et al. 2012). The mechanisms underlying the effects of persistent cannabis use in adolescents are unclear, but could be explained by effects of cannabinoid compounds on the still developing brain (Jager and Ramsey 2008; Rubino et al. 2009).

Cannabis use has been associated with the development of psychoses and schizophrenia by a multitude of studies performed in the last 50 years (Andréasson et al. 1987; Fergusson et al. 2006; Malone et al. 2010). However, whether cannabis use causes psychotic symptoms, precipitates them (in ‘schizophrenically vulnerable’ individuals) or can be seen as a form of self-medication by individuals prone to psychosis is still under debate. There is no consensus over the mechanisms that could be responsible for these negative effects of cannabis use, that may occur especially around adolescence. It has been suggested that the involvement of the eCB system in moderating neurodevelopmental processes could play a role (Malone et al. 2010).

The eCB system and CNS development The development of the CNS involves dynamically regulated cellular processes such as cell generation, differentiation, migration, maturation and proper wiring. Developmental neurobiological studies are increasingly able to identify the molecular mechanisms underlying the specification and differentiation of particular neuronal cells. Several studies have shown that prenatal exposure to cannabinoids affects behavioural aspects regarding the control of emotions and cognitive responses and these results have further increased the interest for cannabinoid-mediated effects on CNS development (D’Souza et al. 2009; Jutras-Aswad et al. 2009; Diaz-Alonso et al. 2012). Early in development, eCBs act as signalling cues in ‘neurogenic niches’, microenvironments where neurons and glia are generated. Activation of the CB1R plays an important role in neuronal fate decisions, such as cell cycle progression and


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proliferation, neural cell specification and cell migration, morphogenesis and apoptosis. Importantly, dysfunction of the eCB system during CNS development may be involved in the onset of epileptogenesis, via an imbalance in excitatory and inhibitory neurotransmission (Katona and Freund 2008; Lutz and Monory 2008). During later, postnatal stages of development, exposure to cannabinoids or altered eCB signalling disrupts neuronal maturation and interferes with neuronal connectivity of developing networks, which could have consequences for neuronal functioning in the adult (Jutras-Aswad et al. 2009). To summarize, the eCB system is present in the CNS from an early stage and plays a key regulatory role in CNS development. Affecting the eCB system during development can have significant consequences for the adult brain, such as the tuning of the appropriate balance between excitatory and inhibitory input and the potential onset of neuropsychiatric disorders (Diaz-Alonso et al. 2012). Cannabinoid receptors CB1R expression Following the discovery of Δ9-THC, the CB1R was cloned and characterized in 1990 (Matsuda et al. 1990). CB1Rs are one of the most abundantly expressed GPCRs in the CNS and are predominantly located on presynaptic terminals (Herkenham et al. 1991). CB1Rs are also expressed at much lower, yet functionally relevant, levels in various peripheral tissues (Hohmann and Herkenham 1999; Navarro et al. 2002; Bensaid et al. 2003; Osei-Hyiaman et al. 2005). In the CNS, particularly high expression levels for CB1Rs are detected in the cerebellum and telencephalon, which is compatible with the effects of Δ9-THC on motor and cognitive functions. The CB1R mediates most psychoactive effects of cannabis (Ledent et al. 1999; Huestis et al. 2001). CB1Rs are expressed at low levels in lower brain stem areas that are responsible for the regulation of cardiovascular and respiratory functions. The relatively low expression of CB1Rs in brain areas involved in vital functions has been linked to the fact that even high doses of cannabinoids are not lethal (Herkenham et al. 1991). Similarly, analgesic, anti-anorexic and antiemetic effects of cannabinoids can be explained by CB1R expression in the spinal dorsal horn, the ventromedial hypothalamic nucleus and the caudal solitary nucleus (Kano et al. 2009). CB1R signalling Since their discovery, the expression and functionality of the CB1R in the CNS has been thoroughly investigated (Devane et al. 1988; Kano et al. 2009; Castillo et al. 2012; Katona and Freund 2012). CB1Rs are coupled to Gi/o proteins and activation of the receptor inhibits adenylyl cyclase and cAMP production in a variety of cells, including neurons expressing CB1Rs. However, under certain conditions, CB1R-coupling to Gs proteins has also been reported, thereby increasing cAMP production (Demuth and Molleman 2006). Furthermore, CB1Rs have been shown to link positively to mitogen-activated protein (MAP) kinase, a key signalling pathway that regulates many cellular functions, such as cell growth and apoptosis (Bouaboula et al. 1995; Demuth and Molleman 2006). MAP kinases can be stimulated through G protein-mediated mechanisms and activate transcription factors, which in turn modulate gene

Chapter 1

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expression. Importantly, CB1R activation also modulates certain ion channels. Activation of CB1Rs induces the opening of A-type K+ channels and G protein-coupled inwardly rectifying K+ (GIRK) channels and it inhibits N- and P/Q type voltage-dependent Ca2+ channels (VDCCs) as well as D- and M-type K+ channels (Hampson et al. 1995; Mackie et al. 1995; Twitchell et al. 1997; Mu et al. 1999; Schweitzer 2000; Kano et al. 2009). The inhibition of VDCCs and activation of GIRK channels upon presynaptic CB1R activation is linked to the most commonly described effect of CB1R activation, the suppression of neurotransmitter release, which will be described below. Cellular response to CB1R activation The principal mechanism by which the eCB system is reported to play a functional role via CB1Rs in neurons, is retrograde signalling (Kreitzer and Regehr 2001; Wilson and Nicoll 2001; Kano et al. 2009). Postsynaptic activity, consisting of Ca2+ influx and/or activation of GPCRs such as group 1 metabotropic glutamate receptors, leads to the synthesis of eCBs. eCBs travel backwards across the synapse and bind to a transmembrane binding site on the presynaptic CB1R. There, activation of presynaptic CB1Rs suppresses neurotransmitter release mainly by inhibition of Ca2+ influx through VDCCs and possibly the activation of GIRK channels (Fig. 1) (Daniel and Crepel 2001; Kreitzer and Regehr 2001; Wilson et al. 2001; Brown et al. 2003; Guo and Ikeda 2004). Neurotransmitters of which the release is modulated by CB1R activation include the main excitatory and inhibitory neurotransmitters glutamate and GABA. CB1R activation also inhibits the release of other neurotransmitters such as glycine, acetylcholine, dopamine, 5-HT and norepinephrine (Kano et al. 2009). CB1R activation is involved in several forms of synaptic plasticity, both short-term and long-term, which will be discussed in more detail later on. CB2R expression The second cannabinoid receptor, the CB2R, was discovered in macrophages in 1993 (Munro et al. 1993). The human CB2R shares 44% amino acid sequence identity with the human CB1R and has a distinct pharmacological profile and expression pattern. The presence of CB2Rs in the brain has been a matter of debate. Initially, CB2Rs were not detected in the CNS. They were found to be predominantly expressed in immune cells and tissues outside the CNS and therefore called ‘peripheral’ cannabinoid receptors (Galiègue et al. 1995). Many conflicting reports on CB2R expression in the brain have been published, varying from reports of widespread expression to complete absence in the CNS. For instance, the presence of CB2R mRNA in the brain and CB2Rs in neurons of the brainstem, cerebellum and hippocampus has been reported by various groups (Van Sickle et al. 2005; Gong et al. 2006; Brusco et al. 2008a, 2008b; Onaivi et al. 2008, 2012; García-Gutiérrez et al. 2010). The abundant presence of CB2Rs was questioned by others, claiming that problems with visualization of CB2Rs by immunohistochemical


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Figure 1. Retrograde signalling mediated by eCBs. Postsynaptic activity leading to Ca2+ influx and/or activation of mGluR results in the synthesis of eCBs, which are released from the plasma membrane and travel across the synapse to activate presynaptic CB1Rs. CB1R activation inhibits presynaptic Ca2+ influx, decreasing the probability of neurotransmitter release. Adapted from Wilson and Nicoll 2002.

stainings compromise reliable detection of CB2R brain expression (Atwood and Mackie 2010; Ashton 2012). Nevertheless, the presence of CB2Rs in microglia cells and other brain immune cells is now generally accepted (Stella 2010). CB2Rs were detected in perivascular microglia in the non-inflamed human brain; the current view supports the expression of functional CB2Rs in neurons upon brain stress or damage (Núñez et al. 2004; Viscomi et al. 2009). CB2R signalling CB2Rs couple to Gi/o proteins and thereby inhibit adenylyl cyclase and cAMP production and stimulate the MAP kinase pathway (Bouaboula et al. 1996; Kobayashi et al. 2001). However, modulation of [Ca2+]i following CB2R activation has also been reported (Beltramo 2009). In calf pulmonary endothelial cells, activation of CB2Rs led to an increase in [Ca2+]i via the phospholipase C- (PLC) dependent production of inositol trisphosphate (IP3) (Zoratti et al. 2003). In contrast, in cardiac cells and adult dorsal root ganglion neurons, CB2R activation is negatively coupled to IP3R-mediated Ca2+ release from intracellular Ca2+ stores (Sagar et al. 2005; Currie et al. 2008). Until recently, CB2Rs were assumed to poorly modulate Ca2+ channels, GIRK channels and other ion channels, unlike CB1Rs (Atwood and Mackie 2010). However, recent papers have demonstrated CB2R-mediated modulation of VDCCs (Atwood, Straiker, et al. 2012). Cellular response to CB2R activation Since CB2Rs were presumed absent from the CNS for many years, much less research has been performed to investigate the role of these cannabinoid receptors in the brain. However, several recent publications have demonstrated the functional presence of CB2Rs in the CNS by applying or administrating CB2R ligands, although the mechanism(s)

Chapter 1

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of action was/were unclear (Elmes et al. 2004; Jhaveri et al. 2008; Liu et al. 2009; Morgan et al. 2009; Xi et al. 2011; Atwood, Straiker, et al. 2012). These studies show varying roles for CB2Rs, for instance in the modulation of pain responses in the thalamus and dorsal horn neurons of rats in pain models (Elmes et al. 2004; Jhaveri et al. 2008). Others show that CB2Rs behave similarly to CB1Rs in the modulation of neurotransmitter release (Morgan et al. 2009; Atwood, Straiker, et al. 2012). Finally, other researchers found that brain CB2Rs modulate the rewarding and locomotor-stimulating effects of cocaine, likely occurring through a dopamine-dependent mechanism (Xi et al. 2011). These recent studies indicate that CB2Rs are functionally present in the CNS, but, so far, lack the description of the CB2R-mediated signalling pathways. Other receptors activated by eCBs In addition to CB1R and CB2R, other receptors that are sensitive to eCBs exist. Recently, an orphan GPCR called the GPR55 receptor has been shown to be activated by eCBs and phytocannabinoids, although this is cell- and tissue type-dependent (Sharir and Abood 2010). This has led to the hypothesis that GPR55 could be a third cannabinoid receptor. Relatively low expression levels of GPR55 have been detected in the brain. GPR55’s pharmacology remains controversial, although it is certainly distinct from that of CB1R and CB2R in terms of sensitivities for cannabinoid ligands (Sharir and Abood 2010; Zhao and Abood 2013). Future research on the GPR55 should determine whether GPR55 can be classified as a genuine cannabinoid receptor. It is important to note that it is not considered a CB receptor currently. Another receptor that can be activated by eCBs is a Ca2+-permeable, non-selective cation channel, which belongs to the transient receptor potential family, subfamily V member 1 (TRPV1). This receptor is peripherally present and is involved in pain signalling. It can be activated by heat greater than 43 °C, capsaicin (found in hot chili peppers) and allyl isothiocyanate (found in mustard and wasabi). Surprisingly, TRPV1 receptors are also expressed in the CNS, where their activation by heat and these chemicals are unlikely. Endogenous ligands that activate TRPV1 receptors are called endovanilloids and include some, but not all eCBs, as discussed below (Kano et al. 2009). In the CNS, these receptors are involved in synaptic plasticity (Gibson et al. 2008). TRPV1 receptors are not considered as components of the eCB system, because they cannot be activated by 2-AG and several synthetic cannabinoid ligands. However, shared ligands and similar tissue distribution, as well as opposing actions on the same intracellular signals imply a cross-talk between the eCB system and the endovanilloid system (Starowicz et al. 2007; Kano et al. 2009). Finally, another class of receptors that can be activated by eCBs are peroxisome proliferator-activated receptors (PPAR). These nuclear receptors function as transcription factors regulating gene expression and are involved in cellular differentiation, lipid metabolism and inflammation (Burstein 2005; O’Sullivan 2007).


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2-AG and anandamide The two most important eCBs are N-arachidonoylethanolamide (anandamide, AEA) and 2-arachidonoylglycerol (2-AG). The synthesis of both AEA and 2-AG, as briefly mentioned earlier, depends on cellular activity. The increase in [Ca2+]i, following neuronal depolarization and/or stimulation of GPCRs that are coupled to Gq/11 proteins, induce synthesis of both AEA and 2-AG, as discussed in more detail below. The first identified eCB, AEA, was isolated from the porcine brain and named ‘anandamide’ after the Sanskrit word ‘ananda’ that means bliss (Devane et al. 1992). AEA is an agonist for CB1Rs and CB2Rs and is also an endogenous ligand for TRPV1 receptors and PPARs. The main pathway for AEA synthesis involves the activity of the enzyme N-acyltransferase (NAT). Activity of (most) NAT enzymes is stimulated by Ca2+ and this is considered the rate-limiting step in AEA synthesis. This enzyme converts arachidonic acid-containing phospholipids in the plasma membrane into N-acyl phosphatidylethanolamine (NAPE). NAPE is then cleaved by N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD), resulting in the formation of AEA (Kano et al. 2009; Luchicchi and Pistis 2012). Alternative pathways for AEA synthesis have also been investigated and described, driven by the observation that NAPE-PLD knock-out (KO) mice showed unchanged levels of AEA (Simon and Cravatt 2010). AEA is broken down predominantly by fatty acid amide hydrolase (FAAH), but - like for the synthesis of AEA - alternative pathways exist. For instance, N-acylethanolaminehydrolizing acide amidase (NAAA), cyclooxygenase-2 (COX-2), lippxigenase isoenzymes (LOX) and the P-450 cytochrome can break down AEA (Bornheim et al. 1993; Ueda et al. 1995, 2010; Yu et al. 1997) (Fig. 2.). The extent to which these alternative degradation pathways are physiologically relevant, remains to be investigated.

The second important eCB, 2-AG, was identified a few years after the discovery of AEA (Mechoulam et al. 1995; Sugiura et al. 1995). Diacylglycerols (DAGs), the major source of 2-AG, are produced via PLC-dependent pathways (Fig. 2) (Bisogno et al. 1997). 2-AG is synthesized predominantly by diacylglycerol lipase α (DAGLα) and β (DAGLβ) from DAGs. Both DAGLα and DAGLβ are sensitive to Ca2+ and dependent on physiological levels of glutathione. Although Ca2+ influx alone can lead to 2-AG synthesis, the simultaneous increase in PLC activity following stimulation of GPCRs that are coupled to Gq/11 proteins and Ca2+ influx is thought to lead to maximal 2-AG synthesis (Min et al. 2010). Alternative, presumably less relevant, routes for 2-AG synthesis have also been described (Fig. 2) (Luchicchi and Pistis 2012). 2-AG is principally hydrolyzed by monoacylglycerol lipase (MGL), with a more recently discovered role for a series of serine hydrolase α-β-hydrolase domain 6 or 12 (ABHD6, ABHD12) (Mechoulam et al. 1995; Sugiura et al. 1995; Dinh et al. 2002, 2004; Marrs et al. 2010; Savinainen et al. 2012).

2-AG and AEA, which are by far the most studied eCBs, were initially regarded as very similar eCBs, in terms of their pharmacological properties. 2-AG and AEA are both found in the CNS as well as the periphery. Despite difficulties with the precise quantification of brain levels for these eCBs, there is consensus with respect to 2-AG

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Figure 2. Depiction of the main pathways involved in synthesis and degradation of 2-AG and AEA. Both 2-AG and AEA are derived from AA-containing phospholipids, but they have distinct formation pathways, see text. AA arachidonic acid, ABHD4 6, 12, alpha, beta hydrolase 4, 6 and 12, COX-2 cyclooxygenase-2, DAGL alpha and beta diacylglycerol lipase alpha and beta, FAAH fatty acid amide hydrolase, GDE1 glycerolphosphodiesterase 1, GP-AEA glycerol-phosphoanandamide, LOX lipoxygenase, LysoNAPE lyso Nacyl phosphatidylethanolamine, LysoPLD lyso phospholipase D, MAG-L monoacylglycerol lipase, NAAA N-acylethanolaminehydrolyzing acid amidase, NAPE N-arachidonoylphosphatidylethanolamine, NAPE-PLD N-acyl phosphatidylethanolamine phospholipase D, pAEA phosphoanandamide, PLA1 phospholipase A1, PLC phospholipase C, PTPN22 protein tyrosine phosphatase N22. Adapted from Luchicchi & Pistis 2012. levels exceeding those for AEA (Stella et al. 1997; Buczynski and Parsons 2010). Since the affinities of AEA and 2-AG for CB receptors are comparable (Table 1), this indicates that 2-AG is, generally speaking, the most relevant eCB. Thanks to the development of mouse lines lacking genes for FAAH or MAGL and pharmacological tools that selectively inhibit eCB-degrading or -synthesizing enzymes, researchers have increasingly disentangled the roles for 2-AG and AEA (Luchicchi and Pistis 2012).


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Since eCBs are lipids, it is generally assumed they cannot be stored in vesicles

like classical neurotransmitters. They are released from the postsynaptic cell after which they cross the synaptic cleft, presumably with the help of chaperone proteins such as the FAAH-like AEA transporter (Fu et al. 2012). As described, eCB levels increase when neurons are stimulated. This has led to the concept of ‘on demand’ synthesis. It has recently been suggested that eCBs, aside from the ‘on demand’ synthesis, are present in a ‘ basal pool’ , being constantly produced and released (Alger and Kim 2011; Di Marzo 2011; Alger 2012). In particular for 2-AG, these pools could be related to signalling, and basal, non-signalling levels. It is now obvious that the synthesis, release and possibly storage of eCBs are organized in a complex manner. Other eCBs Other (putative) eCBs have also been discovered, such as dihomo-γ-linolenoyl ethanolamide, docosatetra enoyl ethanolamide, 2-arachidonyl glycerol ether (noladin ether), O-arachidonoylethanolamine (virodhamine), N-arachidonoyldopamine (NADA), palmitoylethanolamide (PEA) and oleamide. These other eCBs have varying affinities and specificities for both cannabinoid receptors. For instance, members of the N-acylethanolamide family (which also contains AEA) like dihomo-γ-linolenoyl ethanolamide and docosatetra enoyl ethanolamide, bind to CB1Rs, but have lower affinities for CB2Rs. The specific functions of these putative eCBs are still to be determined. Noladin ether also has a lower affinity for CB2Rs than for CB1Rs and its presence in the CNS is disputed (Oka et al. 2003; Richardson et al. 2007). However, a recent study demonstrated that noladin ether increased food intake in rats, possibly by increasing the incentive value of food (Jones and Kirkham 2012). Virodhamine, which was isolated from rat brain, acts as a full agonist at CB2Rs, as an antagonist or partial agonist at CB1Rs and as a partial agonist at putative cannabinoid receptor GPR55 (Sharir et al. 2012). It produces hypothermia in mice, similar to AEA (Porter et al. 2002). The eCB NADA binds to both cannabinoid receptors as well as TRPV1 receptors and has been shown to induce TRPV1-dependent cell death (Davies et al. 2010). Although the affinity of the fatty acid amide PEA for CB1R and CB2R is still under scrutiny, PEA is considered by some as a bone fide eCB (Mackie and Stella 2006). PEA plays a role in protection against inflammation and pain (Re et al. 2007). For oleamide, in vitro efficacy at the CB1R has been shown, although this is controversial (Fowler 2004; Leggett et al. 2004). Oleamide induces sleep in rats, independent of its effects on body temperature, blood pressure and heart rate (Huitrón-Reséndiz et al. 2001). A complication in the investigation of the role of oleamide as an eCB is the leaching of this compound from disposable laboratory plasticware (McDonald et al. 2008). For some of these potential eCBs, a debate regarding their qualification as eCBs is still ongoing. Additional research is needed to elucidate the pharmacological profiles and functions of these putative eCBs. Here, we will focus on the roles of the eCBs 2-AG and AEA. For binding affinities, expressed as Ki values, of commonly used cannabinoid agonists and antagonists, see Table 1 and Table 2.

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eCB-mediated modulation of synaptic transmission Experiences and memories are presumed to be represented in the brain by a vast amount of interconnected networks of synapses. Changes in the strengths of these synapses, synaptic plasticity, are thought to underlie processes like learning and memory. The activation of CB1Rs is associated with several forms of synaptic plasticity, both long-term plasticity and short-term plasticity. Short-term plasticity, mediated by activation of CB1Rs lasting a few seconds, depends on the retrograde neurotransmission process of eCBs and has been described for many brain areas, e.g. hippocampus, cerebellum, basal ganglia and cortex. Two short-term plasticity phenomena called depolarization-induced suppression of inhibition (DSI) and depolarization-induced suppression of excitation (DSE) are CB1R-dependent (Kreitzer and Regehr 2001; Ohno-Shosaku et al. 2001; Wilson and Nicoll 2001). DSI and DSE, typically lasting less than a minute, rely on postsynaptic activity triggering Ca2+ influx through VDCCs. In addition, other Ca2+ sources, such as activated NMDARs and release of Ca2+ from internal stores can play a role as well Table 1. Ki values for various, commonly used cannabinoid agonists. Ratios of Ki values indicate the selectivity for either cannabinoid receptor. Large variations are due to variable experimental conditions, e.g. different mammalian species, tissue origin, cell culture background etc. For eCBs, an additional complicating factor is the rapid metabolism of these compounds. Their potencies depend on whether their metabolic pathways are inhibited. Adapted from Fowler (2008), Pertwee et al. (2010) and Pertwee (2008).

Table 2. Ki values for various, commonly used cannabinoid antagonists. Ratios of Ki values indicate the selectivity for either cannabinoid receptor. Adapted from Lunn et al. (2008).


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(Castillo et al. 2012). The influx of Ca2+ into the cell results in the production of eCBs. Alternatively, or in addition to Ca2+ influx-dependent eCB production, eCB synthesis can be induced by the activation of postsynaptic Gq/11-coupled receptors. Gq/11-coupled receptors induce eCB-mediated short-term plasticity include group I metabotropic glutamate receptors (mGluR 1/5), M1/M3 muscarinic receptors, glucocorticoid receptors, oxytocin receptors and orexin receptors (Kano et al. 2009). Activation of Gq/11-coupled receptors stimulates PLCβ production. PLCβ, component of the pathway for 2-AG synthesis, is thought to act as a coincidence detector for postsynaptic activity and Ca2+ influx (Castillo et al. 2012). eCB-mediated long-term synaptic plasticity has been described in several brain areas, such as the nucleus accumbens, cortex, hippocampus and cerebellum (Kano et al. 2009). The induction of eCB-mediated plasticity starts with increased glutamate release from afferents which induces the postsynaptic synthesis of eCBs, which then

Figure 3. Molecular mechanisms that underlie eCB-mediated short- and long-term plasticity. (A) Postsynaptic activity causes Ca2+ influx through VDCCs (VGCCs). Increased Ca2+ induces eCB synthesis via activation of DAGLα (DGLα). Presynaptic activity can also induce eCB synthesis via activation of Gq/11-coupled GPRCs, such as group I mGluRs, which stimulates PLCβ production. PLCβ can act as a coincidence detector that integrates pre- and postsynaptic activity. DAGLα promotes 2-AG synthesis, which retrogradely activates CB1Rs to reduced neurotransmitter release. (B) Glutamate released from presynaptic neurons induces postsynaptic production of eCBs, which retrogradely activate presynaptic CB1Rs on the original afferent (homosynaptic eCB-LTD) or other nearby afferents (heterosynaptic eCB-LTD). A Gαi/o-dependent decrease in adenylyl cyclase (AC) and PKA activity reduces neurotransmitter release. At inhibitory synapses, reduced PKA activity together with activation of Ca2+-sensitive phosphatase calcineurin (CaN) alters the phorphorylation status of an unidentified presynaptic target (T) necessary for iLTD. The presynaptic active zone protein RIM1α and Rab3B are necessary for iLTD. Adapted from Castillo et al. 2012.

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retrogradely activate CB1Rs on the original afferent (homosynaptic eCB-LTD) or other nearby afferents (heterosynaptic eCB-LTD) (Castillo et al. 2012). In eCB-LTD, presynaptic CB1Rs are activated for several minutes. Their activation is only required during the induction of eCB-LTD, not for the remaining duration of eCB-LTD. The predominant mechanism underlying eCB-mediated long-term plasticity involves the αi/o effector limb of the CB1R G protein signalling cascade, which inhibits adenylyl cyclase and the cAMP/protein kinase A (PKA) pathway (Heifets and Castillo 2009) (Fig. 3). Other forms or LTP and LTD are also known to depend on the modulation of the cAMP/PKA pathway, which can change the transmitter release machinery. For eCB-LTD, a presynaptic active zone protein called RIM1α was identified as a key protein (Chevaleyre et al. 2007). RIM proteins are multidomain molecules that form an important part of the cytomatrix at the presynaptic active zone. In the nucleus accumbens, prolonged inhibition of the cAMP/PKA pathway and presynaptic P/Q-type VDCCs are necessary for eCB-LTD to occur (Mato et al. 2008). eCB-LTD has been heavily investigated in recent years and is now considered one of the best examples of presynaptic forms of long-term plasticity (Castillo et al. 2012). Most results suggest that 2-AG is the main eCB responsible for the regulation of synaptic plasticity, with a minor role for AEA. However, the ability of AEA to activate other receptors, such as TRPV1 receptors, means that AEA might modulate synaptic plasticity via different routes. Interestingly, a recent study showed that both 2-AG and AEA can induce eCB-mediated long-term depression (LTD) via CB1R activation in the striatum. Different stimulation protocols, 100 Hz and 20 Hz stimulation, induced the synthesis of AEA and 2-AG, respectively (Lerner and Kreitzer 2012). Cannabinoid receptors in glia and astrocytes Both cannabinoid receptors are expressed by astrocytes and glial cells and their activation regulates differentiation, function and viability of these cells (Stella 2010). Resting microglia in the healthy brain do not express (many) cannabinoid receptors, but activated microglia can express both types of receptor (Stella 2010). Activation of these receptors in tissues under cell-culture conditions (when microglia are activated) regulates some of the immune-related functions that these cells are responsible for. In astrocytes, CB1R activation controls metabolic functions and reduces the ability of astrocytes to produce inflammatory mediators (Stella 2010).

Interestingly, astrocytic CB1Rs couple to PLC via Gq/11, thereby increasing intracellular Ca2+ and inducing glutamate release from astrocytes (Navarrete and Araque 2008). These results demonstrate eCB-mediated neuron-astrocyte signalling and imply that astrocytes are involved in cannabinoid-mediated non-synaptic interneuronal communication. In the hippocampus, eCBs released by pyramidal neurons increased the probability of transmitter release at CA3-CA1 synapses. This occurs via CB1R-dependent glutamate release from astrocytes that activates presynaptic glutamate receptors. In short, eCBs can both suppress neurotransmitter release from neurons, over a short distance (< 20 µm), and enhance neurotransmitter


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Figure 4. Different forms of eCB signalling at the synapse. (A) Retrograde eCB signalling. (B) Non-retrograde signalling via TRPV receptors or postsynaptic CB1Rs. (C) Neuron-astrocyte eCB signalling, eCBs released by the postsynaptic neuron activate astrocytic CB1Rs, inducing gliotransmission. Glu: glutamate. Adapted from Castillo et al. 2012. release, via astrocytes over a larger distance (Fig. 4) (Navarrete and Araque 2010). Both of these forms of short-term plasticity are CB1R-dependent. In addition, astrocytic CB1Rs were found to be necessary and sufficient for spike timing-dependent LTD (Min and Nevian 2012). eCBs released from neocortical pyramidal neurons activated CB1Rs on astrocytes, evoking the release of glutamate which activates presynaptic NMDARs. These findings demonstrate that the eCB system can influence brain functioning through activation of cannabinoid receptors on both neuronal and non-neuronal cells.

CB2Rs are mostly known for their immunosuppressive effects in the periphery. However, CB2R expression in astrocytes and glial cells indicate that this receptors plays a role in the CNS immune response as well. Several recent studies demonstrate a key role for the CB2R in the regulation of macrophage/microglia functions, both under physiological and pathological conditions (Benito et al. 2008; Zurolo et al. 2010). The fact that their expression levels are increased by inflammatory stimuli indicates their involvement in pathogenesis and/or the endogenous response to injury (Maresz et al. 2005; Benito et al. 2008). Data from in vitro experiments and animal models demonstrate that CB2Rs decrease glial reactivity and are part of the general neuroprotective action of the eCB system. In the human CNS, CB2R expression was also found in immune cells. Here too, upregulation of the CB2R serves as a response against various types of chronic CNS injury (Benito et al. 2008). A recent study confirmed induced CB2R expression in macrophages/microglia in human epileptogenic lesions (Zurolo et al. 2010). The anti-inflammatory effects following CB2R activation suggest that this receptor could be a target for the development of novel anti-inflammatory therapies.

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The prefrontal cortex A specific region of the neocortex called the prefrontal cortex (PFC) consists of 5 layers since layer IV is lacking in most species, although not in humans and primates. In this brain region, the anterior part of the frontal lobe, many thalamocortical inputs come from the mediodorsal nucleus in the thalamus and terminate onto dendritic spines of layer III and layers V/VI neurons. From layer III, neurons can project locally to layers II, III and V, but also further away, to layer III neurons in the contralateral cortical area. From layers V/VI, neurons project back to the thalamus (Fig. 5) (Kuroda et al. 1998). More generally, input into layer I of the neocortex is considered to be cortical, but thalamic input also converges here (Douglas and Martin 2007; Rubio-Garrido et al. 2009).

Organization of the cortex The cerebral cortex is a highly complex area of the brain and plays a key role in sensory processing, movement control and cognitive function. The human cerebral cortex can be classified into four lobes, based on gross topographical conventions, the temporal, occipital, parietal and frontal lobe. Most of the cerebral cortex is part of the, from an evolutionary perspective, more novel neocortex. The neocortex is a sheet-like brain structure that is generally made up of six horizontal layers, which differ in terms of the neurons that are present and their connectivity (Mountcastle 1997). In large mammals, the neocortex is folded and makes up a large portion of the brain. The cortex receives input from the thalamus and these thalamocortical inputs are the most thoroughly studied afferents into the neocortex (Thomson and Bannister 2003; Van Essen 2005). Projections from the cortex go back to the thalamus, but also to the striatum, pons and other cortical areas (Douglas and Martin 2004). The neuronal population in the neocortex consists of 80% excitatory, pyramidal neurons and 20% inhibitory interneurons (Peters and Kara 1985). Pyramidal neurons can be found in layers II/III, V and VI of the cortex and mainly use the excitatory neurotransmitter glutamate in their projections to other cortical pyramidal neurons and cells in other areas. Interneurons, which are mainly inhibitory, are present in all cortical layers and generally make use of the inhibitory neurotransmitters γ-amino butyric acid (GABA) and, to a lesser extent, glycine. Neocortical interneurons are hugely diverse in their morphological, physiological and molecular characteristics and do not have afferents that project to distant brain areas, but regulate the local cortical circuit (Peters and Kara 1985; Somogyi et al. 1998; Markram et al. 2004; Isaacson and Scanziani 2011). Despite the relatively small number of interneurons, the relative contribution of excitatory and inhibitory input conductances (balance between excitation and inhibition, E/I) is, across different layers, dynamically maintained at 20% excitation and 80% inhibition (Le Roux et al. 2006, 2007, 2008; Zhang et al. 2011). The great diversity of interneurons in terms of their functional properties allows them to regulate inhibition to dynamically control complex excitation (Markram et al. 2004). Disturbances in the E/I balance are associated with a broad spectrum of neuropsychiatric and neurological diseases, such as autism, schizophrenia and epilepsy (Cobos et al. 2005; Lewis et al. 2005; Rubenstein 2010).


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The PFC consists of a number of interconnected neocortical areas and sends and receives projections from almost all cortical sensory systems, motor systems and many subcortical structures (Miller and Cohen 2001). In humans, the PFC can be subdivided in three major areas: orbital, medial and lateral. The orbital and medial regions in humans are involved in emotional behaviour and the lateral region plays a role in cognitive processes (Groenewegen and Uylings 2000; Fuster 2001). More specifically, dorsolateral regions of the PFC are involved in the integration of sensory and mnemonic information, the regulation of cognitive functions and actions, working memory, planning, problem solving and predicting forthcoming events (Seamans et al. 2008). The dorsolateral PFC is connected to another brain area that plays an important role in the monitoring of actions and outcomes to guide decisions, the anterior cingulate cortex (ACC, the frontal part of the cingulate cortex). In rodents, the PFC can be subdivided into regions with different connectivities, a medial, a lateral and a ventral, orbital region (Uylings et al. 2003). In terms of anatomy and Figure 5. Simplified example of the cortical circuit of the rodent mPFC. Excitatory terminals (open triangle) from the mediodorsal thalamic nucleus (MD) predominantly end onto dendritic spines of layer III and layers V/VI pyramidal neurons. Non-pyramidal cells in layer III also receive input from the MD. From layer III, pyramidal neurons project to other layer III pyramidal neurons. In layers V/VI, some pyramidal neurons project back to the MD. Adapted from Kuroda et al. 1998.

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electrophysiological properties, the rat medial PFC (mPFC) is related to both the primate and human anterior cingulate cortex (ACC), and the dorsolateral PFC. Overall, it has been suggested that the rat mPFC combines elements from human and primate ACC and the dorsolateral PFC at a more rudimentary level (Uylings et al. 2003; Seamans et al. 2008). The PFC is much more elaborately developed in primates than in other mammals. Phylogenetically, it is one of the most recent cortices to develop. This neocortical area is associated with many cognitive functions, such as working memory, attention, decision making and inhibitory control of actions (Miller and Cohen 2001; Koechlin et al. 2003). The PFC is thought to underlie the orchestration of thoughts and actions and it is believed that this brain area provides the foundation for complex behaviour in primates (Damasio 1995; Miller et al. 2002). Since addiction is a brain disorder that is associated with impairment of many of these PFC functions, it is easily understood why researchers have focused on the PFC in understanding addiction (Wistanley 2007; Perry et al. 2011). The eCB system is involved in many of the cognitive functions that are linked to the PFC (Pattij et al. 2008). Although eCB-mediated signalling in the PFC is largely unexplored, the presence of components of the eCB system has been reported by several studies. For instance, the presence of functional CB1Rs, FAAH, MGL and DAGL in the PFC has been demonstrated (Herkenham et al. 1991; Eggan and Lewis 2007; Hansson et al. 2007; Lafourcade et al. 2007; Chiu et al. 2010; Volk et al. 2010; Yoshino et al. 2011). Intake of cannabinoid ligands, such as cannabis, affects PFC regional blood flow, metabolism and immediate early gene expression, which indicates that cannabinoid compounds can affect neural activity (Egerton et al. 2006). In both humans and rats, studies have shown that interference with the eCB system impairs several forms of working memory, attentional function and reversal learning (Jentsch et al. 1998; Lichtman et al. 2002; Arguello and Jentsch 2004; Egerton et al. 2005, 2006). The mechanisms underlying the effects of cannabinoids on such cognitive processes are thought to involve the altered release of neurotransmitters and synaptic plasticity (Egerton et al. 2006; Puighermanal et al. 2012). More research is needed to understand the role of eCB system in the PFC and, eventually, the effects of disruption of this system on cognitive processes. Aim and approach As mentioned earlier, the research presented in this thesis was performed within a research line of a TIPharma consortium, which focused on the role of the eCB system in the development of psychological disorders, in particular addiction. Several research groups contributed to this broad goal by trying to answer various specific research questions. Since impaired functioning of the PFC is associated with the brain disorder addiction, amongst other disorders, the PFC constitutes an interesting target brain area. Furthermore, it is known that the eCB system is present here, although many questions concerning effects of cannabinoid ligands on PFC functioning remain. The main aim of this research is to elucidate the role of these receptors in neuronal communication in a subregion of the rodent PFC, the mPFC.


27

Here, we used in vitro electrophysiological and biochemical techniques to investigate the role of CB receptors at the cellular and local network level in the mPFC. Firstly, using in vitro electrophysiological tools allowed for the recording of the activity of a neuronal population in this cortical area. Secondly, recordings were made of various electrophysiological properties of cells as well as input onto these cells. Cannabinoid ligands could be applied to brain slices in order to investigate the effects of CB receptor activation/blockade on these parameters. In addition, biochemical tools such as Western blotting, immunohistochemistry and radioactive binding assays enabled us to detect CB receptor proteins and their binding sites. Outline of this thesis This thesis represents a culmination of experimental research over a period of four years. Aside from this general introduction, the thesis contains four experimental chapters and a general discussion.

In chapter two, we examined the effects of CB1R- and CB2R-activation on general network excitability in the mPFC. We first characterized local field potentials recorded in layer II/III. We then used a mixed CB1R/CB2R agonist to evaluate the concentration-dependent effects on field potential amplitudes. In addition, specific cannabinoid ligands were used to investigate whether the observed effects were mediated by CB1Rs and/or CB2Rs.

Following the results of the second chapter, we continued our investigation by examining the functional presence of CB2Rs in the mPFC at the cellular level, in chapter three. This study was specifically based on the previous observation that not all effects of a mixed CB1R/CB2R agonist on field potentials in the mPFC could be explained by the activation of CB1Rs. We used a combination of electrophysiological techniques, Western blotting and a radioactive binding assay to assess the presence and functional role of CB2Rs in this neocortical area. In addition, we performed experiments to determine effects of a CB2R agonist on neuronal excitability.

In chapter four, we specifically investigated the activation of CB2Rs in layer II/III of the mPFC by endogenous ligands. We hypothesized that stimulation protocols used to evoke eCB synthesis and subsequent CB1R activation, could also result in CB2R activation. First, we determined the effects of treating slices with 2-AG or a metabolically stable analog of AEA (methanandamide) and compared these effects. In addition, we performed experiments with a stimulation protocol that evokes action potential (AP) firing to induce eCB synthesis. Finally, we investigated CB2R-mediated effects following repeated AP firing on neuronal excitability.

In chapter five, we investigated the role of the eCB system in neurotransmission and the maintenance of the E/I balance in the mPFC. We started our investigation by recording miniature synaptic currents, excitatory and inhibitory, to confirm the supposed presynaptic localization of CB1Rs. We then visualized CB1Rs using immunohistochemistry in healthy animals and animals that were repeatedly injected with a CB1R/CB2R agonist to corroborate their presynaptic localization and investigate the natural distribution of CB1Rs and their internalization into cell bodies. Finally, we made use of a method to decompose synaptic input conductances onto layer II/III

Chapter 1

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pyramidal cells into its excitatory and inhibitory component to assess the role of CB1Rs in the regulation of the E/I balance.

In chapter six, we summarize the main findings of these studies and discuss the results of the research.

Chapter 2

Cannabinoid receptor activation reverses short-term synaptic plasticity in the

rat medial prefrontal cortex

Femke S. den Boon, Pascal Chameau,

Taco R. Werkman, Wytse J. Wadman

Cannabinoid receptor activation reverses short-term plasticity

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Abstract The role of the endocannabinoid (eCB) system in the modulation of neurotransmission has been described for several cortical areas. However, not much is known about the eCB-mediated regulation of general network excitability in the cortex, as can be measured by the recording of field potentials. Here, we made field potential recording in brain slices of the rat medial prefrontal cortex (mPFC). We show that the application of the mixed CB1R/CB2R agonist WIN55212-2 resulted in a concentration-dependent (IC50 ~ 50 nM) decrease of mPFC network excitability. Furthermore, the activation of cannabinoid receptors reversed the existing short-term plasticity from paired-pulse depression to paired-pulse facilitation. These results show that, in the mPFC, the eCB system exerts a powerful control over both network excitability and synaptic plasticity. The modulation of network excitability and synaptic plasticity may be crucial for the regulation of higher order cognitive functions associated with the mPFC.

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Introduction The endocannabinoid (eCB) system is a modulatory signalling system that consists of at least two G protein-coupled receptors (GPCRs, the type-1 cannabinoid receptor, CB1R, and the type-2 cannabinoid receptor, CB2R), endogenous ligands called endocannabinoids (eCBs) and various enzymes responsible for the synthesis and degradation of eCBs (Kano et al. 2009). The eCB system is abundantly present in both the central nervous system (CNS) and the periphery and is involved in variety of physiological functions. In the periphery, the eCB system plays a role in the immune response, via CB2Rs in immune cells (Munro et al. 1993; Galiègue et al. 1995). In the brain, CB1Rs are one of the most abundantly expressed GPCRs and mediate various forms of synaptic plasticity (Heifets and Castillo 2009; Kano et al. 2009; Castillo et al. 2012). In the CNS, the eCB system is mostly known for retrograde signalling processes of eCBs activating CB1Rs (Kreitzer and Regehr 2001; Ohno-Shosaku et al. 2001; Wilson and Nicoll 2001; Wilson et al. 2001). Briefly, eCBs Ca2+-dependently synthesized upon postsynaptic activity, travel backwards across the synapse and bind to presynaptic CB1Rs to inhibit Ca2+ influx through voltage-dependent Ca2+ channels and, possibly, to activate G protein-coupled K+ channels (Ohno-Shosaku et al. 2001). The activation of CB1Rs can, in this manner, reduce both excitatory and inhibitory neurotransmitter release. Some studies have investigated the effects of CB1R activation on synaptic transmission in the rodent prefrontal cortex (PFC), an area associated with higher cognitive functions (Auclair et al. 2000; Miller and Cohen 2001; Koechlin et al. 2003; Yoshino et al. 2011). In contrast with the heavily investigated CB1Rs, the presence of CB2Rs in the brain has been a matter of debate for a long time. The expression of this receptor by glial cells and even neurons is now becoming more generally accepted (Van Sickle et al. 2005; Stella 2010). Two studies report that CNS CB2Rs may function similarly to CB1Rs by showing that CB2R activation results in the modulation of neurotransmission, presumably mediated from a presynaptic locus (Morgan et al. 2009; Atwood et al. 2012).

In this in vitro study, we have investigated the consequences of cannabinoid receptor activation for the network excitability in the medial PFC (mPFC), a brain area associated with many higher order cognitive functions (Pattij et al. 2008; Seamans et al. 2008). For this, field potentials were evoked (by electrical stimulation) and recorded in rat mPFC brain slices. Such recordings reflect the activity of large numbers of neurons simultaneously and can provide information on the general (evoked) activity of this cortical area. Materials and Methods Electrophysiology Coronal slices (400 µm) of the mPFC were obtained from male Wistar rats (Harlan, the Netherlands) aged 5-8 weeks. Animals were killed by decapitation, their brains rapidly


35

removed and placed in oxygenated (95% O2 – 5% CO2) ice-cold (4 °C) artificial cerebrospinal fluid (ACSF, containing in mM: 120 NaCl, 3.5 KCl, 25 NaHCO3, 25 D-glucose, 2.5 CaCl2, 1.3 MgSO4, 1.25 NaH2PO4). Slices were cut in ACSF on a vibratome (VT1200S, Leica, Germany) and placed for 30 min in ACSF at 32 °C. Slices were kept at room temperature for at least 1 h prior to recording. Subsequently, slices were transferred to a liquid-air interface chamber (Scientifica) and bathed in continuously dripping (2.5-3 ml/min) oxygenated ACSF. Glass recording pipettes were pulled from borosilicate glass (Science Products, Germany) and were filled with ACSF. Extracellular field potentials were recorded via the glass recording pipette that was placed in layer II/III of the prelimbic area of the mPFC. Field potentials were evoked by applying biphasic current injections with a total duration of 0.2 ms and ranging from 40-250 μA into layer V via a concentric stimulation electrode. For most experiments two consecutive stimulations were applied with an interpulse interval (IPI) of 25 ms. Field potentials consisted of several components, a fast antidromic spike, a population spike and field excitatory postsynaptic potential (fEPSP) (Fig. 1A). Input-output curves of fEPSP amplitudes were constructed by increasing the stimulation intensity from 0% (smallest stimulation intensity that produced minimal but just detectable fEPSPs) to 100% (stimulation intensity that produced maximal fEPSP amplitudes) in 11 steps (see e.g. Fig. 2B); the time interval between sweeps was 8 s. In this study, repetitive current injections that evoke field potentials were applied at relatively low frequencies (0.125 Hz). Therefore, we assume that these stimulation protocols did not evoke eCB synthesis, which is induced by higher frequency action potential firing (e.g. 20, 40 Hz) (Fortin et al. 2004; Lemtiri-Chlieh and Levine 2007; Yoshino et al. 2011). Due to difficulties with washing out bath-applied cannabinoid ligands, every slice was treated with one or two cumulative concentrations. Concentration-response curves for cannabinoid ligands were fitted with the following logistic equation:

(eq.1) IC / [Ligand]1

amplitude fEPSP Maximalamplitude fEPSP50+

=

Recordings were made using an in-house built amplifier under control of in-house software running under Matlab (MathWorks, USA). Field potentials recordings were digitized (sampling rate 5 kHz) and stored for off line analysis. Data analysis Data were compared with t-tests. In the figures the significance is indicated with asterisks (*P<0.05, ** P<0.01 and *** P<0.001). Results are given as mean ± standard error of the mean.

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Drugs WIN55212-2, arachidonyl-2'-chloroethylamide (ACEA) and JWH-133 were generous gifts from Abbott Healthcare Laboratories (the Netherlands). Bicuculline and CGP-54626 were purchased from Tocris. Cannabinoid receptor ligands were dissolved in DMSO or ethanol to 50 mM and diluted in ACSF with a final concentration of DMSO or ethanol that was always lower than 0.1%. Results Characterization of mPFC field potentials To investigate the effects of cannabinoid ligand application on the local network activity in the mPFC, we first characterized field potentials recorded in layer II/III. Upon electrical stimulation of layer V, complex field potentials could be recorded in layer II/III. Field potentials contained several components beside the stimulation artefact (which is not shown); a (non-synaptic) antidromic spike, a population spike and the fEPSP (Fig. 1A). The population spikes and fEPSPs were sensitive to the application of glutamate receptor antagonists (20 µM CNQX and 10 µM AP5), whereas the antidromic spike was not (data not shown). In the present study, we focused on the amplitudes of the synaptic components of the field potential, in particular, we analysed the fEPSP amplitudes. Input-output curves of fEPSP amplitudes were constructed and effects of cannabinoid drugs were determined on this component of the field potential (Fig. 2B). When a second stimulation pulse (25 ms after the first pulse) was applied via the stimulation electrode, similarly shaped, but smaller field potentials could be recorded, which is indicative of the activation of inhibitory processes (Fig. 1A). In order to further investigate these inhibitory processes, we performed experiments using stimulation protocols with varying IPIs (ranging from 20 ms to 1 sec), with a stimulation intensity of 80% (Fig. 1B). Plotting the ratio of the fEPSP amplitudes (fEPSP amplitude second stimulus / fEPSP amplitude first stimulus) demonstrated that inhibition occurs at all IPIs between 20 ms and 1s, but it is strongest between 100 - 200 ms, which fits with the timescale of GABAB receptor-mediated inhibition. We applied low concentrations of the GABAA and GABAB receptor blockers bicuculline (0.5 μM) and CGP (1 μM), respectively, to demonstrate the involvement of GABA receptors in the observed inhibition. In the presence of these compounds, the ratios of the fEPSP amplitudes were increased for IPIs from 30 to 200 ms (Fig. 1B). We performed additional experiments with a constant IPI (25 ms), but with a varying stimulation intensity for the first pulse (input-output protocol; the stimulation intensity for the second pulse was set at 50% of the maximal stimulation amplitude (see inset Fig. 1C). These experiments demonstrate that when the stimulation intensity of the first stimulus is smaller than 40% of the maximal stimulus intensity, inhibition was not obviously present. However, when the stimulation intensity of the first stimulation was increased, recruitment of inhibitory processes ensured that the fEPSP amplitude following the second stimulation was decreased (Fig. 1C).


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Figure 1. Characterization of field potentials recorded in layer II/III of the rat mPFC. (A) Representative trace of field potentials recorded in layer II/III following electric stimulation in layer V (2 pulses, 25 ms IPI). The complex field potentials consist of various components, the non-synaptic antidromic spike, the population spike and the fEPSP. (B) The ratio of fEPSP amplitudes at various IPIs under control conditions and in the presence of the GABAA and GABAB receptor blockers bicuculline and CGP. (C) fEPSP amplitudes in response to an increasing stimulus amplitude of the first stimulus (0 – 100%) and a constant stimulus amplitude of the second stimulus (50%) (see inset). Effects of cannabinoid receptor ligands on field potentials We next investigated the effects of cannabinoid receptor activation on field potentials in the mPFC (Fig. 2A). After the application of the mixed CB1R/CB2R agonist WIN55212-2 (WIN, 0.01 – 0.3 μM), fEPSP amplitudes in response to the first stimulation pulse were reduced, in a concentration-dependent manner (Fig. 2B). This effect was observed for the whole stimulation intensity range. The fEPSP amplitudes recorded in response to the maximal stimulation amplitude were plotted as a function of WIN concentration and fitted with a logistic equation (eq. 1), yielding an IC50 value of 51±5 nM (Fig. 2C). fEPSP amplitudes in response to the second stimulation were also reduced, but only for the three highest concentrations of 0.03 μM, 0.1 μM and 0.3 μM WIN (Fig. 2D). We observed a WIN-mediated reduction in fEPSP amplitude for the whole stimulation intensity range. We plotted the ratio of the fEPSP amplitudes for stimulation intensities from 50-100% and we found that, in line with our earlier results, under control conditions, this ratio was always below 0.5 for an IPI of 25 ms (Fig. 1B). The application of WIN concentration-dependently increased the ratio (Fig. 2E). In order to further investigate the effects of WIN on inhibitory processes, we continued to perform experiments where we varied the IPI. After the application of WIN (0.3 μM), ratios were increased for IPIs ranging from 20 – 50 ms and, interestingly, decreased for IPIs ranging from 500-1000 ms (Fig. 2F).

Additional experiments were performed with agonists that are selective for CB1Rs or CB2Rs, ACEA (1 – 10 μM) and JWH-133 (0.1 – 3 μM), respectively. Application of ACEA resulted in a decreased fEPSP amplitude in response to the first stimulus for all tested concentrations, but the fEPSP amplitude in response to the second stimulus was decreased only for the highest concentration of ACEA (Fig. 3A, B). Application of JWH-133 reduced the fEPSP amplitudes evoked by both pulses (Fig. 3C, D). Interestingly, the ratio of the fEPSP amplitudes was not increased after the application

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of either ACEA or JWH-133 (data not shown). This is in contrast with the concentration-dependent effects of WIN on this ratio. Together, these data show that modulation of the cannabinoid system by activation of cannabinoid receptors reduces field potential responses in the rat mPFC.

Figure 2. Effects of the application of the mixed CB1R/CB2R agonist WIN on fEPSP amplitudes. (A) Representative traces of field potentials in layer II/III recorded under control conditions and in the presence of 0.3 μM WIN. (B) Input/output curves for normalized fEPSP amplitudes in response to the first stimulus pulse under control conditions and in the presence of WIN (0.01 – 0.3 μM). WIN concentration-dependently reduced the fEPSP amplitude (P < 0.001, n = 16). (C) Concentration-response curve for WIN, constructed from normalized fEPSP amplitudes in response to the first pulse at maximal stimulation amplitude (16 slices per concentration). The data points were fitted with eq. 1. The estimated EC50 was 51±5 nM. (D) Input/output curves of normalized fEPSP amplitudes in response to the second stimulus pulse under control conditions and in the presence of WIN (0.01 – 0.3 μM). fEPSP amplitudes evoked with the maximal stimulus amplitude were reduced in the presence of WIN (0.03, 0.1 and 0.3 μM, P < 0.05, P < 0.01 and P < 0.05, respectively, n = 16). (E) fEPSP amplitude ratios (fEPSP second stimulus/fESPS amplitude first stimulus, IPI 25 ms) were increased at the maximal stimulus intensity in the presence of WIN (0.1 and 0.3 μM, P < 0.01 and P < 0.001, respectively, n = 16). Similar effects were observed for the stimulus intensities in the range of 50-100%. (F) fEPSP amplitude ratios (fEPSP second stimulus/fEPSP amplitude first stimulus) for different IPI (20 – 1000 ms) were changed in the presence of WIN (0.3 μM). For IPI 20, 30 and 50 ms, ratios were increased at the maximal stimulation amplitude in the presence of WIN (P < 0.05, P < 0.001 and P < 0.001, respectively, n = 13). For IPI 500 and 1000 ms, ratios were decreased at the maximal stimulation amplitude in the presence of WIN (both P < 0.001, n = 13).


39

Figure 3. Effects of the application of selective cannabinoid receptor ligands on fEPSP amplitudes. (A) Input/output curves of normalized fEPSP amplitudes in response to the first stimulus pulse under control conditions and in the presence of the selective CB1R agonist ACEA (1 – 10 μM). Application of all concentrations of ACEA resulted in a decreased fEPSP amplitude at the maximal stimulation amplitude (all P < 0.001, n = 14-18). (B) Input/output curves of normalized fEPSP amplitudes in response to the second stimulus pulse under control conditions and in the presence of ACEA (1 – 10 μM). Application of the highest concentration ACEA (10 μM) resulted in a decreased fEPSP amplitude at the maximal stimulation amplitude (P < 0.001, n = 14). (C) Input/output curves of normalized fEPSP amplitudes in response to the first stimulus pulse under control conditions and in the presence of the selective CB2R agonist JWH-133 (0.1 – 3 μM). Application of all concentrations of JWH-133 resulted in a decreased fEPSP amplitude at the maximal stimulation amplitude (0.1, 0.3, 1 and 3 μM, P < 0.01, P < 0.05, P < 0.05 and P < 0.001, respectively, n = 9, 11, 9 and 10, respectively). (D) Input/output curves of normalized fEPSP amplitudes in response to the second stimulus pulse under control conditions and in the presence of JWH-133(0.1 – 3 μM). Application of most concentrations of JWH-133 resulted in a decreased fEPSP amplitude at the maximal stimulation amplitude (0.1, 0.3 and 1 μM, P < 0.05, P < 0.05 and P < 0.01, respectively, n = 9, 11 and 10, respectively). Discussion In the current study, we demonstrated that complex field potentials can be recorded in the mPFC in layers II/III upon electrical stimulation in layer V to investigate the role of the eCB system at the network level. These field potentials, with synaptic components

Chapter 2

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sensitive to glutamate receptor blockers, are indicative of a highly complex cortical network. Here, we focused on the fEPSP component of the signal, especially since the CB1R (which is the most predominant cannabinoid receptor present in the CNS) is known to – as a presynaptic receptor – modulate neurotransmitter release. As a consequence, its activation may have clear effects on postsynaptic potentials.

Using various experimental protocols, we identified the activation of inhibitory processes in this network. Since the blockade of GABAA and GABAB receptors decreased the inhibition, the underlying processes were probably (at least in part) mediated by GABA. The ratios of the fEPSP amplitudes were decreased for IPIs from 30 to 200 ms, which is in line with the time courses of GABAA and GABAB receptors (Dutar and Nicoll 1988; Mody et al. 1994; Barnard et al. 1998; Bowery et al. 2002; Tamás et al. 2003). We performed experiments where the first stimulus intensity was increased from 0 - 100% of the maximal stimulation intensity and where the second stimulus intensity was kept constant at 50%. Inhibitory processes ensured that only when the stimulation intensity of the first pulse was also 50% (or more) of the maximal stimulation intensity, inhibition was powerful enough to reduce the fEPSP amplitude in response to the second stimulus.

We used several cannabinoid ligands to evaluate the effects of combined and separate CB1R and CB2R activation. Application of the mixed CB1R/CB2R agonist WIN concentration-dependently reduced the fEPSP amplitudes, in particular in response to the first stimulus. It is known that CB1R activation decreases the release of excitatory neurotransmission in various regions of the CNS (Lévénés et al. 1998; Auclair et al. 2000; Kreitzer and Regehr 2001; Kano et al. 2009). If WIN activates CB1Rs, the reduction of fEPSP amplitude could be explained by a CB1R-mediated decrease in glutamate release following electric stimulation in layer V. However, since WIN binds to CB2Rs as well as CB1Rs (Fowler 2008; Pertwee et al. 2010), the possibility exists that (some of) the WIN-mediated effects on fEPSP amplitudes could occur via (simultaneous) CB2R activation. As mentioned earlier, CB2Rs were thought to be absent from the CNS for a long time, but several recent publications have reported that CB2Rs could play a role in various aspects of CNS functioning (Jhaveri et al. 2008; Morgan et al. 2009; Xi et al. 2011; Atwood et al. 2012). For instance, CB2R activation in cultured excitatory autaptic hippocampal neurons resulted in the reduction of excitatory neurotransmission, likely from a presynaptic localization (Atwood, Straiker, et al. 2012). Others have shown that brain CB2Rs modulate cocaine’s rewarding and locomotor-stimulating effects in mice (Xi et al. 2011). Clearly, these recent developments indicate that CB2Rs are functionally present in the CNS and suggest that cannabinoid research in the brain should be extended to include these receptors. If CB2Rs behave in a similar manner to CB1Rs, as reported recently (Atwood, Straiker, et al. 2012), CB2R-mediated reduction of glutamate release could explain the effect of WIN on the fEPSP amplitude. However, the mentioned study was conducted under very different experimental circumstances


41

(in culture, in another brain area) and we have previously not been able to detect effects of CB2R activation on the spontaneous release of neurotransmission in the mPFC (unpublished results).

We next investigated the effects of WIN on the inhibitory processes in this preparation. The application of WIN reduced the fEPSP amplitude in response to the second stimulus, although the reduction was less pronounced than observed for the first stimulus and significant only for the three highest concentrations of WIN (0.03, 0.1 and 0.3 µM). This is reflected by the dramatic increase in fEPSP ratios, changing from below 0.5 (control conditions) to ~1.5 with the highest WIN concentration (0.1 µM) for the maximal stimulation intensity. These data show that, with an IPI of 25 ms, the paired-pulse depression under control conditions turned into paired-pulse facilitation in the presence of a high WIN concentration. In other words, WIN reversed the short-term plasticity present in this network. When the IPI was increased up to 1000 ms, we observed that the increase in ratio was limited to IPIs smaller than 100 ms. For IPIs of 500 and 1000 ms, the application of WIN actually decreased the ratio, via an unknown mechanism.

In addition to decreasing excitatory neurotransmission, the activation of CB1Rs has also been shown to inhibit the release of inhibitory neurotransmission (Hoffman and Lupica 2001; Wilson et al. 2001; Kano et al. 2009). If WIN activates the presynaptic CB1Rs on GABAergic synapses, a decreased release of GABA could result in a reduction in inhibition. Paired-pulse facilitation is generally assumed to involve the build-up of Ca2+ in cells, which gives rise to an increased response to the second stimulus. A decreased inhibition could unmask such paired-pulse facilitation. For IPIs of 500 and 1000 ms, the reduced glutamate release due to CB1R activation could possibly explain the decreased ratio. Again, recent developments in the eCB field indicate that it is possible that CB2R-activation could contribute to the response to WIN. A study that investigated the involvement of CB2Rs in inhibition reported that CB2R activation in the entorhinal cortex decreased the charge transfer of inhibitory neurotransmission (Morgan et al. 2009). However, we have not been able to detect a reduction in spontaneous inhibitory neurotransmission in the mPFC following CB2R activation (unpublished results). Inhibition in cortical circuits following electrical stimulation consists of both feedback inhibition and feed forward inhibition (Isaacson and Scanziani 2011), which complicates studies investigating cortical network activity.

In order to investigate the effects of CB1R and CB2R activation separately, we next investigated the effects of the selective CB1R agonist ACEA and the selective CB2R agonist JWH-133. Like WIN, both cannabinoid ligands evoked a decrease in fEPSP amplitudes in response to the first stimulus, although these reductions were markedly less pronounced as produced by WIN. Overall, the fEPSP amplitudes in response to the second stimulus were also decreased by the two cannabinoid ligands. In contrast with the WIN experiments, the ratio of the fEPSP amplitudes was not changed, which is

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indicative of a different mechanism of action. If the WIN effects were predominantly mediated by CB1Rs, one would expect that the CB1R selective agonist ACEA mimics the WIN-effects. However, the penetration of ACEA into a brain slice is possibly problematic, due to its highly lipophilic nature. Therefore, the possibility remains that CB1Rs were not sufficiently activated by the application of (even high concentrations) of ACEA. Another possibility is that the WIN-effects on fEPSP amplitudes were predominantly mediated by CB2Rs. In this case, the application of JWH-133 should result in changes in the fEPSP amplitudes similar to changes induced by WIN. However, JWH-133 was not able to increase the ratio of fEPSPs, although fEPSP amplitudes were reduced, similar to, but to a lesser extent than, when WIN was applied. Finally, we should note that since WIN is a mixed CB1R/CB2R agonist, the simultaneous activation of CB1Rs and CB2Rs could be necessary for the profound reduction of fEPSP amplitudes and increase in fEPSP ratio to occur. Additional experiments with selective CB receptor antagonists could help with the unravelling of the described CB receptor-mediated effects. However, it is important to note that the use of CB receptor antagonists, many of which behave as inverse agonists, at the network level, could complicate the interpretation of the data (Pertwee et al. 2010; Meye et al. 2012).

Altogether, these data demonstrate that the modulation of the eCB system by the mixed CB1R/CB2R agonist WIN resulted in dramatic changes in the field potential amplitudes and inhibitory processes in the mPFC. We report that the application of WIN leads to a reversal of short-term synaptic plasticity in this area. This modulation of network excitability and synaptic plasticity may be crucial for cannabinoid-mediated regulation of higher order cognitive functions of the mPFC. Future studies, performed at the cellular level, may shed more light on the precise role of each cannabinoid receptor in these changes. Acknowledgements This work was supported by Dutch Top Institute Pharma Grant T5-107-1. We thank Willem van Aken for help with the experimental work.

Chapter 3

Excitability of prefrontal cortical pyramidal neurons is modulated by activation of intracellular

type-2 cannabinoid receptors

Femke S. den Boon, Pascal Chameau, Qiluan Schaafsma-Zhao, Willem

van Aken, Monica Bari, Sergio Oddi, Chris G. Kruse, Mauro

Maccarrone, Wytse J. Wadman and Taco R. Werkman.

Proceedings of the National Academy of Sciences of the United States,

2012 Feb 28;109(9):3534-9. doi: 10.1073/pnas.1118167109.

Excitability of cortical pyramidal neurons is modulated by activation of intracellular CB2Rs

47

Abstract The endocannabinoid (eCB) system is widely expressed throughout the central nervous system (CNS) and the functionality of type-1 cannabinoid receptors (CB1R) in neurons is well documented. In contrast, there is little knowledge about type-2 cannabinoid receptors (CB2R) in the CNS. Here we show that CB2R are located intracellularly in layer II/III pyramidal cells of the rodent medial prefrontal cortex (mPFC) and that their activation results in IP3R-dependent opening of Ca2+-activated Cl- channels. To investigate the functional role of CB2R activation, we induced neuronal firing and observed a CB2R-mediated reduction in firing frequency. The description of this novel CB2R-mediated signalling pathway, controlling neuronal excitability, broadens our knowledge of the influence of the eCB system on brain function.

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Introduction The eCB system is involved in many functions of the CNS, including executive functions associated with the prefrontal cortex, such as decision-making and working memory (Pattij et al. 2008). The eCB system consists of at least two G-protein coupled receptors (GPCR), CB1R and CB2R, lipid endogenous ligands (e.g. anandamide and 2-arachidonoylglycerol) and various enzymes responsible for the synthesis and degradation of the endogenous ligands (Devane et al. 1992; Mechoulam et al. 1995; Bracey et al. 2002; Dinh et al. 2002; Kano et al. 2009). CB1R are one of the most abundantly expressed GPCR in the rat brain and their role, predominantly as presynaptic receptors, in modulating neurotransmission is clearly established (Herkenham et al. 1991; Wilson and Nicoll 2001; Kano et al. 2009).

In contrast with CB1R, the presence and function of CB2R in the brain has long been a matter of debate (Atwood and Mackie 2010). CB2R are found primarily in the immune system and were initially regarded as the ‘peripheral’ cannabinoid receptor (Munro et al. 1993; Galiègue et al. 1995). This generally accepted idea is challenged by the description of CNS CB2R gene expression in rats and wild-type mice (Gong et al. 2006; Liu et al. 2009; García-Gutiérrez et al. 2010) and the identification of functional CB2R receptors on glial cells and neurons (Carlisle et al. 2002; Van Sickle et al. 2005; Onaivi et al. 2006; Stella 2010). In addition to the current view that supports the expression of functional CB2R in neurons upon brain stress or damage (Viscomi et al. 2009), it has been reported that CB2R could play a role in general CNS physiology (Elmes et al. 2004; Morgan et al. 2009; Xi et al. 2011). These developments emphasize the importance of understanding how CB2R activation affects neuronal functioning. To demonstrate the presence of functional CB2R in the rodent mPFC and to elucidate their functional role we used Western blotting, a radioactive binding assay and electrophysiological techniques (whole-cell current and voltage clamp) on layer II/III pyramidal neurons. Materials and Methods Western Blotting Experiments were approved by the animal welfare committee of the University of Amsterdam. mPFC, brain stem, spleen and skeletal muscle of 14-day old male Wistar rats (Harlan, The Netherlands) were rapidly dissected out in ice-cold homogenization buffer (0.05 mM phosphate buffered saline (PBS)) with 320 mM sucrose and protease inhibitor mixture (Complete, Roche, pH 7.4) and homogenized with a glass douncer. For subcellular fractionation of the mPFC the same buffer was used. The homogenate was centrifuged at 800 x g for 10 minutes to discard undisrupted tissue. The supernatant was centrifuged at 100000 x g for 1 hr to separate plasma membrane fractions (pellet) from intracellular fractions (supernatant). Equal amounts of protein, 10 or 20 µg, were loaded and separated by 10% SDS-PAGE and transferred onto nitrocellulose membrane. The membranes were washed in 10% Tris buffered saline (TBS) and blocked in TBS with 1% Tween (TBST) containing 4% non-fat milk and incubated overnight at 4 °C with antibodies against CB2R (Abcam, 1:800) alone or in combination with antibodies against


49

the plasma membrane marker Na+/K+-ATPase α3 (Santa Cruz, 1:500) and against the nuclear envelope marker nucleoporin p62 (Beckton Dickinson, 1:800). In some cases the CB2R antibody was preincubated with the immunogen peptide (Abcam, 1:40). Membranes were washed with TBST and incubated for 1 h with HRP-conjugated goat anti-rabbit antibody (Bio-Rad, 1:3000), goat anti-mouse antibody (Bio-Rad, 1:3000) and donkey anti-goat antibody (Santa Cruz, 1:5000) and processed for immunoreactivities using enhanced chemiluminesence (ECL) Plus Western Blotting detection reagents (Amersham, USA). Bands were visualized with Hyperfilm ECl (Amersham) or the Odyssey Infrared Imaging System (Licor, USA). Radioactive binding assay CB1R and CB2R binding was assessed by rapid filtration assays, using 400 pM [3H]CP55.940 as reported (Maccarrone et al. 2008). mPFC tissue of 14-day old male Wistar rats (Harlan, The Netherlands) was used in rapid filtration assays, after subcellular fractionation as described for the Western blot samples in 2 mM Tris–EDTA, 320 mM sucrose, 5 mM MgCl2 (pH 7.4). Unspecific binding was determined in the presence of “cold” agonist (1 µM CP55.940), and was further corroborated by selective antagonists (0.1 µM SR141716 (SR1) for CB1R, or 0.1 µM SR144528 (SR2) for CB2R). Binding data were expressed as fmol ligand bound per mg of protein. Electrophysiology Coronal slices (300 µm) of the mPFC were obtained from male Wistar rats (Harlan, The Netherlands) aged 14-19 days postnatal and male C57BL/6 Wt mice or male C57BL/6 CB2R KO mice (The Jackson Laboratory, USA) aged 14-19 days postnatal. Animals were killed by decapitation, their brains rapidly removed and placed in oxygenated (95% O2 – 5% CO2) ice cold (4 °C) adapted artificial cerebrospinal fluid (aaCSF, containing in mM: 120 choline chloride, 3.5 KCl, 0.5 CaCl2, 6 MgSO4, 1.25 NaH2PO4, 25 D-glucose, 25 NaHCO3). Slices were cut in aaCSF on a vibratome (VT1200S, Leica, Germany) and placed for 30 min in aCSF (containing in mM: 120 NaCl, 3.5 KCl, 25 NaHCO3, 25 D-glucose, 2.5 CaCl2, 1.3 MgSO4, 1.25 NaH2PO4; [Cl-]out = 128.5 mM) at 32 °C. Slices were kept at room temperature for at least 1 h prior to recording. Glass recording pipettes were pulled from borosilicate glass (Science Products, Germany) and had a resistance of 2-3 MΩ when filled with pipette solution (containing in mM: 110 KGluconate, 30 KCl, 0.5 EGTA, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 4 Mg-ATP, 0.5 Na-GTP). For local application of JWH-133, a Picospritzer II device (General Valve Corporation, USA) was used to deliver the compound to the recorded neurons with brief pressure pulses (500 ms). Modified aCSF with K+-, Ca2+-, and Na+-channel blockers contained in mM: 70 NaCl, 3.5 KCl, 25 NaHCO3, 25 D-glucose, 2.5 CaCl2, 1.3 MgSO4, 1.25 NaH2PO4, 25 TEACl, 5 4-AP, 0.2 CdCl2, 0.0005 TTX; [Cl-]out = 103.7 mM. Modified pipette solution with 30 mM [Cl-]i contained in mM: 110 CH3O3SCs, 30 CsCl, 0.5 EGTA, 10 HEPES, 4 Mg-ATP, 0.5 Na-GTP). Modified pipette solution with 4.95 [Cl-]i contained: 135.05 CH3O3SCs, 4.95 CsCl, 0.5 EGTA, 10 HEPES, 4 Mg-ATP, 0.5 Na-GTP. Introduction of CB2R ligands into neurons was achieved by backfilling the recording pipettes. Whole-cell current and voltage clamp recordings were made at 32 °C from the soma of layer

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II/III pyramidal neurons. In the whole-cell current clamp configuration, we used a slow feedback system that guaranteed that current clamp recordings started at a membrane voltage of -80 mV (Fig. 1 and 3) or -70 mV (Fig. 4). In the whole-cell voltage clamp configuration with [Cl-]i = 30 mM, currents were evoked, every 30 seconds until JWH-133-mediated currents were recorded, by series of rectangular voltage steps (200 ms-duration) from a holding potential of -80 mV to voltage potentials ranging from -90 to -20 mV in 10 mV increments. For experiments with [Cl-]i = 4.95 mM, the currents were evoked from a holding potential of -50 mV. Input resistance was calculated from current responses to hyperpolarizing steps of -5 mV. Frozen filtered Gaussian noise (time constant = 10 ms) was injected via the patch pipette with a variance adjusted for each neuron to result in a mean spike frequency (~0.85 Hz). The firing frequency was calculated using 1- min bins. Prior to drug application (or at least 10 min after preincubation with Sch.356036), 5-min recordings were used as control. Drug effects were determined 3-15 min after application. Recordings were made using an EPC9 patch-clamp amplifier controlled by PULSE software (HEKA Electronic GmbH, Germany) and in-house software running under Matlab (MathWorks, USA). Signals were filtered at 2.9 kHz and sampled at 10 kHz. Series resistance ranged from 5-15 MΩ and was compensated to ~65%. Signals were corrected for liquid junction potential. Current densities were calculated using cell capacitance and expressed in pA/pF. Data analysis Data were statistically tested with t-tests unless otherwise stated. In the figures the significance is indicated with asterisks (*P<0.05, ** P<0.01 and *** P<0.001). Drugs For the radioactive binding assay, the synthetic cannabinoid CP55.940 (5-(1,10-dimethyheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl)-cyclohexyl] phenol) was purchased from Sigma Chemical Co. (USA). [3H]CP55.940 (126 Ci/mmol) was from PerkinElmer Life Sciences (USA). SR141716 (N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide), and SR144528 (N-[(1S)-endo-1,3,3-trimethy-1-bicyclo[2.2.1]-heptan-2-yl]5-(4-choro-3-methyl-phenyl)-1-(4-methyl-benzyl)-pyrazole-3-carboxamide) were kind gifts from Sanofi-Aventis Recherche (France). For the electrophysiological experiments, JWH-133, HU-308 and Sch.356036 were generous gifts from Abbott Laboratories (The Netherlands). Cannabinoid receptor ligands were dissolved in DMSO to 50 mM and diluted in aCSF that never contained a final concentration of DMSO higher than 0.1%. BAPTA, 2-APB and DIDS were all purchased from Sigma-Aldrich (The Netherlands). TTX (0.5 µM, Latoxan, France) was present during all recordings, with the exception of the experiments shown in Fig 4. Results Functional CB2R in the mPFC The presence of CB2R in the rat mPFC was demonstrated by a Western blot performed on homogenated mPFC samples (Fig. 1A). A band of the expected molecular weight for


51

CB2R was detected, which was absent when the primary antibody was incubated with immunizing peptide (Fig. 1A). Immunoblots on tissues from spleen and brainstem (positive controls) and skeletal muscle tissue (negative controls) confirmed the specificity of the CB2R immuno detection (Fig. 1A).

To investigate whether CB2R activation can modulate ion conductances, we performed whole-cell current clamp recordings in layer II/III pyramidal neurons of the mPFC at an experimental membrane potential (Vm = -80 mV) that differed from all experimental ion equilibrium potentials. We observed that bath applications of the selective CB2R agonist JWH-133 (1 µM and 5 µM) resulted in a transient depolarization in most (86%) layer II/III pyramidal neurons of the mPFC, after a delay that lasted several minutes (Fig. 1B-C). The depolarization induced by bath application of 1 µM JWH-133 was significantly smaller after preincubation of the slice with a selective CB2R antagonist, Sch.356036 (5 µM) (Fig. 1C). We confirmed the pharmacological evidence that the depolarization is mediated by CB2R activation by performing similar experiments in CB2R knock-out (KO) and wild type (Wt) mice. We did not observe a delayed depolarizing response in mPFC neurons from KO mice, whereas in Wt mice such responses could be evoked (Fig. 1C).

To investigate the origin of the depolarization delay, we repeated the experiments but replaced the bath application by fast local pressure ejection of 5 µM JWH-133 onto the soma of patched layer II/III pyramidal neurons. As the depolarization delay was hardly affected (Fig. 1C-D), we concluded that it did not originate from diffusion limitations in the bath application method. We hypothesised that CB2R are located intracellularly in these neurons and that the depolarization delay most likely originates from the time required for the lipophilic ligands to pass the plasma membrane and to reach the intracellularly located target. To test this, we introduced 5 µM JWH-133 into the cell via the patch pipette and we still observed a depolarization (Fig. 1C), but with a much reduced delay (~1.5 min) after going whole-cell (Fig. 1D). Introduction of the antagonist Sch.356036 (1 µM) into the cell via the patch pipette largely prevented the membrane depolarization induced by bath application of 1 µM JWH-133 (Fig. 1C). When the antagonist Sch.356036 was bath applied, 5 µM was needed to antagonize the JWH-133 (1 µM) effect (Fig. 1C). Taken together, the combined electrophysiological and pharmacological experiments suggest an intracellular localization of CB2R. CB2R are intracellularly localized in the mPFC To obtain further evidence for an intracellular localization for CB2R, we performed additional Western blot experiments. Whole mPFC samples were fractionated into a plasma membrane and an intracellular fraction. The identity of the fractions was established using antibodies against the plasma membrane marker Na+/K+-ATPase and the intracellular marker nucleoporin. The Western blot on fractionated samples showed that CB2R are abundantly present in the intracellular fraction, whereas they are hardly detectable in the plasma membrane fraction (Fig. 2A). Experiments using a radio-active binding assay on similarly fractionated mPFC samples corroborated the

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Figure 1. CB2R are present in the rodent mPFC and their activation evokes a delayed depolarization. (A) Western blot detection of CB2R in the rat mPFC (top). Primary antibody preincubated with immunogen peptide, showed no CB2R bands (bottom). Positive controls: Spleen (Spl) and brainstem (Brs), negative control: skeletal muscle (SkM). (B) Bath applied JWH-133 (1 µM, arrow indicates the start of the application) induced a delayed (10 min) depolarization of a layer II/III pyramidal neuron. (C) Bath application of 1 and 5 µM JWH-133 induced similar depolarizations (ΔV 29±3 mV and ΔV 25±2 mV, both p < 0.001). After preincubation with 5 µM Sch.356036, application of 1 µM JWH-133 resulted in a significantly reduced depolarization (p < 0.01, Mann-Whitney U test). JWH-133 (1 µM) depolarized neurons of Wt mice, but not of CB2R KO mice (ΔV 15±4 mV, ΔV 0±0.3mV, p < 0.01). Local application of JWH-133 (5 µM) with a Picospritzer device and introduction of JWH-133 (5 µM) into neurons via the patch pipette also depolarized neurons (ΔV 38±4 mV and ΔV 22±6 mV, p < 0.0001 and p < 0.05). Sch.356036 (1 µM), introduced into neurons via the patch pipette, largely prevented the depolarization by JWH-133 (1 µM, ΔV 4±2 mV). (D) The delays of onset of depolarization following bath application of JWH-133 (1 and 5 µM) and local application of JWH-133 (5 µM) were 855±177 s, 490±76 s and 529±125 s, respectively. Introduction of JWH-133 (5 µM) via the patch pipette reduced the delay of onset of the effect compared to the other application methods with 5 µM JWH-133 (103±50 s, both p < 0.01, Mann-Whitney U tests). Numbers of observations are indicated in bars (C, D), error bars represent s.e.m.


53

intracellular localization of CB2R (Fig. 2B). Intracellular binding of [3H]CP55.940 (a mixed CB1R/CB2R agonist) was reduced by ~50% in the presence of the selective CB2R antagonist SR2 (SR144528), but not when the selective CB1R antagonist SR1 (SR141716) was present. Incubating the plasma membrane fraction with SR1 or SR2 reduced the binding of [3H]CP55.940. However, this reduction in binding was much larger in the presence of SR1 (~70% reduction) than in the presence of SR2 (~20% reduction). These results confirm that CB2R binding sites are predominantly located intracellularly in the rat mPFC, while CB1R binding sites appear to be mainly present in the plasma membrane. In additional fluorescence imaging experiments on a neuronal cell line (human SH-SY5Y neuroblastoma cells transiently transfected with GFP-tagged CB2R), we demonstrated that CB2R are almost exclusively localized in intracellular membranous structures and that they are not present on the plasma membrane (Fig. S1 and Table S1). Figure 2. CB2R are localized intracellularly in the rat mPFC. (A) Western blot on subcellular mPFC fractions, consisting of an intracellular fraction (Intr) and a plasma membrane fraction (PM), demonstrated a predominantly intracellular localization of CB2R. Membranes were probed with antibodies against CB2R, the intracellular marker nucleoporin and the plasma membrane marker Na+/K+-ATPase. (B) Radio-active binding experiments show that intracellular binding of the mixed CB1R/CB2R agonist [3H]CP55.940 (CP, 46±3 fmol/mg) was reduced in the presence of SR2 (a CB2R antagonist, 24±1 fmol/mg, p < 0.01) and not in the presence of SR1 (a CB1R antagonist, 44±3 fmol/mg). Plasma membrane binding of [3H]CP55.940 (126±6 fmol/mg) was reduced after incubation with SR1 (36±1 fmol/mg, p < 0.001) or SR2 (102±4 fmol/mg, p < 0.05). The reduction of plasma membrane binding was significantly larger in the presence of SR1 compared to SR2 (71±1% and 19±4%, respectively, p < 0.001). Numbers of observations are indicated in bars, error bars represent s.e.m.

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CB2R activation opens Ca2+-activated Cl- channels via IP3R The signalling cascade following CB2R activation can lead, through phospolipase C production, to Ca2+ release via IP3Rs (Zoratti et al. 2003) and thus to the potential activation of Ca2+-activated conductance (Frings et al. 2000). Introduction of the fast Ca2+ chelator 1,2-Bis(2-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) at a high concentration (10 mM) into the cell via the patch pipette, strongly reduced the amplitude of the delayed depolarization (Fig. 3A). Bath application of the IP3R blocker 2-aminoethyl diphenylborinate (2-APB, 0.1 mM) also reduced indicate that a rise in [Ca2+]i following IP3R activation is necessary for the CB2R-mediated depolarization. A series of voltage clamp experiments was performed to determine which conductance underlies the CB2R-mediated depolarization. Application of JWH-133 (1 µM) decreased the input resistance (Rinput; Fig. 3B), implying that the signal transduction pathway following CB2R activation opens ion channels in the plasma membrane. Using a series of step potentials before and during this JWH-133 application allowed the construction of a current-voltage (I/V) relationship that reversed near the calculated reversal potential (Erev) for Cl- (ECl-) (Fig. 3C). Blocking K+, Ca2+, and Na+ channels with 4-aminopyridine (4-AP), tetraethylammonium-Cl (TEACl), Cd2+ and tetrodotoxin (TTX), hardly had an effect on the magnitude and the reversal potential of the currents evoked by JWH-133 (Fig. 3D). In addition, decreasing [Cl-]i to 4.95 mM shifted Erev towards the newly established ECl- (Fig. 3D). We observed similar currents when we used a different selective CB2R agonist, HU-308 (1 µM) (Fig. 3D, inset). Finally, the JWH-133-evoked currents were markedly reduced in the presence of the Cl- channel blocker 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) (Fig. 3E). In summary, these results show that CB2R activation leads to the opening of Ca2+-activated Cl- channels (CaCCs), that may contribute to the control of the membrane potential of layer II/III pyramidal neurons of the rat mPFC. CB2R activation reduces neuronal excitability The hypothesis that the CB2R-mediated signalling pathway contributes to neuronal excitability was further investigated in a series of current clamp experiments under physiological Cl- conditions (ECl- = -70 mV). A ‘slow’ automatic feedback system ensured that recordings always started at a membrane potential of -70 mV. Neuronal firing was evoked by Gaussian current input into the soma, via the patch pipette, that evoked fluctuations around resting membrane potential. For each neuron, the variance of the input signal was adjusted to cause a stable spiking rate (around 0.85 Hz). Application of 1 µM JWH-133 reduced the firing rate by 45% and this reduction could be prevented by preincubation (of at least 10 min) with the CB2R antagonist, Sch.356036 (5 µM) (Fig. 4). In a separate experiment we tested the effect of Sch.356036 by itself on the firing rate. Application of 5 µM Sch.356036 increased the firing rate (mean baseline firing


55

Figure 3. CB2R activation evokes an IP3R-dependent opening of Ca2+-activated Cl- channels. (A) In the presence of either 10 mM BAPTA in the patch pipette or 0.1 mM 2-APB in the bath, 5 µM or 1 µM JWH-133 evoked significantly reduced (both p < 0.001) depolarizations (ΔV 4±1 mV and ΔV 8±2 mV). The average response evoked with 5 µM JWH-133 (in the absence of BAPTA) is the same as is depicted in Fig. 1C. (B) The input resistance (Rinput) was reduced by JWH-133 (182±20 MΩ, 128±25 MΩ, p < 0.05). (C) I/V relationship of CB2R-mediated currents (evoked with 1 µM JWH-133) that reverse at -40±2mV, close to ECl- (-38.3 mV); the inset shows representative current traces. (D) In the presence of 4-AP, TEACl, Cd2+ and TTX (to block K+, Ca2+ and Na+

channels) and recorded with [Cl-]i = 30 or 4.95 mM, Erev of CB2R-mediated currents (-26±2 mV and -76±4 mV) followed ECl- (-32.6 mV and -80 mV). Inset: another selective CB2R agonist, HU-308 (1 µM), evoked similar currents (Erev: -31±2 mV, ECl-: -32.6 mV). (E) The Cl- channel blocker DIDS (0.2 mM) reduced the amplitude of CB2R-mediated currents. Numbers of observations are indicated in bars/brackets, error bars represent s.e.m. frequency of 0.86±0.09 Hz was normalized over slices to 100±8% and increased to 121±8% in the presence of Sch.356036, p < 0.05, n = 12). These results indicate an endogenous tonus of eCBs and/or constitutive activity of the receptor and show that, when firing is evoked with an input that could resemble spontaneous background synaptic activity, further CB2R activation modulates the firing rate of mPFC neurons.

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Figure 4. CB2R activation decreases firing activity of rat mPFC layer II/III pyramidal neurons. (A) Application of 1 µM JWH-133 led to a reduction of neuronal firing rate. Firing was induced by Gaussian current input into the soma. Mean baseline firing frequency of 0.88±0.10 Hz was normalized over slices to 100±3% and reduced to 55±6% in the presence of 1 µM JWH-133 (p < 0.01). After preincubation (of at least 10 min prior to going whole-cell) with and continuous presence of 5 µM Sch.356036, baseline firing frequency 0.83±0.15 Hz, normalized to 100±3%, JWH-133 (1 µM) could not induce a response (92±6%, JWH-133 plus Sch.356036). (B) Representative traces of current clamp recordings showing action potential firing of layer II/III neurons in the absence (baseline) and presence of 1 µM JWH-133 and in the presence of 5 µM Sch.356036 (Sch.356036 baseline) and 1 µM JWH-133 plus 5 µM Sch.356036. Numbers of observations are indicated in bars, error bars represent s.e.m. Discussion Our results provide the first evidence for functional neuronal CB2R that are located intracellularly, in the rodent mPFC. Their activation results, via IP3R activation, in the opening of CaCCs. Furthermore, this opening of CaCCs is most likely responsible for the observed reduction in neuronal excitability upon CB2R activation. The presence of CB2R mRNA in the brain and CB2R in neurons of the brainstem, the cerebellum and the hippocampus has been reported (Van Sickle et al. 2005; Gong et al. 2006; Brusco et al. 2008a, 2008b; Onaivi et al. 2008, 2012; García-Gutiérrez et al. 2010), but their presence in the cortex remained to be further characterized. The current view on the presence of functional CB2R in the CNS supports the expression of CB2R in neurons essentially upon brain stress and damage (Viscomi et al. 2009). Commonplace problems with the visualization of CB2R by immunohistochemical stainings have been described. These problems may arise from the difficulty of producing reliable antibodies against CB2R, slight differences in diaminobenzidine (DAB) staining protocols, species-specific isoform


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expression patterns and complications with negative controls such as CB2R-KO (Liu et al. 2009; Atwood and Mackie 2010; Ashton 2012; Onaivi et al. 2012). Therefore, we used a combination of biochemical (Western blotting and radio-active binding assay) and functional (in vitro electrophysiology and pharmacology) techniques to provide evidence that functional CB2R are expressed intracellularly in cortical neurons of the healthy brain. The results of the radio-active binding assay did not depend on the quality of antibodies and provided additional support for the intracellular presence of CB2R in the mPFC where this technique confirmed that CB1R are primarily located in the plasma membrane. In addition, by means of fluorescence imaging we could demonstrate that in a neuronal cell line (human neuroblastoma cells; see Supplemental Information) CB2R are predominantly located in intracellular membranous structures and not in the plasma membrane. In accordance with our results, a few publications support the idea that, like other functional GPCR (Lee et al. 2004a; Jong et al. 2005a), functional CB2R may have an intracellular localization. A report by Currie et al. (Currie et al. 2008) shows the intracellular localization of functional CB2R in guinea pig heart cells. Others report that CB2R are associated with the rough endoplasmic reticulum and Golgi apparatus in hippocampal pyramidal neurons, but in these studies the functional role of intracellular CB2R was not investigated (Brusco et al. 2008a, 2008b; Onaivi et al. 2012)..

The CB2R signalling pathway shows great diversity and complexity (Beltramo 2009) and has not been fully elucidated in neurons. The main downstream signalling pathway of CB2R activation involves Gi/o and the subsequent modulation of adenylate cyclase and MAP kinase activity (Demuth and Molleman 2006), but coupling to the modulation of [Ca2+]i has also been reported (Beltramo 2009). In calf pulmonary endothelial cells, activation of CB2R by anandamide resulted in an increase in [Ca2+]i, mediated by PLC activation and IP3 production (Zoratti et al. 2003). In cardiac cells (Currie et al. 2008) and in adult dorsal root ganglion neurons (Sagar et al. 2005), CB2R activation was reported to be negatively coupled to IP3R-mediated Ca2+ release. In addition to the known CB2R-mediated pathways, we demonstrate that in rodent layer II/III cortical pyramidal neurons, the CB2R signalling cascade involves IP3R activation and results in the opening of CaCCs. CaCCs are known to control excitability in various types of peripheral and central neurons (Frings et al. 2000; Hartzell et al. 2005), but have not been described before in pyramidal neurons of the mPFC.

Although some publications report a functional role for CB2R in the CNS (Elmes et al. 2004; Morgan et al. 2009; Xi et al. 2011), the precise mode of action is not known. In this study we demonstrate that CB2R activation – under physiological conditions – leads to a decrease in neuronal firing rate, probably via the opening of CaCCs. If ECl- is close to the resting membrane potential of cortical neurons, the activation of CB2R will stabilize or even clamp the membrane potential around that level. This reduction of neuronal excitability upon CB2R activation is reminiscent of shunting inhibition, described for GABAA receptors (Andersen et al. 1980; Staley and Mody 1992), that also mediate a Cl- conductance. Furthermore, we observed that the application of a CB2R antagonist slightly increased the firing rate, indicative of a certain eCB tonus and/or a constitutive activity of the receptor. These results are in line with both the emerging

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idea that the eCB system consists of a basal and an “on demand” pool of endocannabinoids and with the possibility of constitutively active CB2R (Alger and Kim 2011; Di Marzo 2011; Atwood, Wager-Miller, et al. 2012). The mode of action we report here could be the underlying mechanism involved in the reduction of firing activity by JWH-133 in wide-range dorsal horn neurons in a rat model of acute, inflammatory and neuropathic pain (Elmes et al. 2004) and in thalamic neurons in a rat model of neuropathic pain (Jhaveri et al. 2008).

The unique aspects of eCB signalling we describe in this study, uncover a novel modulatory role for the eCB system in mPFC function, through the regulation of neuronal excitability by CB2R activation. As high frequency stimulation leads to eCB synthesis (Brown et al. 2003), activation of CB2R following increased neuronal activity may prevent excessive neuronal firing via an intracellularly organised feedback system. Through this mechanism CB2R could play a protective role in the brain (Pacher and Mechoulam 2011). More generally, the differential (sub)cellular localization of CB1R and CB2R and their downstream pathways diversify the response repertoire of the neuronal eCB system beyond the generally accepted modulation of neurotransmission processes. Acknowledgements We would like to thank Dr. N.L.M. Cappaert and Dr. J.A. van Hooft for critical reading of the manuscript and Dr. T.Z. Baram for critical suggestions in an early phase of the project. This study was supported by a grant from the Dutch Top Institute Pharma (T5-107-1). M.B., S.O. and M.M. were supported by Fondazione TERCAS (grant 2009-2012).


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Supplemental information Materials and Methods Reagents. DiIC16 was purchased from Molecular Probes. Attractene was from QIAGEN. Culture media, sera, and supplements were from Euroclone. All other chemicals were purchased from Sigma Chemical, unless stated otherwise. GFP Fusion Construct To generate green fluorescent fusion protein, human CB2R was amplified and cloned in frame to a GFPtag in a pVL-GFP vector (AB Vector) (Oddi et al. 2011). Cell Culture and Transfection Human SH-SY5Y neuroblastoma cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium, supplemented with 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 100 Units/mL penicillin, and 50 Units/mL streptomycin, at 37 °C in a 5% CO2 humidified atmosphere (Oddi et al. 2011). A monolayer of native cells at 70–80% confluence in eight-well chamber slides (Ibidi) or in collagen-coated (20 μg/mL) coverslips (10 mm diameter) were transiently transfected with CB2R-GFP by using the Attractene reagent as suggested by the manufacturer (QIAGEN). Imaging A Leica TCS SP5 DMI6000 confocal microscope (Leica Microsystems) was equipped with Leica HCX plan apo 63× (numerical aperture 1.4) oil immersion objective. Excitation laser lines were 488 nm (argon laser) and 561 nm (diode pumped solid state laser). GFP-tagged CB2R was excited at 488 nm, and the corresponding fluorescence was detected by using a 525 ± 25 nm bandpass filter. DiIC16 was excited by using a 561-nm laser line, and the corresponding fluorescence was detected by using a 580- to 620-nm bandpass filter. In the codetection experiments of GFP and DiIC16, cells were fixed, and green fluorescence emission and red fluorescence emission were acquired sequentially upon excitation with 488-nm and 561-nm laser light, respectively. Pictures were taken by using the Leica Application Suite Advanced Fluorescence (LAS AF) software (Leica Microsystems). For presentation purposes, LAS AF pictures were exported in TIFF format and processed with Adobe Photoshop CS2, for adjustments of brightness and contrast. Quantification of the mean fluorescence intensity in selected regions was carried out by using the ImageJ software (http://rsb.info.nih.gov/ij/). For image analysis, five fields from at least three independent experiments were examined.

For assessing membrane targeting of GFP-tagged CB2R, we imaged transfected cells that were costained by extracellular application of the red emitting lipophilic dye DiIC16 (Oddi et al. 2011). Briefly, transfected cells were washed three times with RPMI without phenol red, incubated with RPMI containing 4 μg/mL DiIC16 for 5 min on ice, rinsed twice with cold PBS, fixed with 2% formaldehyde in PBS, and examined by confocal fluorescence imaging. For quantification of the membrane/total (M/T) CB2R ratio, we measured mean fluorescence density values (M and T),

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corresponding to the membrane (measured from the edge of the cell to 300 nm inside, M) and total cell area (surface plus cytoplasm, T), respectively. A more accurate estimate of the level of localization of GFP-tagged CB2R at the plasma membrane was obtained through the overlap coefficient, which measures the degree of overlap between green (Fig. S1A, GFP-tagged CB2R) and red (Fig. S1B, DiIC16) fluorescence (Marchant et al. 2002). Overlap coefficients were measured by using the ImageJ plugin JACoP that groups together the most important colocalization tools (Bolte and Cordelières 2006). Apparent colocalization due to random staining, or very high intensity, in one window will have values of overlap coefficient near to zero, whereas if the two signal intensities are interdependent (colocalized), these values will be positive with a maximum of 1. Fluorescence microscopy analysis of the subcellular localization of GFP-tagged CB2R in human neuroblastoma revealed that CB2R staining was almost completely intracellular, and was distributed in several dotted membranous structures that were widely diffused in the cytoplasm, with scarce or absent association to the plasma membrane (Fig. S1). The virtual exclusion of CB2R from the plasma membrane of SH-SY5Y cells was quantitatively assessed by the very low values (close to zero) of the overlap coefficient and the membrane/total (M/T) CB2R fluorescence ratio (Table S1).

It is noteworthy that the same analysis of the subcellular localization of GFP-tagged CB1R in SH-SY5Y cells showed an overall expression of the receptor approximately twofold higher than that of GFP-CB2R on the cell surface (M/T ratio = 0.120 ±0.020) (1), but a quantitative colocalization with the plasma membrane marker DiIC16 that was ≈19-fold higher (overlap coefficient = 0.75 ± 0.04) (Oddi et al. 2012). Taken together, these data clearly demonstrate that CB2Rs are much less associated with the plasma membrane than CB1Rs.


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Figure S1. Surface expression and co-localization of hCB2-GFP and DiC16 in human SH-SY5Y cells. Details are given under Materials and Methods, and parameter values are summarized in Table S1. (A and B) Double staining of SH-SY5Y cells expressing hCB2-GFP (A, a’) with DiIC16 (B, b’), as a plasma membrane marker. Merged image of the magnified regions is shown in the panel a’/b’. For quantification of the overlap coefficient, the colocalization analysis of the two stainings was restricted to the region of interest which delimitates the plasma membrane (B and b’). For quantification of the membrane/total (M/T) ratio, the mean fluorescence (M and T) values of CB2 were calculated within the regions of interest, shown in A and B (and magnified in panels a’ and b’). Images are representative of three independent experiments, for a total of 30 cells. (Scale bar: 10 µm)

Chapter 4

Endocannabinoids produced upon action

potential firing evoke a Cl- current via type-2 cannabinoid receptors in

the medial prefrontal cortex

Femke S. den Boon, Pascal Chameau, Kas Houthuijs, Nicolina

Mastrangelo, Chris G. Kruse, Mauro Maccarrone,

Wytse J. Wadman and Taco R. Werkman

Submitted to Cerebral Cortex

Endocannabinoids produced upon action potential firing evoke a Cl- current via CB2Rs

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Abstract The presence of type-2 cannabinoid receptors (CB2Rs) in neurons of the central nervous system (CNS) has long been controversial. We recently demonstrated their functional presence in layer II/III pyramidal neurons of the rat medial prefrontal cortex (mPFC), by using synthetic cannabinoids. In the present study, we show that the application of the endocannabinoids (eCBs) 2-arachidonoylglycerol (2-AG) and methanandamide (a stable analog of the eCB anandamide (AEA)) can activate CB2Rs of mPFC layer II/III pyramidal neurons, which subsequently induces a Cl- current. In addition, we show that action potential (AP) firing evoked by 20-Hz current injections results in an eCB-mediated opening of Cl- channels via CB2R activation. This AP-evoked synthesis of eCBs is dependent on the Ca2+ influx through N-type voltage-gated calcium currents. Our results indicate that 2-AG is the main eCB involved in this process. Finally, we demonstrate that under physiologically relevant intracellular Cl- conditions, 20-Hz AP firing leads to a CB2R-dependent reduction in neuronal excitability. Altogether, our data suggest that the eCB system plays a modulatory role in the mPFC in the prevention of excessive neuronal firing via the activation of CB2Rs after an initial period of high activity.

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Introduction The endocannabinoid (eCB) system comprises at least two G protein coupled receptors (GPCRs), type-1 and type-2 cannabinoid receptors (CB1Rs and CB2Rs), various lipid endogenous cannabinoids (eCBs), eCB transporters and enzymes responsible for the synthesis and degradation of eCBs (Kano et al. 2009). The eCB system is mostly known for mediating the effects of cannabinoids from the marijuana plant (cannabis sativa). Cannabinoid receptors are activated by phytocannabinoids found in marijuana, synthetic cannabinoids and eCBs, such as 2-arachidonoylglycerol (2-AG) and anandamide (AEA). AEA was the first eCB to be isolated (Devane et al. 1992) and it is thought to be synthesized from its membrane precursor N-acylphosphatidylethanolamine (NAPE) by a specific phospholipase D (NAPE-PLD) and for the most part hydrolyzed by fatty acid amide hydrolase (FAAH). 2-AG, identified a few years later, is synthesized by diacylglycerol lipase α (DLG α) and β (DGL β) from DAG-containing phospholipids and is principally hydrolyzed by monoacylglycerol (MGL) (Mechoulam et al. 1995; Sugiura et al. 1995; Dinh et al. 2002, 2004). The eCB system is involved in many cognitive functions that are associated with the medial prefrontal cortex (mPFC) (Pattij et al. 2008). The expression pattern and functional role of CB1Rs as presynaptic receptors modulating neurotransmitter release in the central nervous system (CNS) are well described (Herkenham et al. 1991; Wilson and Nicoll 2001; Castillo et al. 2012; Katona and Freund 2012). However, there is relatively little knowledge about the functionality of CB2Rs in the CNS. Initially, CB2Rs were not detected in the brain, but their presence in microglia cells and other brain immune cells is now generally accepted (Stella 2010). We have recently shown, using a combination of biochemical and electrophysiological techniques, that CB2Rs are present intracellularly in layer II/III pyramidal neurons of the rat mPFC (Den Boon et al. 2012). Furthermore, we demonstrated that their activation with synthetic, selective agonists leads to an IP3R-dependent opening of Ca2+-activated Cl- channels (CaCCs) located in the plasma membrane. Under physiologically relevant intracellular Cl- conditions, the opening of Cl- channels will stabilize the membrane potential around resting membrane potential, thereby reducing neuronal excitability (Frings et al. 2000; Hartzell et al. 2005). Although we have shown that CB2Rs are involved in the modulation of mPFC neuronal excitability, presumably through the opening of CaCCs, these findings were obtained with synthetic cannabinoids (Den Boon et al. 2012). The questions how and when CB2Rs are activated by endogenously released eCBs in mPFC layer II/III neurons remain to be answered. The downstream signalling pathway following CB2R activation suggests a modulatory role of neuronal excitability for CB2Rs. We hypothesize that in response to increased AP firing, mPFC layer II/III neurons synthesize and release eCBs to prevent excessive neuronal activity via a CB2R-mediated feedback system.

Endogenous ligands for cannabinoid receptors are synthesized ‘upon demand’ from membrane phospholipids following an increase in [Ca2+]i, and/or activation of GPCRs such as metabotropic glutamate receptors or M1/M3 muscarinic acetylcholine receptors (Kano et al. 2009). Aside from the ‘on demand’ pool, eCBs are believed to be present in a ‘basal pool’, being constantly produced, accumulated and released (Maccarrone et al. 2010; Alger and Kim 2011; Di Marzo 2011). It is known that adequate


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stimulation (10-50 Hz) can lead to eCB synthesis and subsequent CB1R-mediated plasticity (Brown et al. 2003; Lafourcade et al. 2007). Furthermore, in the mouse PFC, repetitive AP firing at an intermediate frequency (20 Hz) was also reported to evoke eCB synthesis and resulted in 2-AG-dependent CB1R-mediated depression of synaptic transmission (Yoshino et al. 2011). Other studies have also demonstrated CB1R-mediated effects in cortical areas following brief trains of APs (Fortin et al. 2004; Lemtiri-Chlieh and Levine 2007). We propose that stimulation protocols that are able to induce synthesis and release of eCBs which induce CB1R-mediated effects, could also lead to CB2R-mediated effects.

To assess the physiological role of CB2Rs, we proceeded to investigate the eCB-mediated activation of CB2Rs and the subsequent opening of CaCCs, which in turn will modulate neuronal excitability. We demonstrate how the exogenous application of 2-AG and mAEA concentration-dependently evokes a Cl- current in layer II/III pyramidal neurons of the rat mPFC. Furthermore, we reveal the activation of CB2Rs by eCBs following 20-Hz suprathreshold current injections, mediated – at least for the most part – by 2-AG. Finally, we show that 20-Hz AP firing can reduce neuronal excitability through the activation of CB2Rs. Materials and Methods Electrophysiology Coronal slices (300 µm) of the mPFC were obtained from male Wistar rats (Harlan, the Netherlands) aged 14-19 days postnatal and male C57BL/6 Wt mice or male C57BL/6 CB2R KO mice (The Jackson Laboratory, USA) aged 14-19 days postnatal. Animals were killed by decapitation, their brains rapidly removed and placed in oxygenated (95% O2 – 5% CO2) ice cold (4 °C) adapted artificial cerebrospinal fluid (aACSF, containing in mM: 120 choline chloride, 3.5 KCl, 0.5 CaCl2, 6 MgSO4, 1.25 NaH2PO4, 25 D-glucose, 25 NaHCO3). Slices were cut in aACSF on a vibratome (VT1200S, Leica, Germany) and placed for 30 min in ACSF (containing in mM: 120 NaCl, 3.5 KCl, 25 NaHCO3, 25 D-glucose, 2.5 CaCl2, 1.3 MgSO4, 1.25 NaH2PO4; [Cl-]out = 128.5 mM) at 32 °C. Slices were kept at room temperature for at least 1 h prior to recording. Glass recording pipettes were pulled from borosilicate glass (Science Products, Germany) and had a resistance of 2-3 MΩ when filled with pipette solution (containing in mM: 110 KGluconate, 30 KCl, 0.5 EGTA, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 4 Mg-ATP, 0.5 Na-GTP). Modified ACSF (mACSF) with K+-, Ca2+-, and Na+-channel blockers contained (in mM): 98 NaCl, 3.5 KCl, 25 NaHCO3, 25 D-glucose, 2.5 CaCl2, 1.3 MgSO4, 1.25 NaH2PO4, 25 TEACl, 5 4-AP, 0.2 CdCl2, 0.0005 TTX; [Cl-]out = 131.7 mM. Modified pipette solution with high [Cl-]i (30 mM) contained (in mM): 110 CH3O3SCs, 30 CsCl, 0.5 EGTA, 10 HEPES, 4 Mg-ATP, 0.5 Na-GTP). Modified pipette solution with low [Cl-]i (8.75 mM) contained (in mM): 131.25 KGluconate, 8.75 KCl, 0.5 EGTA, 10 HEPES, 4 Mg-ATP, 0.5 Na-GTP. For recordings of Ca2+ currents carried by voltage-dependent Ca2+ channels (VDCCs), a VDCC ACSF was used, containing in mM: 105 NaCl, 3.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1.25 NaH2PO4, 25 D-glucose, 25 NaHCO3, 10 TEA-Cl, 5 4-AP, 0.005 TTX. A VDCC pipette solution was used for these experiments, containing in mM: 135

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CsMeSO4, 10 1,2-Bis(2-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, 10 Hepes, 4 MgATP, 0.4 NaGTP. Whole-cell current and voltage clamp recordings were made at 32 °C from the soma of layer II/III pyramidal neurons. Currents were evoked by 200 ms voltage steps (2 s interval) from a hyperpolarizing prepulse potential (400 ms, -120 mV) to test pulses potentials ranging from -90 mV to 60 mV. Sustained current amplitudes were measured as the average current during the last 30 ms of the current. To allow the detection of CB2R-mediated currents, currents were evoked in the voltage clamp configuration every 15 s by series of rectangular voltage steps (200 ms-duration) from a holding potential of -80 mV to voltage potentials ranging from -90 or -105 mV to -20 or -55 mV in 10 mV increments (Fig. 1A). To estimate CB2R-mediated currents, currents evoked by rectangular voltage steps before and after bath application of cannabinoids or a stimulation protocol (in the current clamp configuration) that evoked APs (Fig. 2A), were compared. Due to difficulties with washing out bath-applied cannabinoid ligands, every cell was treated with one concentration. Concentration-response curves for cannabinoid ligands were fitted with the following logistic equation:

(eq.1) [Ligand] / EC1

densitycurrent MaximaldensityCurrent 50+

=

Under current clamp conditions, a stimulation protocol was used to evoke APs with 60 suprathreshold current injections (1 nA, 5 ms duration, 45 ms interval) into the soma via the patch pipette (Fig 2A). For experiments with synaptically evoked APs and/or EPSPs, currents ranging from 300-600 μA (5 ms) were injected (at 20 Hz) via a separate stimulation electrode that was placed in layer I (Fig. 2D inset). Experiments determining eCB-mediated effects on cell excitability were done in the whole-cell current clamp configuration. We used a slow feedback system that guaranteed that current clamp recordings started at a defined membrane voltage of -80 mV (Fig. 3) or -70 mV (Fig. 4). Frozen filtered Gaussian noise (time constant = 10 ms) was injected via the patch pipette with a variance adjusted for each neuron to result in a mean spike frequency of ~0.95 Hz. Firing frequency was calculated using 1-min bins. Prior to the stimulation protocol, 5-min recordings were used as control. Maximal effects of 20-Hz AP firing were observed within 2-6 min after the stimulation protocol was applied. Recordings were made using an Axopatch 200b (Axon, USA) and in-house software running under Matlab (MathWorks, USA). Signals were filtered at 2.9 kHz and sampled at 10 kHz. Series resistance ranged from 5–15 MΩ and was compensated to ~65%. Signals were corrected for liquid junction potential. Current densities were calculated using cell capacitance and expressed in pA/pF. Data analysis Data were compared with t-tests. When data was not normally distributed a Mann-Whitney test was used. In the figures the significance is indicated with asterisks


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(*P<0.05, ** P<0.01 and *** P<0.001) and the number of observations are given between brackets (Figs. 1 and 2) or in the bar charts (Figs. 3 and 4). Results are given as mean ± standard error of the mean. Drugs mAEA, 2-AG, RHC-80267 and ω-conotoxin were purchased from Tocris (the Netherlands). Sch.356036 and JWH-133 were generous gifts from Abbott Healthcare Laboratories (the Netherlands). 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate (DIDS) and nefidipine were purchased from Sigma-Aldrich (the Netherlands), TTX was purchased from Latoxan (France). URB602 and URB597 were purchased from Cayman Chemical (USA). Cannabinoid receptor ligands were dissolved in DMSO or ethanol to 50 mM and diluted in ACSF with a final concentration of DMSO or ethanol that was always lower than 0.1%. Some cells were filled with biocytin (Sigma, 1 mg/ml dissolved in pipette solution) during whole-cell patch recordings. Biocytin was visualised using previously described methods (Chameau et al. 2009). Results 2-AG and mAEA induce a Cl- current To investigate whether eCBs can induce the previously described Ca2+-activated Cl- current (Den Boon et al. 2012), we first performed whole-cell voltage clamp recordings of layer II/III pyramidal neurons. These experiments were done in the presence of K+, Ca2+ and Na+ channel blockers (mACSF) and with a pipette solution containing a high Cl- concentration and no K+ (modified pipette solution with 30 mM Cl-). Using a series of step potentials before and during the bath application of 2-AG (25 out of 35 cells responded) or mAEA (the stable analog of AEA, 24 out of 33 cells responded) allowed the construction of a current-voltage relationship that reversed (Erev) close to the calculated reversal potential for Cl- (ECl-) (Fig. 1B). Both 2-AG (0.1-50 µM) and mAEA (1-30 µM) concentration-dependently evoked a Cl- current. The current density values determined at -90 mV were plotted as a function of ligand concentration and fitted with a logistic equation (eq. 1), yielding EC50 values of 1.2±1.0 µM and 4.9±1.4 µM for mAEA and 2-AG, respectively (Fig. 1C). The maximal current density recorded at -90 mV for 2-AG exceeded that for mAEA (Fig. 1B,C), which demonstrates that 2-AG is a more efficacious agonist than mAEA. However, the estimated EC50 values indicate that mAEA is a slightly more potent agonist than 2-AG (Fig. 1C). When 2-AG (30 μM) and mAEA (10 µM) were applied simultaneously, the maximal current density was not different from that evoked by mAEA (10 μM) alone, which suggests that both ligands compete for the same binding site (Fig. 1B). The selective CB2R agonist JWH-133 (1 μM) induced a Cl- current that was comparable to that elicited by 2-AG, suggesting that the current

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Figure 1. 2-AG and mAEA induce a Cl- current in layer II/III mPFC pyramidal neurons. (A) Representative traces of voltage steps (A1), evoking currents during baseline (A2), and currents including Cl- currents during cannabinoid-ligand application (A3); the isolated Cl- currents (A4) are obtained by subtracting the traces of A2 from the traces of A3. (B) I/V relationships of 2-AG- and mAEA-induced Cl- currents at their maximal effective concentrations (30 μM and 10 μM, respectively). Erev for 2-AG (-38.7±1 mV) and mAEA (-42.2±2.4 mV) is close to ECl- (-38.9 mV). When 2-AG (30 μM) and mAEA (10 μM) were applied simultaneously, current densities were not different than for mAEA (10 μM) alone (Erev = -40.4±1.2 mV). Application of the CB2R-selective agonist JWH-133 (1 μM) induced a current (Erev = -41.1±2.1 mV) that was comparable to the current induced by 2-AG (30 μM). (C) Concentration-response curves for 2-AG and mAEA, constructed from the current densities recorded at -90 mV (4-10 cells per concentration). The data points were fitted with eq. 1. The estimated maximal current density at -90 mV was larger for 2-AG (3.1±0.3 pA/pF, 30 µM) than for mAEA (1.3±0.3 pA/pF, 10 µM, p < 0.01). The estimated EC50 value for mAEA (1.2±1 µM) was smaller than that for 2-AG (4.9±1.4 µM, p < 0.01).


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evoked by 2-AG and mAEA is mediated by CB2Rs (Fig. 1B). Together, these experiments show that 2-AG and mAEA can induce a Cl- current, presumably via the activation of CB2Rs, and that 2-AG is a more efficacious, yet slightly less potent agonist for the CB2R. AP firing (20 Hz) results in a CB2R-mediated Cl- current We hypothesized that a stimulation protocol that induces eCB synthesis and CB1R activation, may also lead to CB2R activation. We tested whether 60 APs fired at 20 Hz (Fig. 2A) could induce the release of sufficient eCBs to activate CB2Rs. In the voltage clamp configuration, we recorded currents evoked by 60 APs firing at 20 Hz. We found that this protocol evoked a current in 10 out of 14 tested neurons and that this reversed close to ECl- (Fig. 2B). The stimulation protocol did not evoke a current when neurons were preincubated with the CB2R antagonist Sch.356036, which indicates that the current is mediated by CB2Rs (Fig. 2B). To exclude the involvement of CB1Rs, we repeated the experiment in the presence of the selective CB1R antagonist rimonabant (5 µM) and detected a current that was comparable to the one recorded in the absence of rimonabant (Fig. 2B). Decreasing [Cl-]i to 8.75 mM shifted the reversal potential of the current (Erev) toward the newly established ECl- (-70 mV, data not shown). This experiment confirmed the identity of the CB2R-mediated current as a Cl- current. Since the synthesis of eCBs is sensitive to the influx of Ca2+ (Di Marzo 2011), we next examined the possible involvement of N-type and L-type voltage-dependent Ca2+ channels (VDCCs). VDCC-mediated currents were recorded in the absence and presence of ω-conotoxin (0.25 µM) or nifedipine (10 µM) to block N-type or L-type VDCCs, respectively. Ca2+ current densities were clearly reduced in the presence of either blocker, which shows the presence of these two channels in layer II/III pyramidal neurons (Fig. 1C inset). In the presence of the N-type VDCC blocker ω-conotoxin (0.25 µM), but not the L-type VDCC blocker nifedipine (10 µM), 60 current injections at 20 Hz resulted in a markedly reduced Cl- current (Fig. 2C). This indicates that Ca2+ influx through N-type VDCCs following AP spiking at 20 Hz contributes to the synthesis of eCBs that mediate CB2R activation. To investigate whether the CB2R-mediated current could be evoked under more physiological conditions, we next applied synaptic stimulation to evoke APs, instead of current injections into the soma. These experiments were performed with normal ACSF and with a pipette solution containing a physiologically relevant Cl- concentration ([Cl-]i = 8.75 mM). With electrical synaptic stimulation in layer I, excitatory postsynaptic potentials (EPSPs) large enough to induce APs could be generated in layer II/III neurons. Following 60 synaptically evoked APs at 20 Hz, we recorded a current that reversed close to ECl- (Fig. 2D). This current could not be induced by the synaptic stimulation protocol if only EPSPs that did not generate APs were evoked (Fig. 2D). If APs were evoked, but recordings were performed after preincubation (of at least 10 minutes) with and in the continuous presence of the CB2R antagonist Sch.356036 (5 µM), we did not detect the current (Fig. 2D). These results show that the synthesis of eCBs resulting in CB2R activation (to open CaCCs) depends on

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Figure 2. 60 APs at 20 Hz result in a CB2R-mediated Cl- current in 71% of layer II/III mPFC neurons. (A) Cropped traces of 60 evoked APs (A1) and injected currents (A2) at 20 Hz. (B) I/V relationships showing that AP firing evoked with 60 current injections at 20 Hz induced a CB2R-mediated Cl- current, Erev = -42.4±2.3 mV (ECl- = -38.3 mV). Such current injections could not evoke a current after preincubation with and in the continuous presence of the CB2R antagonist Sch. 356036 (5 µM). After preincubation with and in the continuous presence of the CB1R antagonist rimonabant (5 µM), a similar current, Erev = -38.7±4.9 mV, could be detected following 60 current injections at 20 Hz. (C) The N-type VDCC blocker ω-conotoxin, but not the L-type blocker nifedipine, markedly reduced the amplitude of CB2R-mediated Cl- currents evoked with 60 current injections at 20 Hz. Erev for currents recorded with ω-conotoxin (-41.4±2.3 mV) and with nifedipine (-42.9±1.4 mV) are close to ECl- (-38.3 mV). The current in the absence of VDCC blockers is the same as the control current depicted in Fig. 2B (closed circles). Inset shows Ca2+ currents recorded in the absence and presence of ω-conotoxin (0.25 µM) and nefidipine (10 µM). VDCC-mediated current densities were clearly reduced in the presence of either blocker, showing the presence of both N- and L-type VDCCs in these neurons. (D) I/V relationship of CB2R-mediated current following 60 synaptically evoked APs at 20 Hz reversed at -71.7±1.2 mV, which is close to ECl- (-70 mV). The Cl- current could not be observed if no APs were evoked or after the preincubation with and in the continuous presence of Sch. 356036 (5 µM). Inset shows a biocytin-filled layer II/III neuron and the schematic representation of a stimulation electrode in layer I and a recording electrode for registering APs or EPSPs in layer II/III neurons.


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AP firing and that the subsequent Ca2+ influx through N-type VDCCs is a major contributor to the response. 2-AG is the main eCB involved in CB2R-mediated effects following 60 APs at 20 Hz We next performed experiments in the current clamp configuration to elucidate whether 2-AG and/or AEA are involved in the activation of CB2Rs following the firing of 60 APs at 20 Hz. In the current clamp configuration, at an experimental membrane potential of -80 mV and with ECl- = -38.3 mV, opening of CaCCs resulted in a transient depolarization of the neurons (Fig. 3A). These experimental conditions allow the determination of the delay, duration and amplitude of the depolarization of CB2R-mediated responses, similar to what we observed with a synthetic CB2R ligand (Den Boon et al. 2012). The transient depolarization of the membrane, evoked by the 20-Hz current injection protocol, was absent in neurons that were preincubated (for at least 10 min) with and in the continuous presence of the selective CB2R antagonist Sch.356036 (5 µM, Fig. 3B). To corroborate the involvement of Cl- channels, we performed a similar experiment in the presence of the Cl- channel blocker DIDS (0.2 mM) and observed a reduced depolarization of the membrane potential (Fig. 3B). Finally, we confirmed the pharmacological evidence that the depolarization is mediated via CB2R activation by using brain slices from CB2R knock-out (KO) and wild type (Wt) mice. Following 60 suprathreshold current injections at 20 Hz we observed a depolarization of Wt layer II/III mPFC neurons, but not of KO neurons (Fig. 3B). In order to differentiate between 2-AG and AEA, we blocked eCB hydrolysis by their most important catabolic enzymes, MGL and FAAH, respectively. We used URB602 to inhibit MGL activity and URB597 to inhibit FAAH activity (Fegley et al. 2005; Hohmann et al. 2005; King et al. 2007; Chávez et al. 2010). In the presence of either the MGL inhibitor URB602 (100 µM) or the FAAH inhibitor URB597 (1 µM), 60 APs at 20 Hz resulted in depolarizations that were not different in amplitude from control recordings (Fig. 3C). However, when the MGL inhibitor URB602 was present, the duration of the depolarization was larger than in control conditions, whereas the FAAH inhibitor URB597 did not affect the duration of the response (Fig. 3D). This suggests that 2-AG, but not AEA, mediates CB2R activation. In control conditions, the depolarization occurred with a delay of ~150 seconds after 60 current injections at 20 Hz. Delays were not different when neurons were tested in the presence of either URB602 or URB597 (Fig. 3E). We further implicated 2-AG as the main eCB involved in the CB2R-mediated depolarization by showing a decreased depolarization in the presence of the diacylglycerol lipase inhibitor RHC-80267 (100 µM), which is known to disrupt 2-AG synthesis (Puente et al. 2011) (Fig. 3C). Taken together, these data indicate that 2-AG is the main eCB involved in CB2R activation following 60 APs at 20 Hz.

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Figure 3. Stimulation with 60 AP-evoking current injections at 20 Hz induces eCB synthesis and subsequent CB2R activation in layer II/III mPFC pyramidal neurons. (A) Representative trace recorded from a neuron that depolarizes following 60 current injections at 20 Hz (arrow); the depolarization generated AP firing. (B) In current clamp recordings with ECl = -38.3 mV, 60 current injections at 20 Hz resulted in a depolarization (24.3±2.2 mV) which was reduced after preincubation with and in the continuous presence of the CB2R antagonist Sch. 356036 (5 µM) (2.2±0.8 mV, P < 0.001) and in the presence of the Cl- channel blocker DIDS (10.8±1.3 mV, P < 0.001). The same stimulation protocol depolarized layer II/III mPFC neurons of Wt mice, but not of CB2R KO mice (16.8±2.4 mV and 1±1.1 mV, respectively, P < 0.001). (C) The magnitude of the depolarization following 60 current injections at 20 Hz was not different in the presence of the MGL inhibitor URB602 (17.7±2.4 mV) or the FAAH inhibitor URB597 (24.6±2.6 mV). Incubation with the DGL inhibitor


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RHC-80267 (100 µM) reduced the depolarization (5.6±2.6 mV, Mann-Whitney, P < 0.001). The average control depolarization following 60 current injections at 20 Hz is the same as the one depicted in Fig. 3B. (D) The duration of the depolarization was increased – compared to the control stimulation – in the presence of URB602 (245±68 s and 550±109 s, respectively, P < 0.05), but not with URB597 (289±71 s). (E) The delay after stimulation until depolarization was not different in the presence of either URB602 or URB597 (147±45 s, 211±64 s and 132±35 s, respectively). CB2R activation following 60 APs at 20 Hz decreases neuronal excitability We have shown that activation of CB2Rs with the synthetic agonist JWH-133 reduces neuronal excitability under physiologically relevant intracellular Cl- conditions (ECl- = -70 mV) (Den Boon et al. 2012), most likely through the opening of CaCCs. We next tested whether endogenous activation of CB2Rs has a similar effect in a series of current clamp experiments. Neuronal firing was evoked by Gaussian current injection, which led to fluctuations around resting membrane potential, resulting in the occasional firing of APs. For each neuron, the variance of the injected current was adjusted to cause a stable firing rate of ~0.95 Hz. Two to six minutes after stimulation with 60 APs at 20 Hz the normalized firing rate was transiently reduced by ~30%. This reduction could be prevented by preincubation (of at least 10 min) with and in the continuous presence of the CB2R antagonist Sch. 356036 (5 µM) (Fig. 4). These results indicate that, when AP firing is evoked with an input that could resemble spontaneous background synaptic activity, CB2R activation following 60 APs at 20 Hz modulates the regular firing rate of mPFC neurons, presumably through the opening of CaCCs.

Discussion We have previously demonstrated that the activation of intracellular CB2Rs by synthetic cannabinoid ligands in layer II/III pyramidal neurons of the mPFC leads to the IP3R-dependent opening of CaCCs (Den Boon et al. 2012). Here, we show that bath application of 2-AG and mAEA (the stable analog of AEA) can, concentration-dependently, induce a similar Cl- current. Both compounds evoked a comparable Cl- current as was evoked by the selective CB2R agonist JWH-133, suggesting that 2-AG and mAEA activate CB2Rs. The estimated EC50 values indicate that 2-AG, although more efficacious, has a slightly lower affinity for CB2Rs than mAEA. Future studies on the binding of eCBs at the CB2R in the rodent mPFC could show how 2-AG and AEA behave at their binding sites. When both agonists are bath applied simultaneously at their respective maximal effective concentrations, the resulting current density does not differ from experiments in which mAEA was applied alone. These data suggest that 2-AG and mAEA compete for the same CB2R binding site and that they are able to activate a common downstream signalling cascade. This is in line with earlier findings that AEA can attenuate the agonistic activity of 2-AG (Gonsiorek et al. 2000; Sugiura 2009). 2-AG and AEA are considered as the two major eCBs and they have been shown to be involved in several neuronal processes via abundantly expressed CB1Rs

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Figure 4. Endogenous CB2R activation decreases firing activity of rat mPFC layer II/III pyramidal neurons. (A) Stimulation with 60 current injections at 20 Hz led to a reduction of the neuronal firing rate. Firing was induced by a Gaussian current input into the soma. Mean baseline firing frequency of 0.95±0.09 Hz was normalized over slices to 100±3% and reduced to 72±6% 2-6 minutes after 60 current injections at 20 Hz (P < 0.01). After preincubation with and in the continuous presence of the CB2R antagonist Sch. 356036 (5 µM) (baseline firing frequency 0.93±0.08 Hz, normalized to 100±2%), 60 current injections at 20 Hz could not induce a response (99±9%). (B) Representative traces of current clamp recordings showing AP firing of layer II/III neurons before and after 60 current injections at 20 Hz, in the absence and presence of Sch. 356036 (5 µM). (Terranova et al. 1995; Stella et al. 1997; Wilson and Nicoll 2001; Lafourcade et al. 2007; Kano et al. 2009). Despite difficulties with the precise quantification of brain eCB levels, it is clear that 2-AG levels exceed those for AEA (Stella et al. 1997; Buczynski and Parsons 2010). Since the discovery of 2-AG and AEA and the development of selective pharmacological tools and transgenic mice strains, the roles of 2-AG and AEA have been increasingly disentangled (Luchicchi and Pistis 2012). So far, collected data suggests that 2-AG is the principal eCB that mediates synaptic plasticity, with a smaller role for AEA (Kano et al. 2009; Kim and Alger 2010). In the PFC specifically, 2-AG, but not AEA, was reported to mediate long term depression (LTD) and depolarization-induced suppression of inhibition (DSI) (Lafourcade et al. 2007; Yoshino et al. 2011). However, AEA has also been shown to be involved in some forms of synaptic plasticity in the rodent PFC (Lourenço et al. 2011). In this study, we confirm earlier findings that 2-AG is a full agonist at the CB2R, whereas mAEA behaves as a partial agonist (Gonsiorek et al.


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2000; Sugiura 2009). This means that CB2R-mediated effects depend on the local concentrations of both 2-AG and AEA. The synthesis and release of eCBs that lead to CB1R-mediated effects are described to follow a wide range of stimulation protocols, such as voltage steps and AP firing (Llano et al. 1991; Brown et al. 2003; Lafourcade et al. 2007). In particular, eCBs can be synthesized upon Ca2+-entry through VDCCs after repetitive firing of the postsynaptic cell and then travel to presynaptic CB1Rs. Alternatively, activation of GPCRs coupled to Gq/11, such as group I mGluRs and M1/M3 muscarinic receptors, can enhance or by itself lead to eCB production (Kano et al. 2009). So far, research has focused on CB1R-medatiated effects following eCB synthesis. We hypothesized that similar protocols that lead to CB1R activation may also result in CB2R activation. We tested this by administering 60 AP-evoking current injections at 20 Hz that were described to lead to CB1R-mediated effects in the mouse PFC (Yoshino et al. 2011). Using this stimulation protocol, we demonstrate that CB2Rs are activated and that this results in the opening of CaCCs. To demonstrate that CB2Rs mediated this effect, we used the selective CB2R antagonist Sch.356036 to block the Cl- current following 60 APs at 20 Hz (Shankar et al. 2005; Lunn et al. 2008). Importantly, we excluded the involvement of CB1Rs by performing similar experiments in which we observed the Cl- current in the presence of the selective CB1R antagonist rimonabant. Both the Erev of the CB2R-mediated current and the reduction in the membrane depolarization in the presence of the Cl- channel blocker DIDS (Akasu et al. 1990; Hogg et al. 1994) identified this current as a Cl- current. In the present study, we did not find evidence for the modulation of other ion channels following CB2R activation. This is in contrast with a recent study which reported a CB2R-mediated reduction of neurotransmission in cultured autaptic hippocampal neurons of CB1R null mice (Atwood, Straiker, et al. 2012). The authors determined that the CB2R exerted its function from a presynaptic localization, presumably through the inhibition of VDCCs. It is possible that this discrepancy can be explained by differences in preparation, brain region and species. Here, we show that blocking N-type VDCCs reduced the amplitude of the Cl- current elicited with our stimulation protocol (repetitive neuronal firing), whereas blocking L-type VDCCs did not. This indicates that Ca2+ influx through N-type VDCCs following 20-Hz AP firing is sufficient to induce the synthesis of eCBs. Blocking 2-AG synthesis reduced the amplitude of the CB2R-mediated effect and inhibiting its degradation prolonged it. This finding indicates that due to a reduction in the degradation of 2-AG, local 2-AG levels are elevated for a longer period of time, which results in a longer lasting depolarization of the membrane potential. In addition, this points to a CB2R-evoked effect that is most likely mediated by 2-AG. Interfering with AEA degradation did not affect the response. However, a minor role for AEA cannot be excluded under these experimental conditions. Although the main route of AEA synthesis involves the conversion of NAPE into AEA, alternative pathways have been

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described (Leung et al. 2008; Katona and Freund 2012; Luchicchi and Pistis 2012). A lack of specific pharmacological tools to inhibit AEA synthesis complicates the exclusion of AEA from playing a role in CB2R-mediated effects. In the striatum, both 2-AG and AEA were reported to induce eCB-mediated LTD through CB1Rs (Lerner and Kreitzer 2012). Interestingly, different stimulation protocols, high- and low-frequency stimulation, induced the synthesis of AEA and 2-AG, respectively. This is in line with the notion that 2-AG and AEA play specialized roles in the modulation of synaptic plasticity (Luchicchi and Pistis 2012). In this study we demonstrate that CB2R activation following eCB release - under physiologically relevant intracellular Cl- conditions - results in a decrease of neuronal firing rate, presumably via the opening of CaCCs. If ECl- is at or close to the resting membrane potential of neurons, the opening of CaCCs following activation of CB2Rs will stabilize or even clamp the membrane potential around that value. We observed a similar reduction in neuronal excitability when CB2Rs were activated with a selective, synthetic cannabinoid (Den Boon et al. 2012). Here we demonstrate that 20-Hz AP firing of layer II/III pyramidal neurons can, with a delay of 2-6 minutes, reduce neuronal excitability via CB2Rs. This suggests that in the mPFC, the eCB system plays a slow modulatory role via the activation of CB2Rs, possibly preventing excessive firing via a feedback mechanism after an initial brief period of high activity. We have previously shown that the application of the CB2R antagonist Sch. 356036 increased the regular firing rate in a similar experiment, which points to a basal pool of eCBs and/or constitutive activity of CB2Rs (Alger and Kim 2011; Di Marzo 2011; den Boon et al. 2012). The activity-induced self-inhibition via CB2Rs we demonstrate here is likely to depend on the on-demand pool. The timescale of the onset of this feedback mechanism (several minutes) is different from well described and generally much faster modulatory eCB-mediated processes via presynaptic CB1Rs (seconds), such as depolarization-induced suppression of excitation (DSE) and DSI. Both cannabinoid receptors may thus modulate neuronal activity, albeit through different mechanisms and on different time-scales. Future studies on the role of CB1Rs and CB2Rs in the cortex could shed more light on the precise role of each receptor and their interplay in cortical network functioning. Funding This work was supported by Dutch Top Institute Pharma (grant number T5-107-1). MM was partly supported by Fondazione Italiana Sclerosi Multipla (grant 2010), and by Ministero dell’Istruzione, dell’Università e della Ricerca (grant PRIN 2011-2012).

Chapter 5

Modulation of the balance between excitation and inhibition in the rat medial prefrontal cortex

by type-1 cannabinoid receptors

Femke S. den Boon, Taco R. Werkman, Qiluan Schaafsma-Zhao,

Kas Houthuijs, Tania Vitalis, Chris G. Kruse,

Wytse J. Wadman, Pascal Chameau.

Submitted to Cerebral Cortex

Modulation of the E/I balance in the rat medial prefrontal cortex by CB1Rs

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Abstract Although type 1 cannabinoid receptors (CB1Rs) are abundantly expressed in many brain areas and well characterized, less is known about their role in the medial prefrontal cortex (mPFC). Using electrophysiological and immunohistochemical techniques, we demonstrate that functional CB1Rs are located on both excitatory and inhibitory inputs to layer II/III pyramidal neurons and that their activation results in a reduction of ~30% of the release of both GABA and glutamate. More importantly, by decomposing the evoked synaptic response into its excitatory and inhibitory components, we show that CB1R activation modulates the balance between excitation and inhibition (E/I balance) by causing a shift of this balance towards excitation, from ~20/80% to 25/75%. Finally, when animals were injected with a cannabinoid receptor agonist, we observed a E/I balance of 30/70%. The modulation of the E/I balance by presynaptic CB1Rs may be fundamental in the regulation of local mPFC network excitability and consequently in the modulation of higher order cognitive functions fulfilled by the mPFC.

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Introduction Cannabinoid type-1 receptors (CB1Rs) are among the most abundantly expressed G protein-coupled receptors (GPCRs) in the central nervous system (CNS) (Herkenham et al. 1991). Together with at least one other GPCR, type-2 cannabinoid receptors (CB2R), they are part of the endocannabinoid (eCB) system. This system also contains lipid endogenous cannabinoids (eCBs), eCB transporters and enzymes responsible for the synthesis and degradation of eCBs (Kano et al. 2009; Fu et al. 2012). The expression and functionality of CB1Rs in the CNS have been heavily investigated (Devane et al. 1988; Kano et al. 2009; Castillo et al. 2012; Katona and Freund 2012). The principal mechanism by which the eCB system is reported to play a functional role is through retrograde signalling (Kano et al. 2009). Briefly, postsynaptic activity, consisting of Ca2+ influx and/or activation of GPCRs such as group 1 metabotropic glutamate receptors, leads to the synthesis of eCBs in the plasma membrane. eCBs can travel backwards across the synapse and bind to presynaptic CB1Rs. There, activation of presynaptic CB1Rs suppresses neurotransmitter release by inhibition of Ca2+ influx through voltage-dependent Ca2+ channels and activation of G protein coupled inwardly rectifying K+ (GIRK) channels (Kreitzer and Regehr 2001; Wilson et al. 2001; Brown et al. 2003; Guo and Ikeda 2004). CB1Rs are expressed in the PFC where their activation suppresses spontaneous IPSCs (sIPSCs), evoked EPSCs (eEPSCs) and which mediates synaptic plasticity of inhibitory synapses (Auclair et al. 2000; Yoshino et al. 2011). However, the expression pattern of CB1Rs in this cortical area remains relatively unknown.

The neuronal population in the cerebral cortex consists of ~20% GABAergic interneurons and ~80% non-GABAergic cells (Peters and Kara 1985; Somogyi et al. 1998; Markram et al. 2004). Despite the small number of interneurons, the relative contribution of excitatory and inhibitory input conductances (balance between excitation (E) and inhibition (I), E/I) is, across different layers, dynamically maintained at approximately 20% excitation and 80% inhibition (Le Roux et al. 2006, 2007, 2008; Zhang et al. 2011). The E/I balance is thought to result from the coordinated activities of direct and recurrent excitation together with feed-forward and feedback inhibition. It determines proper cortical network rhythms responsible for higher order cognitive functions (Shu et al. 2003; Haider et al. 2006a). Disturbances in the E/I balance are associated with a broad spectrum of neuropsychiatric and neurological diseases, such as autism, schizophrenia and epilepsy (Cobos et al. 2005; Lewis et al. 2005; Rubenstein 2010). In particular, in the mPFC, an elevated E/I balance elicits impairments in cellular information processing and social dysfunction (Yizhar et al. 2011). Since dysregulation of the eCB system is associated with various psychiatric disorders that could be linked to a disturbed E/I balance (Ishiguro et al. 2010; Parolaro et al. 2010; Roche and Finn 2010), we set out to investigate the effects of CB1R activation on the E/I balance in the mPFC.

In the present study, we have determined the effects of CB1R-activation in the mPFC on miniature synaptic currents in order to confirm the supposed presynaptic localization of these receptors. Furthermore, we made use of immunohistochemical stainings to determine the preferential distribution of CB1Rs on GABAergic cells and non-GABAergic (principle) cells in the mPFC. Based on the expression of CB1Rs, we


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investigated the effects of CB1R-activation by the superfusion of a cannabinoid agonist on the E/I balance in the mPFC. We made use of a method that enabled us to decompose the total synaptic conductance into excitatory and inhibitory components (Borg-Graham et al. 1998; Wehr and Zador 2003). These methods allow for the estimation of the E/I balance, without altering the functional interactions between glutamatergic and GABAergic cells, since the use of pharmacological tools is avoided (Monier et al. 2008). In addition, the total, excitatory and inhibitory conductances can be monitored dynamically over the course of the synaptic response. Finally, we used the same method to determine the E/I balance in slices obtained from animals that had been treated with a cannabinoid agonist. Materials and Methods Animals Wistar rats (Harlan, the Netherlands) between the age of postnatal day 14 (P14) and postnatal day 20 (P20) were used for this study. All experiments were performed in accordance with the committee on animal bioethics of the University of Amsterdam. Electrophysiology recordings Coronal slices (300 µm) of the mPFC were obtained from male rats aged 17-20 days postnatal. Animals were killed by decapitation, their brains rapidly removed and placed in oxygenated (95% O2 – 5% CO2), ice-cold (4 °C) adapted artificial cerebrospinal fluid (aACSF, containing in mM: 120 choline chloride, 3.5 KCl, 0.5 CaCl2, 6 MgSO4, 1.25 NaH2PO4, 25 D-glucose, 25 NaHCO3). mPFC slices were cut in aACSF on a vibratome (VT1200S, Leica, Germany) and placed for 30 min in ACSF (containing in mM: 120 NaCl, 3.5 KCl, 25 NaHCO3, 25 D-glucose, 2.5 CaCl2, 1.3 MgSO4, 1.25 NaH2PO4; [Cl-]out = 128.5 mM) at 32 °C. Slices were kept at room temperature for at least 1 h prior to recording. Glass recording pipettes were pulled from borosilicate glass (Science Products, Germany) and had a resistance of 2-3 MΩ when filled with pipette solution used for recordings of miniature synaptic currents (containing in mM: 110 KGluconate, 30 KCl, 0.5 EGTA, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 4 Mg-ATP, 0.5 Na-GTP). Whole-cell voltage clamp recordings were made at 32 °C from the soma of layer II/III pyramidal neurons. For the experiments determining the effects of cannabinoid ligands on the balance between excitatory and inhibitory input, a pipette solution with a physiologically relevant Cl- concentration was used, containing (in mM): 131.25 KGluconate, 8.75 KCl, 0.5 EGTA, 10 HEPES, 4 Mg-ATP, 0.5 Na-GTP. Recordings were made using an Axopatch 200b (Axon, USA) and in-house software running under Matlab (MathWorks, USA). Signals were filtered at 5 kHz and sampled at 10 kHz. Series resistance ranged from 5–15 MΩ and was compensated to ~65%. Signals were corrected for liquid junction potential. Miniature synaptic currents analysis The amplitude and instantaneous frequency were determined for every miniature synaptic event and analysed. Distributions of binned mEPSC/mIPSC instantaneous

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frequencies and amplitudes (40-50 bins) were constructed for every cell and then averaged. For mEPSCs, at least 250 events were analysed per cell, for mIPSCs, at least 100 events were analysed per cell. Synaptic response analysis Data were analysed off-line with in-house software running under Matlab. The method that was used to decompose the excitatory and inhibitory synaptic input is based on the continuous measurement of conductance dynamics of evoked synaptic responses, first described in vivo in cat cortex (Borg-Graham et al. 1998; Monier et al. 2003). This method has since then been validated by several groups and in different cortices (Shu et al. 2003; Wehr and Zador 2003; Higley and Contreras 2006; Le Roux et al. 2006, 2007, 2008; Cruikshank et al. 2007). For synaptic stimulation, a stimulation electrode was placed in layer I, through which currents (100-600 μA, 0.1 ms) were injected. Stimulation amplitudes were adjusted to be large enough to evoke postsynaptic responses of half-maximal amplitude and which did not evoke action potentials (APs) in the current clamp configuration. Evoked synaptic responses were recorded in layer II/III and averaged (four traces) at different Vm levels (-90 to -50 mV, in 500 ms steps of 5 mV). This allowed for the construction of an average I/V relationship during rest (50 ms) and an I/V relationship for each delay (t) per 1 ms after synaptic stimulation. The evoked synaptic conductance (gsyn(t)) was calculated by subtracting the slope of the best linear fit (mean least square criterion) of the average I/V curve prior to stimulation (grest) from the slope of the best linear fit for the I/V curve for each delay (t) after synaptic stimulation (Fig. 4B). The voltage abscissa of the intersection point between the average rest I/V curve and a I/V curve at time (t) was taken as the reversal potential of the synaptic current (Esyn(t)). In order to decompose the evoked synaptic conductance (gsyn(t)) into its excitatory (ge(t)) and its inhibitory (gi(t)) component, we used the following simplifications, as described by others (Borg-Graham et al. 1998; Wehr and Zador 2003):

Esyn(t) =ge(t)×Ee+gi(t)×Ei

ge(t)+gi(t)

gsyn(t)=ge(t)+gi(t)


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And solving for ge(t) and gi(t):

gi(t)= gsyn(t)×(Ee-Esyn(t))

Ee-Ei

ge(t)= gsyn(t)-gi(t) where ge(t) and gi(t) are the excitatory and inhibitory conductances, respectively; Ee and Ei are the reversal potentials for excitatory and inhibitory conductances, respectively. These latter values were determined by the intracellular and extracellular solutions. The value for Ee was set at 0 mV, the value for Ei was set at -86 mV. The integral (int) of the conductances over a time window of 250 ms after synaptic stimulation was used to quantify changes in conductances. The contributions of the excitatory and inhibitory components were expressed by the ratio of their integrals (integral for excitatory conductances, inte and integral for inhibitory conductances, inti) to the integral of the total conductance (intt), as previously described (Borg-Graham et al. 1998; Wehr and Zador 2003). Since we performed somatic recordings, the conductance measurements are representative of the proximal excitatory and inhibitory inputs to the neuron and may underestimate the contribution of distal synaptic events. Nevertheless, they reflect the relative changes in excitatory and inhibitory conductances read out at the soma (Haider et al. 2006b; Le Roux et al. 2006). CB1R internalization and immunohistochemistry To determine the expression of CB1Rs in the mPFC of P14 male pups, some animals were injected with the mixed CB1R/CB2R agonist CP-55940 (0.7 mg/kg, i.p., dissolved in DMSO and a physiological saline solution) 24, 12 and 2 hours prior to being sacrificed. Animals, treated and untreated (vehicle injections), were deeply anaesthetized with pentobarbital (80 mg/kg, i.p.) and ketamine (60 mg/kg, i.p.) and perfused transcardially with 4% paraformaldehyde (PFA). Brains were dissected out and left in PFA overnight for postfixation. 60-μm slices of the mPFC were cut on a vibratome (Leica VT1000S) and washed with saline phosphate buffer (0.01 M, pH 7.4; PBS). Slices were permeabilized for 30 min in PBS-0.25% Triton-X (PBST), kept for one hour in PBST + 10% normal goat serum (NGS) and incubated overnight at 4 °C with primary antibody in PBST + 5% NGS. The next day, slices were washed with PBS and incubated for 2 hours at room temperature in secondary antibody in PBST + 5% NGS. Slices were washed again in PBS and mounted on slides using Vectashield Hard Set Mounting Medium (Vector Laboratories, Peterborough, UK). Primary antibody against the CB1R was a rabbit polyclonal antibody raised against the C-terminal portion of the CB1R (1:1000) that was produced by Eurogentec (Seraing, Belgium) and has been characterized and used previously (Leterrier et al. 2004; Vitalis et al. 2008). Primary antibodies against the mouse glutamate decarboxylase (GAD) 65 (1:5000, ab26113, Abcam) and 67 (1:1000, MAB5406, Millipore) were coincubated with some slices. Secondary antibodies (goat

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anti-mouse IgG Alexa fluor 488, 1:200 and goat anti-rabbit IgG Alexa fluor 594, 1:200) were obtained from Invitrogen (the Netherlands). Images from stained slices were made using a confocal microscope (LSM 510, Zeiss) with 488- and 543- nm lines of an Argon/Krypton laser. Cannabinoid agonist treatment For experiments that investigated the effects of in vivo cannabinoid receptor activation on the in vitro E/I balance, animals were injected with the mixed CB1R/CB2R agonist CP-55940 (0.7 mg/kg, i.p., dissolved in dimethyl sulfoxide (DMSO) and a physiological saline solution) 60 min prior to being sacrificed and their brains being sliced. Control animals were injected with a vehicle DMSO solution. Drugs CP-55940, DL-AP5 (AP5), CNQX disodium salt, bicuculline methochloride and DMSO were purchased from Tocris (the Netherlands). TTX was purchased from Latoxan (France). WIN55212-2, rimonabant (SR141716) and Sch.356036 were generous gifts from Abbott Healthcare Laboratories (the Netherlands). Statistical analysis Data were statistically tested with t-tests and the Kolmogorov-Smirnov test. In the figures the significance is indicated with asterisks (*P<0.05, ** P<0.01 and *** P<0.001). Results are given as mean ± standard error of the mean. The number of observations are given in the bar charts (Figs. 1 and 2) or in the figure legends (Figs. 5 -7).

Results CB1R activation reduces mEPSC and mIPSC frequency With a pipette solution with [Cl-]i = 30 mM and our standard ACSF in the recording chamber (reversal potential for Cl-: -38.3 mV), both excitatory and inhibitory postsynaptic currents could be easily detected as inward currents when cells were clamped at -70 mV (Fig. 1A, 2A). We isolated miniature postsynaptic currents by superfusion of TTX (0.5 μM). CNQX (20 μM) and AP5 (10 μM) were used to isolate miniature inhibitory postsynaptic currents (mIPSCs), bicuculline (20 μM) was used to isolate miniature excitatory postsynaptic currents (mEPSCs). To determine the effects of cannabinoid ligands on mEPSCs and mIPSCs, control recordings (5 min) were made at least 5 min after going whole-cell. Prior to determining the effects on mEPSCs and mIPSCs (5 min), cannabinoids were washed in for 15 min. The application of the mixed CB1R/CB2R agonist WIN55212-2 (WIN) changed the cumulative probability distribution plot of mEPSC instantaneous frequencies (Fig. 1C), but not the cumulative distribution plot of the amplitudes (Fig. 1D). After preincubation (of at least 10 min) and in the continuous presence of the CB1R antagonist rimonabant (5 μM), we did not detect a change in the frequency or


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Figure 1. CB1R activation reduces the frequency, but not the amplitude of mEPSCs. (A) Representative current trace of mEPSCs (control). Inset shows an example current trace of a single mEPSC. (B) Representative current trace of recorded mEPSCs after the application of WIN (5 µM). (C) Cumulative distribution plot of mEPSC frequency showing that bath-applied WIN (5 µM) caused a left-ward shift of the distribution (Kolmogorov-Smirnov test, P < 0.01). (D) Cumulative distribution plots of mEPSC amplitude were not different before and after bath application of WIN (5 µM). (E) Cumulative distribution plot of mEPSC frequency demonstrating that the frequency of mEPSC recorded after preincubation with, and in the continuous presence of rimonabant (5 µM), is not different from the frequency of mEPSCs recorded after preincubation with and in the

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continuous presence of rimonabant (5 μM) and WIN (5 µM). (F) Cumulative distribution plots of mEPSC amplitude in the presence of rimonabant (5 μM) and rimonabant (5 μM) plus WIN (5 μM) are not different. (G) Normalized mEPSC frequency is reduced by ~30% in the presence of WIN (5 µM) (P < 0.01). This reduction by WIN was prevented after preincubation with, and in the continuous presence of rimonabant (5 µM). (H) Normalized mEPSC amplitude is not changed after the application of WIN (5 µM). After preincubation with, and in the continuous presence of rimonabant (5 µM), normalized mEPSC amplitude was also not different after application of WIN (5 µM). amplitude of mEPSCs following WIN application (Fig. 1E, F), indicating that this effect was mediated by CB1Rs. We show that the frequency, but not the amplitude, of mEPSCs was reduced in the presence of WIN by ~30% (Fig. 1G). We next investigated the effects of WIN on mIPSCs. WIN changed the cumulative probability distribution plot of mIPSC instantaneous frequencies (Fig. 2C), but not the cumulative distribution plot of the amplitudes (Fig. 2D). Preincubation (of at least 10 min) and continuous presence of rimonabant (5 μM) could prevent this effect on mIPSC frequency, which indicates that the reduction was mediated by CB1Rs (Fig. 2E, F). The frequency, but not the amplitude, of mIPSCs was reduced in the presence of WIN by ~30% (Fig. 2G). These data demonstrate that CB1R activation leads to a reduction in frequency of both excitatory and inhibitory miniature synaptic events. The frequency of mEPSCs and mIPSCs was affected, but not the amplitude, which indicates that WIN acted on CB1Rs at presynaptic terminals. Immunohistochemical detection of CB1Rs in the mPFC To confirm the presynaptic localization of CB1Rs in the rat mPFC, we performed immunohistochemical stainings. We stained mPFC slices of P14 pups to demonstrate the presence of CB1Rs. CB1R immunostaining was detected throughout the mPFC, but higher concentrations were observed in layer II/III and in the deeper layer V (Fig. 3A). High immunoreactivity was found on axonal fibers surrounding the unstained cortical cell bodies (Fig. 3B). In order to determine whether both GABAergic and non-GABAergic cells express CB1Rs, we performed immunohistochemistry on slices obtained from animals that were treated with three injections (24, 12 and 2 hours prior to sacrifice) of a mixed CB1R/CB2R agonist (CP-55940, 0.7 mg/kg). Internalization of CB1Rs induced by treatment with CP-55940 combined with immunohistochemistry for markers of GABAergic cells, glutamate decarboxylase (GAD) 65 and 67, allowed for the detection of CB1Rs on inhibitory GABAergic cells (Fig. 3C). We found that a small number of CB1R-expressing cells also expressed GAD65/67, whereas most CB1R-expressing cells did not (Fig. 3C, Table 1). Although it is likely that a portion of GABAergic cells failed to display a detectable amount of GAD65/67, we assume that a majority of CB1R-positive cells that were GAD65/67 negative are non-GABAergic cells. Together, this data shows that CB1Rs are mainly expressed on axonal fibers and that they are expressed by both GABAergic and non-GABAergic cells, which is in line with the effects of CB1R activation on miniature synaptic events (Fig. 1 and 2).


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Figure 2. CB1R activation reduces the frequency, but not the amplitude of mIPSCs. (A) Representative current trace of mIPSCs (control). Inset shows an example current trace of a single mIPSC. (B) Representative current trace of recorded mIPSCs after the application of WIN (5 µM). (C) Cumulative distribution plot of mIPSC frequency showing that the application of WIN (5 µM) resulted in a left-ward shift of the distribution (Kolmogorov-Smirnov test, P < 0.05). (D) Cumulative distribution plots of mIPSC amplitude are not different before and after bath application of WIN (5 µM). (E) Cumulative distribution plot of mIPSC frequency which demonstrates that the frequency of mIPSCs recorded after preincubation with, and in the continuous

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presence of rimonabant (5 µM), is not different from the frequency of mIPSCs recorded after preincubation with, and in the continuous presence of rimonabant (5 µM) and in the presence of WIN (5 µM). (F) Cumulative distribution plots of mIPSC amplitude in the presence of rimonabant (5 μM) and rimonabant (5 μM) plus WIN (5 μM) are not different. (G) Normalized mIPSC frequency is reduced by ~30% in the presence of WIN (5 µM) (P < 0.001). The reduction by WIN was prevented after preincubation with, and in the continuous presence of rimonabant (5 µM). (H) Normalized mIPSC amplitude is not changed after the application of WIN (5 µM). Normalized mIPSC amplitude was not different following application of WIN (5 µM), if slices were preincubated with, and in the continuous presence of rimonabant (5 µM). Characterization of the E/I balance in layer II/III pyramidal neurons of the mPFC in response to synaptic stimulation in layer I We investigated the E/I balance of synaptic input from layer I to pyramidal neurons in layers II/III, using previously described methods. We used a pipette solution with a physiologically relevant [Cl-]i (8.75 mM), in combination with our standard ACSF. Electrical stimulation of layer I evoked complex current responses in layer II/III cells (Fig. 4A1). We used CNQX (20 µM), bicuculline (20 µM) and AP5 (10 µM) to block AMPA/kainate, GABAA and NMDA receptor-mediated currents, respectively, showing that the current responses were mediated by glutamate and GABA (Fig. 4A2). Figure 4B shows, as example, the average rest I/V curve and the I/V curve at 8 ms after electrical stimulation, which is around the peak of the synaptic response. We analysed traces over a total duration of 250 ms as to cover the whole range of the synaptic response. Figure 4C shows the decomposition of the total synaptic conductance (g total) into inhibitory (g inhibition) and excitatory (g excitation) components. The contributions of the excitatory and inhibitory components were expressed by the ratio of their integrals to the integral of the total conductance. The E/I balance typically consisted of ~80% inhibition and ~20% excitation (inset Fig. 4C). This is consistent with previous findings in the neocortex and it shows that excitability in layer II/III of the mPFC is mainly controlled by inhibition (Le Roux et al. 2006, 2007, 2008). We next used cannabinoid ligands to investigate whether CB1R activation affected conductance amplitudes and the ratio between excitation and inhibition. CB1R receptor activation changes the E/I balance of synaptic input We investigated the effects of CB1R activation on the ratio between excitation and inhibition of synaptic input onto layer II/III mPFC pyramidal neurons (Fig. 5A-D). Control recordings, performed at least 5 min after going whole-cell, show that the balance between excitation and inhibition under control conditions is maintained at approximately 20-80% (Fig. 5C, E). Separate control recordings were performed in which only vehicle was applied for 15 min and in which no changes in E/I balance or conductances were observed (data not shown). To investigate CB1R-mediated effects on the E/I balance, the cannabinoid receptor agonist WIN was washed in for 15 min after which (still in the presence of the ligand) recordings were performed to determine WIN effects (Fig. 5B, D, E, F). Bath application of WIN (5 μM) resulted in a reduction of the inhibitory component relative to the total synaptic conductance and a corresponding relative increase in the excitatory component, shifting the balance


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Figure 3. CB1R immunoreactivity was found on axonal fibers in layers II/III and layer V, belonging to GABAergic and non-GABAergic cells. (A) Overview of CB1R expression in the various layers of the mPFC in an untreated brain. CB1R-positive immunoreactivity was predominantly found in layers II, III and the deeper layer V. Five slices from three animals showed similar staining patterns. (B) CB1R-positive immunoreactivity in more detail in layer III, particularly high immunoreactivity was found in axonal fibers surrounding the unstained cortical cell bodies. (C) When animals were repeatedly treated with the cannabinoid agonist CP-55940, CB1R internalization could be induced. This internalization, combined with staining for markers of GABAergic GAD65/67, allowed for the detection of CB1R-positive interneurons. Only a small number of CB1R-expressing cells also expressed GAD65/67, see also Table 1. Table 1. Distribution of CB1R in GABAergic and non-GABAergic neurons of the mPFC. The detection of CB1R-positive interneurons showed that in both superficial (I-III) and deeper (V-VI) layers only a small percentage of CB1R-postive cells also expressed GAD65/67. A total of 4 slices obtained from 3 animals were used to determine co-localisation of CB1R- and GAD65/67-positive immunostaining. The values between brackets indicate the % of neurons that co-localise CB1R and GAD65/67 immunoreactivity.

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Figure 4. Electrical stimulation of layer I of mPFC brain slices resulted in synaptic response currents in layer II/III pyramidal neurons that could be decomposed into excitatory and inhibitory components. (A) Representative current traces of synaptic responses to electrical stimulation (arrow) in layer I at various holding potentials (-90 - -50 mV) under control conditions (A1) and in the presence of CNQX (20 µM), bicuculline (20 µM) and AP5 (10 µM) (A2). The black and grey vertical bars indicate two 1-ms time points at which current amplitudes were measured for plotting I/V relationships (see panel B) at rest and at the peak of the synaptic response, respectively. (B) Average rest I/V relationship (50 ms) and I/V relationship at time (t) (1 ms), taken at the peak of the synaptic response. The voltage intersection point of the average rest I/V curve is taken as the resting membrane potential. The slopes of the linear fits of the average rest I/V curve and the I/V curve at time (t) are the rest conductance and synaptic conductance, respectively. The voltage abscissa of the intersection point between the average rest I/V curve and the I/V curve at time (t) represents the reversal potential of the synaptic current. (C) Decomposition of the total conductance (black) into excitatory (light grey) and inhibitory (dark grey) components. Inset: the percentage of the integrals of inhibitory and excitatory conductance compared to the integral of total conductance, typically existing of ~80% inhibition and ~20% excitation. towards excitation (Fig. 5E). When total, excitatory and inhibitory conductances were normalized to control (before WIN application), we observed a decrease in all conductances following the application of WIN (Fig. 5F). However, the reduction in inhibitory conductance exceeded the reduction in excitatory conductance, which explains the shift of the balance towards excitation. To confirm the involvement of CB1Rs and to exclude CB2R-mediated effects, we repeated the experiments in the presence of the selective CB1R antagonist rimonabant (5 µM) or the selective CB2R


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Figure 5. Effects of CB1R activation on balanced synaptic input. (A) Representative current traces of synaptic responses recorded in a layer II/III mPFC pyramidal neuron to electrical stimulation in layer I at various holding potentials (-90 - -50 mV) under control conditions. (B) Representative current traces of synaptic responses in the same cell as in A, 15 min after application of WIN (5 µM). (C) Decomposition of total (black), excitatory (light grey) and inhibitory (dark grey) conductances of the current traces displayed in A. (D) Decomposition of the conductances of the current traces displayed in B after application of WIN (5 µM). (E) Average integral values of inhibitory and excitatory conductances (as percentage of the integral value of the total conductance) were changed after application of WIN (5 µM), control: 82.1±0.8% and 17.9±0.8%, respectively; WIN: 75.3±1.5% and 24.7±1.5%, respectively (P < 0.001, n = 16). (F) WIN reduced total (black), inhibitory (dark grey) and excitatory (light grey) normalized conductances by 36.2±9.1%, 38.6±9.4% and 19.5±7.9%, respectively (P < 0.01, P< 0.001 and P < 0.05, respectively, n = 16).

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Figure 6. Effects of CB1R activation on balanced synaptic input after preincubation (of at least 10 min) with, and in the continuous presence of selective CB1R and CB2R antagonists. (A) Integral values of inhibitory and excitatory conductances (as percentage of the integral value of the total conductance) were not changed after application of WIN (5 µM) following preincubation with, and in the continuous presence of 5 μM rimonabant (rim); control: 83.0±1.7% and 17.0±1.7%, respectively; WIN: 80.6±1.2% and 19.4±1.2%, respectively (n = 7). (B) WIN did not change total (black), inhibitory (dark grey) and excitatory (light grey) normalized conductances in the presence of rimonabant (5 μM), -4.1±11.5%, -6.1±12.3% and +9.7±11.4%, respectively (n = 7). (C) Integral values of inhibitory and excitatory conductances (as percentage of the integral value of the total conductance) were changed following application of WIN (5 µM) after preincubation with and in the continuous presence of 5 μM Sch.356036 (Sch); control: 79.8±1.8% and 20.2±1.8%, respectively; WIN: 73.5±2.4% and 26.5±1.2%, respectively (n = 8). (D) In the presence of Sch.356036 (5 μM), WIN (5 µM) reduced total (black), inhibitory (dark grey) and excitatory (light grey) normalized conductances by 38.25±7.9%, 43.7±8.6% and 26.9±5.4%, respectively (P < 0.01, n = 8). antagonist Sch.356036 (5 µM) (Fig. 6). After preincubation with and in the continuous presence of rimonabant, the application of WIN did not affect the E/I balance (Fig. 6A) or the normalized conductances (Fig. 6B). In contrast, after preincubation with and in the continuous presence of Sch.356036, the application of WIN changed the E/I balance (Fig. 6C). This effect on the E/I balance was comparable to the effects of WIN, when it was applied alone (Fig. 5E). In addition, when CB2Rs were blocked, the normalized total, excitatory and inhibitory conductances were decreased after the application of WIN


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(Fig. 6D). These data show that the effects observed after WIN application are mediated by CB1Rs. In vivo cannabinoid receptor activation We next investigated whether slices obtained from animals acutely treated with a cannabinoid receptor agonist showed a change in the E/I balance compared to vehicle-injected animals. Rats were injected with the mixed CB1R/CB2R agonist CP-55940 (0.7 mg/kg, i.p.) or vehicle 60 minutes prior to being sacrificed. mPFC slices obtained from these animals were tested and the decomposition method revealed that the E/I balance was different compared to control slices, in favour of excitation, in CP-55940-treated animals (Fig. 7). We did not observe such a shift in vehicle-treated animals (Fig. 7), which showed ratios comparable to non-injected animals (Fig. 5). Discussion In this study, we have investigated the effects of the activation of CB1Rs on miniature synaptic currents and the E/I balance in the mPFC. We show that CB1R activation reduced the frequency, but not the amplitude, of both mEPSCs and mIPSCs. This indicates a presynaptic localization of CB1Rs on excitatory and inhibitory terminals in the rodent mPFC, which is in line with earlier studies which have reported the presynaptic presence of CB1Rs on pyramidal neurons and various classes of inhibitory interneurons (Katona et al. 1999, 2006; Tsou et al. 1999; Bodor et al. 2005; Hill et al. 2007). In addition, we performed immunohistochemical stainings for CB1Rs that confirmed their predominant presence on axonal fibers. Previously, treatment of pregnant females with CP-55940 was shown to result, in their embryos, in the internalization of CB1Rs to cell bodies (Vitalis et al. 2008). Another study showed the internalization of CB1Rs by repeated injections of CP-55940 in adult rats (Thibault et al. 2012). In the present study, when CB1Rs were internalized following several injections with CP-55940, we observed that CB1Rs were present on both GABAergic and non-GABAergic neurons. This is in agreement with our electrophysiological findings (see above), showing that CB1R activation affects spontaneous release of excitatory and inhibitory neurotransmitters.

To better understand the repercussions of the activation of presynaptic CB1Rs, we investigated the effects of CB1R activation on the E/I balance in the mPFC. We first characterized the E/I balance of synaptic input onto layer II/III pyramidal neurons after stimulation of layer I. Pyramidal neurons in layer II/III are thought to be key players in the cortical network, receiving cortical and subcortical input and projecting to other cortical areas. Input into layer I is thought to be crucial for feedback processes that are involved in cognitive functions (Gilbert and Sigman 2007). The excitatory input into this layer is considered to be cortical, but thalamic input also converges here (Douglas and Martin 2007; Rubio-Garrido et al. 2009). Although glutamatergic cells, like layer II/III pyramidal neurons, constitute more than 80% of the neuronal population in the neocortex, GABAergic interneurons are highly connected to glutamatergic neurons, allowing inhibitory conductances to dominate (Peters and Kara 1985). Inhibitory interneurons, which vary greatly in their morphology and physiology

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Figure 7. In vivo cannabinoid receptor activation changed the in vitro E/I balance. (A) Representative current traces of synaptic responses recorded in a layer II/III mPFC pyramidal neuron of a vehicle-treated rat to electrical stimulation in layer I at various holding potentials (-90 - -50 mV). (B) Decomposition of total (black), excitatory (light grey) and inhibitory (dark grey) conductances of the current traces displayed in A. (C) Representative current traces of similarly recorded synaptic responses in a layer II/III mPFC pyramidal neuron of a rat treated with CP-55940. (D) Decomposition of total (black), excitatory (light grey) and inhibitory (dark grey) conductances of the current traces displayed in C. (E) Average integral values of inhibitory and excitatory conductances (as percentage of the integral value of the total conductance) were different for cells from two vehicle- and two CP-55940-treated animals; vehicle: 80.7±1.4% and 19.3±1.4% (n = 15), respectively; CP-55940 (CP): 70.0±3.2% and 30.0±3.2% (n = 18), respectively (P < 0.01).


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(Markram et al. 2004), regulate local cortical circuit activity. It has been suggested that it is the great diversity of interneurons that enables them to regulate inhibition to dynamically match and balance complex excitation (Markram et al. 2004; Isaacson and Scanziani 2011). The E/I balance in the neocortex is maintained at around 20% excitation and 80% inhibition over different layers and a variety of stimuli and deviations from this ratio are associated with a range of neuropsychiatric disorders (Cobos et al. 2005; Lewis et al. 2005; Rubenstein 2010). Here, we show that the balance between excitation and inhibition following stimulation in layer I is similar to previously reported values, at ~20% - ~80%, respectively (Le Roux et al. 2006, 2007, 2008). When CB1Rs are activated by the bath application of WIN, the E/I balance is shifted towards excitation. The decomposition method we applied in this study allowed us to compare the conductance amplitudes before and after CB1R activation for total, excitatory and inhibitory conductances, separately. We report a decrease in all conductance amplitudes, which is in line with our results that demonstrate the modulation of both excitatory and inhibitory neurotransmitter release following the application of the mixed CB1R/CB2R agonist WIN. The most prominent reduction in conductance amplitude was found for inhibition, which explains the shift in favour of excitation in the E/I balance. WIN induced a similar range of effects in the presence of a selective CB2R antagonist, but not in the presence of a selective CB1R antagonist. This indicates that these effects are mediated by CB1R activation and were not dependent on CB2Rs, which – as postsynaptic receptors – are involved in regulating layer II/III pyramidal cell excitability (den Boon et al. 2012). Our immunohistochemical data and miniature synaptic current recordings demonstrate that functional CB1Rs are located presynaptically. Therefore, we conclude that the WIN-mediated reduction of the conductances, following synaptic stimulation, is mediated by the presynaptic modulation of neurotransmitter release by CB1R activation. Altogether, this points to a general role of the eCB system in the modulation of neurotransmission via presynaptic CB1Rs.

In order to investigate the effects of in vivo cannabinoid receptor activation on the E/I balance we used slices obtained from animals that were treated with the cannabinoid receptor agonist CP-55490. Slices from these animals showed a shift in E/I balance in favour of excitation, compared to slices from control animals. This shift was comparable to the shift induced by bath application of WIN. These data show that when cannabinoid receptors are acutely activated in vivo by exogenous cannabinoids, changes in the E/I balance are robust and long-lasting enough to be determined with the decomposition method used on in vitro data, collected during a period of recording lasting several hours. To compare, it is known that effects of cannabis administration may last up to 24 hours, which is relatively long compared to other drugs of abuse (Leirer et al. 1991; Grotenhermen 2003). The duration of effects mediated by endogenous ligands, such as eCB-mediated short-term synaptic plasticity, last much shorter (< 1 min) (Kreitzer and Regehr 2001; Ohno-Shosaku et al. 2001; Wilson and Nicoll 2001). Our results could suggest that (part of) the effects of cannabinoid ligands on mPFC functioning may be mediated by a change in the E/I balance. Changes in the

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E/I balance can have profound consequences not only for network excitability, but also for the tuning of neurons to specific stimuli and in shaping of their activity in time (Kavalali et al. 2011). Several studies showed that in sensory cortical areas, the E/I balance also depends on the property of the sensory stimulus (Wu et al. 2008; Poo and Isaacson 2009; Liu et al. 2011; Tan et al. 2011). The modulation of the E/I balance by presynaptic CB1Rs may be a key determinant in the regulation of higher order cognitive functions associated with the mPFC. The shift in the E/I balance we observed, is associated with a relative increased excitatory input onto cells. A recent paper reported that Ca2+ transients evoked by backpropagating APs were potentiated by the activation of CB1Rs, which was mediated by suppression of GABAergic transmission (Hsieh and Levine 2012). This could reinforce coincidence detection of feedback and feedforward processes .

Interestingly, an increase in E/I ratio in the mPFC, obtained using optogenetic tools, results in impairments in cellular information processing and social dysfunction (Yizhar et al. 2011). In that study, evidence that supports a causal relationship between an increased E/I balance and behavioural deficits was uncovered. Future studies should also determine whether intake of cannabinoids, e.g. the smoking of marijuana, can induce its behavioural effects through a changed E/I balance and whether long-term effects of cannabis use during adolescence are mediated by the same mechanism.

Taken together, the data reported in the present study indicate that presynaptic CB1Rs are involved in the modulation of both evoked and spontaneous neurotransmission. Furthermore, in vitro CB1R-activation and in vivo cannabinoid receptor activation changed the E/I balance in favour of excitation. Future studies could shed more light on the regulation of neuronal communication by the eCB system, both under control conditions and during excessive stimulation of the eCB system, e.g. following cannabis use. Acknowledgements We thank Willem van Aken for help with the immunohistochemical experiments. This study was supported by Dutch Top Institute Pharma Grant T5-107-1.

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General discussion

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Scope of this thesis This thesis focusses on the role of the endocannabinoid (eCB) system with respect to interneuronal communication and neuronal excitability in the neocortex. The studies described here were performed within a research project of a TIPharma consortium (grant T5-107-1). This research project investigated the role of the eCB system in the regulation of brain functions involved in psychopathological syndromes with high medical need, e.g. addiction and cognitive disorders. Summary The eCB system is a powerful modulator of communication between cells. Since the discovery of its constituents in the 90s, much work has been done to unravel the workings of this neurotransmitter system in various physiological processes. One of the key features of the eCB system is its involvement in the modulation of neurotransmitter release. Many different studies have shown that the activation of type-1 cannabinoid receptors (CB1Rs), which are abundantly present in the central nervous system (CNS), results in the attenuated release of GABA, glutamate, glycine, acetylcholine, dopamine, 5-HT and norepinephrine (Kreitzer and Regehr 2001; Ohno-Shosaku et al. 2001; Wilson and Nicoll 2001; Kano et al. 2009). Furthermore, CB1Rs have been shown to be involved in several forms of synaptic plasticity (Heifets and Castillo 2009; Castillo et al. 2012). In contrast, the role of type-2 cannabinoid receptors (CB2R) in the CNS has been relatively unclear, although more and more research groups are now focusing their efforts on this component of the eCB system in order to determine its role in the brain.

In chapter two, we examined the effects of CB1R- and CB2R-activation on local evoked field potentials in the medial prefrontal cortex (mPFC). We demonstrated that activation of these receptors resulted in a concentration-dependent reduction of the amplitude of the fEPSP component of the evoked mPFC field potential amplitude. This shows that the eCB system is present in this brain area and that activation of CBRs results in the modulation of neuronal activity, as detected by the recording of field potentials.

In chapter three, we focused on investigating the presence of CB2Rs in layer II/III pyramidal cells of the mPFC. We were the first to demonstrate that CB2Rs are expressed intracellularly in layer II/III pyramidal neurons. We showed that CB2R-activation, by application of synthetic, selective agonists, resulted in the opening of Ca2+-activated Cl- channels (CaCCs). The opening of CaCCs was mediated by the release of Ca2+ from intracellular IP3-sensitive Ca2+ stores, since this could be prevented by blocking IP3Rs, which could be located e.g. on the endoplasmic reticulum. Furthermore, to investigate the functional role of CB2R activation, we induced action potential (AP) firing with the injection of noisy current and observed a CB2R-mediated reduction of neuronal firing rate.

In chapter four, we hypothesized that stimulation protocols suitable to evoke eCB synthesis and subsequent CB1R activation, could also result in CB2R activation. We first determined that the application of the eCB 2-arachidonoylglycerol (2-AG) and methanandamide (mAEA, a stable analog of anandamide, AEA) could result in a similar

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Cl- current as was evoked with synthetic cannabinoid ligands. We found that although both 2-AG and mAEA can evoke a Cl- current, 2-AG is a more efficacious agonist for the CB2R. In addition, we performed experiments to show that a stimulation protocol that evokes 20 Hz AP firing can induce eCB synthesis and result in CB2R-activation. We performed additional experiments that demonstrate the involvement of Ca2+ influx through N-type VDCCs in this process. Further experiments demonstrated that 2-AG is the main eCB involved in this process. Finally, we showed that under physiologically relevant Cl- conditions, AP firing could lead to a CB2R-dependent reduction of neuronal excitability.

In chapter five, we investigated the role of the eCB system in neurotransmission and the maintenance of the excitation/inhibition (E/I) balance in the mPFC. We showed that CB1R activation reduced both inhibitory and excitatory neurotransmission, indicative of a presynaptic localization of these receptors. With immunohistochemical stainings for CB1Rs we confirmed the presynaptic localization and found the CB1Rs to be expressed by both excitatory and inhibitory cells. We furthermore described that the application of a commonly used, mixed CB1R/CB2R agonist, could alter the E/I balance in the mPFC, in favour of excitation. We showed that this shift in E/I balance is mediated by CB1Rs. Importantly, we showed that a similar shift in the E/I balance due to in vivo treatment with a cannabinoid agonist could be detected using the decomposition method to analyse in vitro data.

In summary, the data presented in this thesis show the functional presence of both cannabinoid receptors, CB1Rs and CB2Rs, in the mPFC. The consequences and physiological relevance of these findings will be discussed next.

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Discussion Cannabinoid receptors in the cortical network In chapter two, we have described effects of cannabinoid receptor activation on excitability of the local mPFC cortical network. Recordings in layers II/III of evoked field potentials in mPFC brain slices are indicative of a highly complex cortical network with components sensitive to glutamate receptor blockers. In experiments with two stimuli separated by varying interpulse intervals (IPIs), we also show that these field potentials are modulated by inhibitory network components through GABAA and GABAB receptors. The technique of field potential recordings has been proven especially successful in recordings in the hippocampus. The organized structure of the hippocampus has traditionally made this technique a suitable method of hippocampal research into a broad range of topics (Leung 1979; Foy et al. 1987; Kerr et al. 1989). In this thesis, we present results from field potential recordings in the cortex, a brain area relatively little investigated with field potential methods.

The data reported in chapter two demonstrates that cannabinoid receptor activation leads to the reduction of fEPSP amplitudes. This reduction was particularly strong when the mixed CB1R/CB2R agonist WIN55212-2 (WIN) was applied to mPFC brain slices, but could also be observed following the application of selective CB1R and CB2R agonists. Interestingly, the application of WIN resulted in the switch from paired-pulse depression to paired-pulse facilitation, when two stimuli were given with an IPI of 25 ms. This reversal in short-term plasticity was not induced by separate CB1R and CB2R activation with selective CB1R and CB2R agonists, respectively. Furthermore, additional experiments with the simultaneous application of the selective CB1R and CB2R ligands could not replicate the effects induced by WIN application (data not shown). These results indicate the complexity of cannabinoid receptor-mediated effects at the level of the cortical network. Furthermore, additional knowledge about the connectivities in this cortical network would be very useful for the interpretation of the field potential data. In recent years, the use of multielectrode arrays enabled researchers to answer ever more detailed questions in other brain areas. The use of such multielectrode arrays could shed more light on the functioning of the local mPFC network.

As discussed in the introduction of this dissertation, the PFC is associated with several executive functions such as working memory, reversal learning and attentional function. Studies have shown that these higher cognitive functions are impaired when the eCB system is modulated (Jentsch et al. 1998; Lichtman et al. 2002; Arguello and Jentsch 2004; Egerton et al. 2005, 2006). The mechanisms that could underlie these effects include the altered release of neurotransmitters and synaptic plasticity (Egerton et al. 2006; Puighermanal et al. 2012). Following the data presented in this thesis, future studies should determine the role of both CB1Rs and CB2Rs in the modulation of network activity and the eventual disturbance of executive functions performed by the PFC by cannabinoid ligands.

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The presence of CB2Rs in the CNS Since the discovery of CB2Rs in macrophages in the marginal zone of the spleen (Munro et al. 1993), CB2Rs were assumed to be exclusively present in the periphery. Their expression was detected in cells of the immune system, such as in the spleen and thymus, as a well as in several circulating immune cell populations (Galiègue et al. 1995; Klein et al. 2003). A few years after the discovery of CB2Rs, a study reported the presence of CB2R mRNA in cerebellar granule cells (Skaper et al. 1996). Later, CB2Rs were discovered on perivascular microglial cells and in cultured cerebrovascular endothelium (Golech et al. 2004; Núñez et al. 2004). More than a decade after their discovery, an important paper was published in which the presence of CB2Rs in the brainstem, cerebellum and cortex of several rodent species was reported (Van Sickle et al. 2005). In that study, the authors also demonstrated a role for CB2Rs in the reduction of emesis in ferrets, showing that these receptors are functional. More recently and in addition to this evidence, several other articles were published that report a variety of functional effects mediated by CB2Rs in the brain (Gong et al. 2006; Brusco et al. 2008a, 2008b; Jhaveri et al. 2008; Onaivi et al. 2008; Morgan et al. 2009; Xi et al. 2011; Atwood, Straiker, et al. 2012). It is now generally accepted that the CB2R is expressed in neurons upon brain stress and damage (Viscomi et al. 2009).

Despite the studies claiming the (functional) presence of CB2Rs in the healthy brain, the existence of such central CB2Rs has remained controversial. One of the main objections raised against a large portion of the evidence for CB2Rs in the brain concerns immunohistochemical stainings of these receptors. It is well known that the visualization of many G protein-coupled receptors (GPCRs) is problematic due to the lack of specific antibodies (Ashton 2012). While the staining of CB1Rs with specific antibodies has been validated and matches the pattern of CB1R expression detected with autoradiography methods (Grimsey et al. 2008), antibodies against the CB2R are presumed to lack specificity (Atwood and Mackie 2010; Ashton 2012). Other problems could arise from slight differences in staining protocols, species-specific isoform expression patterns and complications with negative controls such as CB2R-KO mice (Liu et al. 2009; Atwood and Mackie 2010; Ashton 2012; Onaivi et al. 2012). In conclusion, this highlights the importance of investigations into CB2R expression in the brain with tools that do not (solely) rely on antibodies.

In chapter three, we have avoided the controversial use of antibodies against CB2Rs in order to visualize their expression with immunohistochemistry. Nevertheless, we did use a different biochemical technique relying on antibodies, Western blotting, to show the presence of these receptors in the mPFC. A lack of specificities of antibodies in one assay (e.g. immunohistochemistry) does not mean that the same antibody will not be specific in another assay (e.g. Western blotting). Fixatives used in immunohistochemistry may mask the epitope of interest so that the antibody no longer correctly binds to the protein (Ramos-Vara 2005). In our Western blotting experiments, we detected a band for mPFC tissue of the correct molecular weight (~45 kD). Subsequently, when mPFC samples were fractionated in a plasma membrane fraction and an intracellular fraction, we were only able to detect a similar band in the intracellular fraction. These unexpected results were confirmed by additional

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experiments performed with a radioactive binding assay on similarly fractionated samples, and corroborated our hypothesis based on electrophysiological data that functional CB2Rs are intracellularly present in the mPFC. In addition to these results, with fluorescence imaging experiments on a neuronal cell line (human neuroblastoma cells transiently transfected with GFP-tagged CB2Rs), we found that CB2Rs were almost exclusively localized in intracellular membranous structures and that they were not present in the plasma membrane. The intracellular presence of several GPCRs was described earlier for receptors such as the mGluR1, mGluR5, apelin receptor, angiotensin AT1 receptor and the bradykinin B2 receptor (Lee et al. 2004b; Jong et al. 2005b, 2007). Functional CB2Rs were also found to be present intracellularly in guinea pig heart cells (Currie et al. 2008). Other studies reported that the CB2R protein was found to be associated with the rough endoplasmic reticulum and Golgi apparatus in hippocampal pyramidal neurons, although the functional role of these intracellular CB2Rs was not elucidated (Brusco et al. 2008a, 2008b; Onaivi et al. 2012).

Some papers have reported CB2R-mediated effects on synaptic currents, that indicate a presynaptic locus for these receptors (Morgan et al. 2009; Atwood, Straiker, et al. 2012). The reduction of spontaneous excitatory postsynaptic currents (EPSCs) described in cultured hippocampal autaptic neurons from CB1R-KO mice could be explained, according to the authors, by a CB2R-mediated inhibition of voltage-dependent Ca2+ channels (VDCCs) (Atwood, Straiker, et al. 2012). In short, these studies suggest that the CB2R has a similar role as the CB1R, i.e. mediating the presynaptic reduction of neurotransmitter release. In our preparation of the mPFC, we were not able to detect CB2R-mediated changes in the frequency or amplitude of miniature excitatory postsynaptic currents (mEPSCs) or miniature inhibitory postsynaptic currents (mIPSCs), (data not shown). One of the mentioned studies that reported presynaptic CB2R-mediated effects was performed on cultured neurons (Atwood, Straiker, et al. 2012). This could explain differences in CB2R functioning, since CB2R expression is thought to be highly inducible by immune response-triggering conditions, such as injury and culturing (Wotherspoon et al. 2005; Viscomi et al. 2009). The other study was performed in acute slices from the entorhinal cortex (Morgan et al. 2009). In this study, no CB2R-mediated effects on mIPSCs were found and changes in sIPSC frequency were inconsistent. Despite these results, the authors conclude that CB2Rs mediate their AP-dependent effects from a presynaptic locus, since they observed a small change in sIPSC decay time. Taken together, the results from various studies into CB2R-mediated effects imply that the CB2R may come to expression at various locations in neurons, presynaptically and postsynaptically, and that their activation can couple to different downstream signalling pathways. Localisation of CB2R Our findings point to a postsynaptic, intracellular localisation for CB2Rs in layer II/III pyramidal neurons of the mPFC. Since we have not been able to use antibodies for immunohistochemical visualization of the receptors, one of the main questions that remain to be answered concerns the exact localisation of CB2Rs (Fig 1). As described in chapter three, CB2R activation involves a IP3R-mediated rise in [Ca2+]i. Most GPCRs that

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mediate a rise in [Ca2+]i can be found on the plasma membrane. The binding of an agonist to such a receptor results in the production of IP3, which diffuses into the cytosol and binds to IP3Rs. IP3Rs are located within the membrane of intracellular Ca2+-stores such as the endoplasmic reticulum, which is a continuum with the perinuclear space (Gerasimenko et al. 1995). Consequently, a rapid rise in [Ca2+]i can be detected. The ability of IP3 in reaching its receptor in a different part of the cell means that the GPCR and IP3R do not necessarily need to be localized close together.

Many other GPCRs that are found intracellularly are specifically localized at the cell nucleus (Gobeil et al. 2006a). Stimulation of such GPCRs can take place intracellularly. Particularly interesting examples of such GPCRs are the receptors activated by bioactive lipids such as prostaglandin, platelette-activating factor and lysophosphatidic acid (Zhu et al. 2006). These ligands are formed from membranes, including the nuclear membrane, in close proximity to their receptors. Alternatively, ligands may be transported from the extracellular space into cells to reach their receptors. This has been described for the polar compounds glutamate and quisqualate and their binding to nuclear mGluR5 in neurons, which is followed by a rise in [Ca2+]i (O’Malley et al. 2003a; Jong et al. 2005b). Other nuclear receptors, such as peroxisome proliferator-activated receptors (PPARs), can be activated by AEA and other lipids that reach their target by, so far, unknown means (O’Sullivan 2007). The possible involvement in this process of chaperone proteins is currently a topic of intense investigation (Fu et al. 2012).

In addition to GPCRs, many components of downstream signal transduction machinery are present at the nuclear membrane, such as G proteins (Gs, Gi/o), enzyme effectors and ion channels (Gobeil et al. 2006b). An interesting point, brought up in the article describing functional, nuclear mGluR5 (O’Malley et al. 2003b), is the orientation of these receptors in the nuclear membrane. Since the luminal side of the endoplasmic reticulum corresponds to the extracellular side of the plasma membrane, the authors predict that ligand binding domains of mGluR5 are on the luminal side. This means that ligands need to pass the nuclear envelope to reach their binding site, which should be possible for lipid eCBs. In turn, this means that the G protein binding domains are on the cytosolic side, where interaction with downstream signalling machinery is possible. In this model, mGluR5 are ideally located to activate nuclear PLC, IP3 and Ca2+ cascades. Possibly, CB2Rs are present at a similar location and in a similar orientation to enable interaction with the G protein signalling machinery. Both the exact localization of CB2Rs and their orientation in the intracellular membrane are interesting topics for future investigations.

CB2R activation eCB synthesis depends on the influx of Ca2+ through VDCCs and/or activation of GPCRs such as metabotropic glutamate receptors or M1/M3 muscarinic acetylcholine receptors (Kano et al. 2009). In chapter four, we have shown that a stimulation protocol (inducing 20 Hz AP firing), used to evoke eCB-mediated CB1R activation, can result also in CB2R activation. We performed additional experiments that demonstrate the involvement of N-type VDCCs and that 2-AG is the main eCB in this process. Ca2+

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influx through VDCCs is thought to be a major contributor to eCB synthesis (Fig. 1), likely through the activation of diacylglycerol lipase (DAGL), the enzyme that synthesizes 2-AG from dialcylglycerol (DAG). Alternatively, it has been suggested that Ca2+ influx could release presynthesized eCBs from intracellular pools so that eCBs are available to then bind to intracellular CB2Rs (Min et al. 2010; Alger and Kim 2011; Alger 2012). With CB2R-activation involving Ca2+ influx-induced eCB synthesis in the same cell, the process of the activation of this receptor and the consequent opening of CaCCs both require an increase in [Ca2+]i. If Ca2+ flows into the cell through VDCCs upon repeated depolarizations, is this Ca2+ itself not sufficient to activate CaCCs? It is possible that compartmentalization in the cell requires the complex machinery of a CB2R-mediated rise in [Ca2+]i. This will provide the cell with a more precise control over the final opening of CaCCs.

An interesting parameter in the described process is the relatively long delay following our stimulation protocol until the depolarization response and the all or nothing nature of this response, which has a Cl- current as underlying conductance. Presumably, the stimulation protocol results in the immediate synthesis of eCBs, mainly 2-AG. The G protein signal transduction pathway, including IP3 synthesis, the activation of IP3Rs and the rise in [Ca2+]i, normally occur within seconds (Berridge 2009). Release of Ca2+ from intracellular stores following activation of IP3Rs or ryanodine receptors (RyRs) is described to be a process that occurs abruptly (Berridge 1998, 2009). In fact, like RyRs, IP3Rs are sensitive to intracellular Ca2+ levels and this gives rise to Ca2+-induced Ca2+ release (CICR), which is a nonlinear cooperative process (Ross 2012). CICR, depending on activation of IP3Rs or RyRs, is assumed to underlie the sudden upstroke of [Ca2+]i typical for Ca2+ release from intracellular stores (Berridge 1998, 2009). The abrupt increase in [Ca2+]i following Ca2+ release from intracellular stores could underlie the all or nothing character of the CB2R-mediated depolarization. However, it is important to mention that experiments should still be performed to investigate the mechanism behind the opening of CaCCs, which is prevented when intracellular Ca2+ is chelated (chapter 3).

In complement with these fast processes, changes in [Ca2+]i can instantaneously induce Cl- currents through CaCCs (Osipchuk et al. 1990). The long delay (several minutes) we reported in chapter four could be due to the time it takes for eCBs to travel to their binding sites on intracellular CB2Rs. This delay could be enhanced by the fact that the whole-cell patch clamp method is associated with the dialysis of the cytosol with pipette solution, effectively reducing the concentration of the intracellular contents. In chapter three, we have shown that the delay following application of synthetic cannabinoids until the CaCC-mediated depolarization of the cell could be reduced by the intracellular application of the ligand. This is indicative of the slow penetration of cannabinoid ligands into the cell. The lipophilic nature of both synthetic cannabinoids and eCBs enable these compounds to enter lipid membranes.

Researchers have investigated whether eCBs exiting membranes is a passive or active process (Ligresti et al. 2004; Chicca et al. 2012; Fowler 2012; Fu et al. 2012). Despite 2-AG being the predominant eCB in the CNS, 2-AG transport has received

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relatively little attention. In contrast, AEA transport has been the topic of intense investigation. Initially, the intracellular degradation of AEA by FAAH was thought to drive AEA uptake, but several studies reported that specific transporters mediate bidirectional transport of this eCB and that a carrier protein can shuttle AEA to intracellular targets (Fowler 2012; Fu et al. 2012). Recently, it was demonstrated that a common eCB membrane transporter controls the cellular AEA and 2-AG trafficking in both directions (i.e. release and uptake) and metabolism (Chicca et al. 2012). The authors indicate that this eCB membrane transporter could play an important role in various aspects of eCB signalling, since eCBs bind to several intracellular targets such as PPARs and intracellular cannabinoid receptors, as well as to extracellular targets. Despite the fact that to date no intracellular carrier protein has been described for 2-AG, the existence of such a protein is an intriguing possibility which could help explain our findings regarding the delay of the CB2R-mediated response. Opening and function of CaCCs Interest in Cl- channels in general has been driven by the finding that multiple human diseases are Cl- channelopathies, like cystic fibrosis (Duran et al. 2010). However, neuronal Cl- channels have received relatively little attention from researchers, compared to cation channels. In immature neurons with high [Cl-]i, GABA produces depolarizing postsynaptic potentials via Cl--conducting GABAA receptors, which could play a role in stabilizing developing synapses (Ben-Ari et al. 2007). In mature neurons, [Cl-]i is much lower, so that Cl- ions are in electrochemical equilibrium across membranes. In these mature neurons, the reversal potential of IPSCs mediated by GABAA receptors, is very close to the resting membrane potential. Maintaining appropriate [Cl-]i following Cl- influx when GABAA receptors are activated is typically attributed to several cation Cl- cotransporters, most notably the K+-Cl- cotransporter (Blaesse et al. 2009). The dramatic changes in [Cl-]i can occur slowly during the development from immature to mature neurons. More acute changes in [Cl-]i may occur in response to (sustained) synaptic activity due to the accumulation of intracellular Cl- and, following that, the local collapse of the Cl- gradient (Kuner and Augustine 2000; Isomura et al. 2003; Berglund et al. 2006).

In chapter three, we have described the opening of CaCCs following the activation of CB2Rs with synthetic cannabinoids. We reported that the opening of these channels is dependent on a rise in [Ca2+]i, mediated by IP3Rs (Fig. 1). It is known that CaCCs can be activated by Ca2+ influx through VDCCs, ligand-operated Ca2+ channels, as well as Ca2+ release from intracellular stores (Frings et al. 2000). Alternatively, some CaCCs may not possess a Ca2+ binding site, but instead are activated by Ca2+/calmodulin-dependent protein kinase II (CaMK II) (Hartzell et al. 2005). In some neurons, CaCCs can be localized in the dendritic membrane, so that their contribution to the processing of synaptic input depends on local Cl- concentrations (Frings et al. 2000). Their activation can amplify or attenuate the cellular response to such input. Recent years have seen important developments in the field of CaCCs (Flores et al. 2009; Hartzell et al. 2009) with the almost simultaneous publications of three papers that reported the description of the transmembrane protein Anoctamin 1 (also known

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as TMEM16A) as a CaCC (Caputo et al. 2008; Schroeder et al. 2008; Yang et al. 2008). At least one other member of the Anoctamin family, Anoctamin 2, generates Ca2+-activated Cl- currents. Aside from Anoctamins, other, more controversial transmembrane proteins could also function as CaCCs (Sun et al. 2002). Reports on the molecular identity of CaCC proteins could help us to understand details of the functioning of these channels. Some important questions concern the exact localization of these channels as well as their sensitivity to [Ca2+]i and CaMK II. In particular, future knowledge on the molecular identity of CaCCs should help with the development of specific antibodies, which in turn could provide us with information on the subcellular localization of these channels.

As described in chapters 3 and 4, CB2R activation under conditions with high [Cl-]i resulted in transient, but relatively long-lasting (several min) depolarizations. In experiments in chapters 3 and 4 we examined CB2R-mediated modulation of neuronal excitability with a physiologically relevant [Cl-]i, where the reversal potential for Cl- was close to the resting membrane potential (-70 mV). Under these experimental conditions, which were designed to more closely mimic the physiological situation for mature neurons, the opening of CaCCs did not (or hardly) mediate a Cl- current. Under these conditions, the opening of CaCCs reduced the input resistance of the cell in a process which is reminiscent of GABAA receptor-mediated shunting inhibition. This could be the process through which CB2R-activation affects neuronal excitability. If CB2Rs are functionally expressed by immature neurons, the activation of these receptors should result in the depolarization of the cell. It would be interesting to investigate whether such CB2R-mediated responses can be detected in immature neurons.

Figure 1. Schematic overview of the proposed eCB signalling pathway. Ca2+-entry through VDCCs induces eCB synthesis. eCBs then bind to intracellular Gi/o-coupled CB2Rs which results, via the activation of PLC, into IP3 production. IP3 activates IP3Rs and this induces release of Ca2+ from intracellular Ca2+ stores, which in turn leads to the opening of CaCCs.

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CB1R-mediated modulation of neurotransmission There is broad consensus regarding the notion that CB1Rs mediate the vast majority of their modulatory effects in the CNS from a presynaptic locus (Chapter 1, Fig. 1). The initial discovery of eCB-mediated retrograde signalling via the activation of CB1Rs was followed by many descriptions of this mechanism in different brain areas (Kano et al. 2009; Castillo et al. 2012). However, non-retrograde, autocrine eCB signalling via CB1Rs has also been reported (Bacci et al. 2004). In cortical interneurons and layer II/III pyramidal neurons, repetitive AP firing can trigger a CB1R-dependent postsynaptic hyperpolarization via the opening of GIRK channels (Bacci et al. 2004; Marinelli et al. 2008, 2009). This process was termed somatodendritic slow self-inhibition (SSI) and provides an alternative route via which CB1Rs can influence neuronal communication.

In chapter five, we demonstrated the presynaptic localization of CB1Rs by means of electrophysiology and immunohistochemistry. We reported a reduction of the frequency of both mEPSCs and mIPSCs following CB1R activation. These findings are consistent with a presynaptic localization for CB1Rs and were confirmed by the immunohistochemical visualization of CB1Rs on axonal fibers. Importantly, both the observed CB1R-mediated reduction in the frequency of mEPSCs and mIPSCs and our immunohistochemical data are in line with results from earlier studies performed in the PFC, which reported modulation of inhibitory and excitatory input (Lafourcade et al. 2007; Yoshino et al. 2011).

In chapter five, we performed CB1R internalization experiments and with immunohistochemistry we showed that most of the CB1R-positive neurons did not express the interneuron marker GAD65/67. Although it is likely that some GABAergic neurons did not display a detectable amount of GAD65/67, we assume that most of the CB1R-positive neurons that were GAD65/67 negative were non-GABAergic, principal neuron. Most investigations into the localization of CB1Rs report their predominant presence on inhibitory interneurons, in particular the subtypes of GABAergic interneurons that contain the neuropeptide cholecystokinin (CCK) or the Ca2+-binding protein calbindin (CaBP) (Katona et al. 1999; Bodor et al. 2005; Eggan et al. 2010; Thibault et al. 2012). However, CB1Rs have also been detected in the rodent brain at excitatory synapses where they were shown to modulate the release of glutamate (Auclair et al. 2000; Katona et al. 2006; Kawamura et al. 2006). It has been reported that the relative CB1R expression in interneurons and principal neurons may depend on the developmental stage of the CNS (Vitalis et al. 2008). In a recent paper, authors describe that, in a mouse model of Huntington’s disease, the functionality of CB1Rs in GABAergic and glutamatergic neurons is differently affected (Chiodi et al. 2012). As a result, the net effect of CB1R activation is profoundly altered in these animals, compared to healthy wild type mice. This could indicate an association between appropriate functioning of CB1Rs in both neuron types and proper CNS functioning.

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CB1R activation modulates the E/I balance Although inhibitory interneurons constitute only ~20% of the neocortical neuronal population, they are heavily connected and are able to match and balance excitatory input conductance derived from the remaining ~80% excitatory neurons (Peters and Kara 1985; Somogyi et al. 1998; Markram et al. 2004). In fact, the relative contribution of excitatory and inhibitory input conductance is dynamically maintained at ~20% excitation and ~80% inhibition (Le Roux et al. 2006, 2007, 2008; Zhang et al. 2011). As discussed in chapter five, we found that CB1R activation in the mPFC reduced both inhibitory and excitatory spontaneous neurotransmission onto layer II/III pyramidal cells by ~30%. This means that, despite our predominant detection of CB1Rs on non-GABAergic cells, CB1R activation affects spontaneous inhibitory and excitatory neurotransmission similarly. This could be explained by the multitude of inhibitory synaptic contacts made by only a small amount of inhibitory interneurons.

When we investigated the E/I balance in layer II/III pyramidal neurons of the mPFC we found that this balance was very close to previously reported values at ~20% excitation and ~80% inhibition (Le Roux et al. 2006, 2007, 2008). The activation of CB1Rs with a synthetic agonist resulted in the reduction of both inhibitory and excitatory conductances. However, the relatively larger reduction in inhibitory conductance meant a shift in the E/I balance towards excitation. If the E/I balance is shifted, one would expect that, overall, neurons are exposed to more excitatory input than normally, which increases the chance of firing APs. In this way, a changed E/I balance could have profound physiological consequences. Deviations from the ‘normal’ E/I balance (~20/~80%) are associated with a range of neuropsychiatric disorders, such as epilepsy, schizophrenia and autism spectrum disorders (Cobos et al. 2005; Lewis et al. 2005; Rubenstein 2010). A causal relationship between an increased E/I balance in the mPFC and behavioural deficits and social dysfunction was uncovered in a recent study (Yizhar et al. 2011). These findings and our data described in chapter five suggest the possibility that CB1R activation following cannabis intake could alter the E/I balance which in turn could result in behavioural deficits. The effects of cannabis intake on executive functioning are thought to be mediated predominantly by CB1Rs and the involvement of this receptor in the modulation of the E/I balance forms an interesting potential mechanism to explain neuropsychological deficits associated with cannabis use (Ledent et al. 1999; Huestis et al. 2001; Grant et al. 2012). In addition to overactivation of CB1Rs by exogenous cannabinoids, physiological activation of CB1Rs (either by induced eCB release or the basal eCB tone) could play a role in the maintenance of the proper E/I balance. CB2R and CB1R interplay The findings represented in chapters three and four show the presence and functionality of intracellular CB2Rs in pyramidal neurons. CB2R activation leads to the

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opening of CaCCs and the reduction of neuronal excitability in the mPFC. The findings in chapter five demonstrate that CB1R activation modulates both excitatory and inhibitory neurotransmission and changes the E/I balance in favour of excitation in the same brain region. Taken together, these studies show the diverse mechanisms by which the eCB system can play a role in the modulation of neuronal communication. The intracellular presence of CB2Rs and the CB2R-mediated effects described in chapters thee and four have not been reported earlier and discredit the general assumption to exclude these receptors from investigations into the CNS eCB system.

Since similar stimulation protocols can induce CB1R and CB2R activation, it is a distinct possibility that both cannabinoid receptors may be activated following certain patterns of neuronal activity, although perhaps at a different time scale. The activation of presynaptic CB1Rs is reported to immediately follow the induced synthesis of eCBs in short-lasting (~1 min) phenomena such as DSI and DSE (Kreitzer and Regehr 2001; Wilson et al. 2001; Fortin et al. 2004; Yoshino et al. 2011). In chapter four, we have demonstrated that, under our experimental conditions, a CB2R-mediated Cl- current could be recorded only after a (highly variable) delay of ~2.5 min. As previously discussed, the delay we reported could be an overestimation of the delay in a more physiological situation, due to our experimental conditions. Nonetheless, there seems to be a marked difference between the timescale at which CB1Rs and CB2Rs are activated following induced eCB synthesis. This means that the CB1R-mediated reduction of the release of glutamate at an excitatory synapse will precede the transiently reduced excitability of the postsynaptic principal neuron through CB2Rs, in the case of eCB synthesis in this postsynaptic neuron. This hypothetical principal neuron would initially experience a reduced activation of its glutamate receptors, followed by a reduction in excitability due to the opening of CaCCs. In the case of longer lasting CB1R-mediated effects, such as eCB-LTD, CB1R-mediated effects and CB2R-mediated effects may exist simultaneously. This would mean that the neuron might receive reduced synaptic input and that it is less sensitive to synaptic input at the same time. In the mPFC, pharmacological activation of CB1Rs leads to a decrease in both excitatory and inhibitory input conductance, summing up to the net effect of a relative decrease in inhibitory input conductance. If CB2Rs are activated simultaneously, the relative increase in excitatory input conductance could be balanced by the CB2R-mediated opening of CaCCs, which leads to a reduction of neuronal excitability. Taking into account the CB1R and CB2R, the eCB system is endowed with powerful means to influence both synaptic input (on a short and long time-scale) and the neuronal sensitivity to synaptic input (on a long time-scale).

Complicating the situation further, a recent article reports that CB1Rs and CB2Rs can form plasma membrane heteromers in transfected neuronal cells and tissue from subcortical brain regions (Callén et al. 2012). The authors show that heteromer formation led to bidirectional cross antagonism (i.e. the ability of a CB1R antagonist to

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block the effect of a CB2R agonist and the ability of a CB2R antagonist to block the effect of a CB1R agonist). The authors suggest that these heteromers could be the basis for several contradicting and controversial reports regarding the expression levels of CB1Rs and CB2Rs and their activation by different levels of ligands. More research is required to elucidate the role of plasma membrane CB1R-CB2R heteromers in the eCB system in the CNS.

Taken together, results from the data presented in this thesis show that CB1Rs and CB2Rs are in a powerful position to modulate neurotransmission, the balance between inhibitory and excitatory input conductance and neuronal excitability. CB1Rs are abundantly expressed in the CNS and most of our knowledge on the eCB system in the brain derives from investigations into CB1R-functioning. Recent developments have highlighted the importance of investigations into CB2R-functioning (Elmes et al. 2004; Jhaveri et al. 2008; Morgan et al. 2009; Xi et al. 2011). The results described in chapters three and four of this thesis are relevant additions to this work and strengthen the importance of exploring the role CB2R in the CNS. Concluding remarks and future directions In this dissertation I have investigated how the eCB system can exert its effects through the two GPCRs currently classified as cannabinoid receptors, in layer II/III pyramidal neurons of the mPFC. The work presented in chapters three and four represent the first descriptions of neuronal CB2Rs as intracellular receptors that, upon activation, can induce a Cl- current. The surprising localization of these receptors brings to mind various questions, some of which have been discussed earlier. One of the most obvious questions concerns the exact localization of intracellular CB2Rs. Since reliable antibodies against CB2Rs for immunohistochemistry are currently unavailable, experiments with fluorescent CB2R ligands could be performed in order to answer this question. In fact, such a fluorescent ligand exists and it has been used to evaluate CB2R binding in high throughput screening in cultured cell lines expressing CB2Rs (Sexton et al. 2011). However, ligands that may be used in slice experiments are still to be discovered.

Another question regarding the localization and downstream signalling pathway of CB2Rs concerns the expression of these receptors by inhibitory interneurons. Since cortical interneurons can synthesize eCBs (Bacci et al. 2004), it would be very useful to investigate whether interneurons show a delayed reduction in excitability due to the opening of Cl- channels, mediated by CB2Rs. Answering the question whether CB2Rs are exclusively expressed by pyramidal neurons or also by inhibitory interneurons would provide valuable information for understanding the role of CB2Rs at the network level. The question of CNS-wide generalizability of the described CB2R-mediated effects is also an important issue. It would be very interesting to perform additional studies to

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determine the presence, as well as the function of CB2Rs in principal neurons in other layers of the cortex and other brain areas.

We have reported, in chapter five, that CB1R activation resulted in the shift of the E/I balance in favour of excitation. These findings raise the question whether CB1R activation by Δ9-THC in cannabis can cause a similar shift in this balance. Future studies into the change of the E/I balance by modulation of the eCB system could increase our understanding of the effects of cannabis use on the brain.

It is known that both cannabinoid receptors play a role during neurodevelopment. In light of the findings presented in chapter five, it would be very interesting to investigate the involvement of the eCB system in the proper wiring of the mPFC network. Such experiments could be of particular importance for evaluating of the risk of cannabis use in pregnant mothers.

The eCB system represents possible therapeutic targets for a wide range of pathologies including neurodegenerative disorders (such as multiple sclerosis and Parkinson’s disease), neuropsychiatric disorders (such as depression and schizophrenia), epilepsy, ischemia, neuropathic and inflammatory pain, autoimmune and cardiovascular and gastrointestinal diseases (Vinod and Hungund 2006; Bisogno and Di Marzo 2007; Di Marzo 2009; Fernández-Ruiz et al. 2010; Pacher and Mechoulam 2011; Skaper and Di Marzo 2012). Within the eCB system, the CB2R could be of particular interest as therapeutic target, since the psychoactive effects of cannabis are believed to be mediated exclusively by CB1Rs. However, it must be noted that, since the eCB system is abundantly present in the CNS and the periphery, the risk of side effects of cannabinoid compounds is large. This is illustrated by the development of the selective CB1R antagonist/inverse agonist rimonabant, which was used as an anti-obesity drug. Rimonabant was redrawn from the market in 2009 due to reports of increased risk of psychiatric events such as depressed mood disorders, anxiety and an increased risk of suicide (Christensen et al. 2007). Clearly, knowledge of the basic properties of the eCB system is essential for the development of new eCB system-modulating therapeutic drugs which display less side effects.

Addendum

References

Nederlandse samenvatting

List of publications

Curriculum Vitae

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Het lichaamseigen cannabis-systeem, oftewel het endocannabinoïde systeem, is vernoemd naar de cannabis plant die een belangrijke rol heeft gespeeld bij de ontdekking van dit neurotransmitter systeem. Werkzame bestanddelen uit cannabis, zoals Δ9-tetrahydrocanabinol (Δ9-THC), veroorzaken hun psychoactieve effecten door een interactie aan te gaan met het endocannabinoïde systeem in het centrale zenuwstelsel. Het endocannabinoïde systeem bestaat uit cannabinoïde receptoren en endogene cannabinoïden, zogenaamde endocannabinoïden, die op deze receptoren aangrijpen. De twee belangrijkste endocannabinoïden zijn 2-arachidonoylglycerol (2-AG) en anandamide (AEA). Daarnaast zijn verschillende enzymen en eiwitten beschreven die een rol spelen bij de aanmaak, afbraak en transport van endocannabinoïden. Het endocannabinoïde systeem is aangetroffen in zoogdieren, vissen, zee-egels en weekdieren en vanuit evolutionair perspectief gezien vele miljoenen jaren oud.

Het endocannabinoïde systeem is van groot belang voor diverse fysiologische systemen in het lichaam waaronder pijn, honger en voortplanting. Bovendien is het systeem betrokken bij cognitieve functies zoals beloning, aandacht, geheugen en het remmen van impulsief gedrag. Deze cognitieve functies worden veelal geassocieerd met een specifiek gedeelte van de cerebrale cortex, de prefrontale cortex. Dit hersengebied is in primaten bijzonder uitgebreid ontwikkeld en vormt dankzij de geassocieerde functies een interessant focus voor onderzoek naar verslaving. De verschillende componenten van het endocannabinoïde systeem komen bijna in het hele centrale zenuwstelsel voor en zijn aanwezig in de prefrontale cortex. Het endocannabinoïde systeem is ook direct geassocieerd met verslaving, omdat de belonende effecten van verslavende middelen als opioïden, alcohol en nicotine in andere hersengebieden gemedieerd kunnen worden door dit neurotransmitter systeem. Daarnaast speelt het endocannabinoïde systeem een belangrijke rol bij de hunkering en terugval naar gebruik van dergelijke verslavende middelen in proefdieren. Over de mechanismen die hieraan ten grondslag liggen is nog relatief weinig bekend. Het doel van het onderzoek beschreven in dit proefschrift is het in kaart brengen van de functionaliteit van het endocannabinoïde systeem in de prefrontale cortex. Hierbij heeft vooral het functioneren van de cannabinoïde receptoren in dit hersengebied centraal gestaan. Cannabinoïde receptoren komen voor in twee variaties, type-1 cannabinoïde receptoren (CB1R) en type-2 cannabinoïde receptoren (CB2R). Beide type receptoren behoren tot de zogenaamde G proteïnen gekoppelde receptoren (GPCR). CB1R zijn in grote aantallen aanwezig in het centrale zenuwstelsel en zijn in neuronen voornamelijk presynaptisch in de plasmamembraan gelokaliseerd. Vanwege deze lokalisering


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vervullen ze vooral een rol bij de modulatie van neurotransmitterafgifte (zie hieronder). Van CB2R wordt gedacht dat ze zich met name in immuuncellen bevinden, zowel in perifere weefsels als in het centrale zenuwstelsel. Recentelijk zijn er verschillende publicaties verschenen waarin effecten van in neuronen gelokaliseerde CB2R beschreven werden. Over de aanwezigheid en de rol van CB2R in neuronale cellen wordt, ondanks deze ontwikkelingen en in tegenstelling tot CB1R, nog gedebatteerd door wetenschappers. Activatie van CB1R door endocannabinoïden, of door exogene cannabinoïde stoffen zoals Δ9-THC, leidt tot een verminderde afgifte van neurotransmitters. De natuurlijke aanmaak van endocannabinoïden wordt gestimuleerd door (over)activatie van neuronale cellen. Endocannabinoïden zijn lipide-achtige stoffen en hun (calcium-afhankelijke) synthese vindt plaats in de plasmamembraan van cellen waarna ze diffunderen naar het extracellulaire en intracellulaire compartiment. Vervolgens kunnen ze binden aan presynaptische CB1R en intracellulair gelokaliseerde receptoren van een andere klasse die ook gevoelig zijn voor endocannabinoïden. Gezien de presynaptische lokalisatie van CB1R, kunnen de effecten van CB1R activatie goed gemeten worden in het postsynaptische neuron, aan de hand van elementaire synaptische responsies (miniature EPSCs/IPSCs). Afhankelijk van het type gemoduleerde neurotransmitter, inhibitoir (bv. GABA) of excitatoir (bv. glutamaat), leidt deze verminderde synaptische activiteit tot respectievelijk een toename of een afname van de (elektrische) activiteit van het postsynaptische neuron. Het is reeds bekend dat CB1R verschillende vormen van synaptische plasticiteit mediëren, waardoor de sterkte van synaptische verbindingen voor korte of langere tijd kan veranderen. Voor CB2R was het tot nog toe onduidelijk hoe ze neuronale activiteit kunnen beïnvloeden. Het onderzoek dat beschreven is in dit proefschrift heeft zich gericht op de functie van beide cannabinoïde receptoren in de mediale prefrontale cortex (mPFC). De mPFC vormt in knaagdieren zoals ratten en muizen een belangrijk onderdeel van de prefrontale cortex. We hebben elektrofysiologische technieken en verschillende biochemische methoden toegepast om dit onderzoek uit te voeren, waarbij we – naast wild type muizen en ratten – ook genetisch gemodificeerde muizen (waarin CB2R ontbreken) gebruikt hebben. Bijna alle experimenten werden in vitro uitgevoerd, waarbij mPFC hersenplakjes werden gemaakt om m.b.v. elektrofysiologische meettechnieken de elektrische activiteit van onderdelen van het mPFC netwerk of die van individuele mPFC neuronen te registreren. In hoofdstuk 2 hebben we met behulp van veldpotentiaalmetingen onderzocht hoe het activeren van cannabinoïde receptoren netwerkactiviteit in hersenplakjes van de mPFC beïnvloedt. Veldpotentialen worden extracellulair gemeten en representeren de synchrone elektrische activiteit van grote groepen neuronen. In de mPFC kunnen veldpotentialen worden opgewekt door bepaalde onderdelen van het netwerk te


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activeren middels een stroominjectie via een stimulatie-elektrode. In de experimenten beschreven in dit hoofdstuk betrof het veldpotentialen gemeten in laag II/III van de cortex die waren opgewekt door laag V te stimuleren. Het toedienen van de synthetische cannabinoïde agonist WIN55212-2 (WIN), die zowel CB1R als CB2R kan activeren, leidde tot een aanzienlijke reductie van de netwerkactiviteit. De amplitude van de (excitatoire) synaptische component van het veldpotentialensignaal was sterk gereduceerd na behandeling met WIN, wat duidt op een afname van (glutamaterge) neurotransmissie. Dit zou verklaard kunnen worden door activatie van presynaptische CB1R van (glutamaterge) eindigingen die de laag II/III piramidale neuronen innerveren. Dubbelpuls experimenten wezen erop dat cannabinoïde receptor activatie in de mPFC ook inhibitoire neurotransmissieprocessen weet te reduceren. Experimenten met andere synthetische cannabinoïde agonisten die selectief CB1R of CB2R activeren, gaven aan dat de activatie van beide receptoren de netwerkactiviteit vermindert. De veldpotentiaalexperimenten toonden aan dat cannabinoïde receptor activatie van invloed is op de mPFC netwerkactiviteit. Echter, de mechanismen die hieraan ten grondslag liggen zijn lastig vast te stellen met deze methode. Om dergelijke mechanismen te onderzoeken kan beter gebruik worden gemaakt van cellulaire meettechnieken, waarbij afleidingen worden gedaan van individuele neuronen. Deze technieken hebben we toegepast in de volgende hoofdstukken. Resultaten van experimenten in hoofdstuk 2 suggereerden dat CB2R bijdragen aan de responsies opgewekt in de mPFC. Daarom hebben we in hoofdstuk 3 de aanwezigheid en functie van CB2R in meer detail onderzocht. In dit hoofdstuk hebben we met biochemische methoden aangetoond dat CB2R intracellulair aanwezig zijn in de mPFC. Dit is een opmerkelijke bevinding, aangezien GPCR meestal gelokaliseerd zijn in de plasmamembraan. Vooralsnog hebben we niet kunnen vaststellen wat precies de intracellulaire locatie van CB2R is. Vervolgens hebben we met de “whole-cell patch clamp” techniek elektrische registraties gemaakt van individuele laag II/III piramidale neuronen. Deze excitatoire neuronen zijn belangrijke spelers in het mPFC netwerk. Onze experimenten lieten zien dat activatie van CB2R met een synthetische, selectieve agonist enige tijd na toediening (enkele minuten) resulteerde in het tijdelijke (enkele minuten) openen van calciumafhankelijke chloridekanalen. Deze ionkanalen worden aangetroffen in verschillende cellen, zowel in de periferie als in het centrale zenuwstelsel, maar hun aanwezigheid in piramidale neuronen in de mPFC was niet eerder beschreven. Verdere experimenten lieten zien dat de activatie van CB2R de prikkelbaarheid van laag II/III neuronen verminderde. Het vermoedelijke onderliggende mechanisme is het openen van chloridekanalen, waardoor negatief geladen chloride-ionen de cel in kunnen stromen en de cel minder makkelijk de voltagedrempel voor het vuren van actiepotentialen kan bereiken. We hebben in hoofdstuk 3 niet alleen gedemonstreerd dat CB2R intracellulair aanwezig zijn in de mPFC, maar ook dat hun activatie gevolgen heeft voor het vuurgedrag van piramidale neuronen in laag II/III.


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Naar aanleiding van de resultaten in hoofdstuk 3 waarbij we gebruik maakten van synthetische CB2R liganden, hebben we in hoofdstuk 4 de effecten van CB2R activatie door endocannabinoïden onderzocht. Uit deze experimenten bleek dat de activatie van CB2R door de endocannabinoïde 2-AG tot een grotere chloridestroom leidde dan activatie door methanandamide (een stof die lijkt op anandamide), de andere belangrijke endocannabinoïde. Vervolgens hebben we onderzocht of CB2R geactiveerd kunnen worden door het stimuleren van de endogene aanmaak van endocannabinoïden. Hiervoor gebruikten we een stimulatiepatroon, waarvan we weten (gebaseerd op gegevens uit de literatuur over CB1R activatie) dat het geschikt is voor het aanzetten tot de aanmaak van endocannabinoïden in de mPFC van de muis. Dit stimulatiepatroon bestond uit het opwekken van een reeks van 60 actiepotentialen met een frequentie van 20 Hz. Uit onze experimenten bleek dat dergelijke stimulaties van laag II/III piramidale neuronen leidde tot het openen van de calciumafhankelijke chloridekanalen, via de activatie van CB2R. Vergelijkbare resultaten werden behaald als de neuronen tot 20 Hz vuren werd aangezet via synaptische activatie (door stimulatie van de aanvoerende zenuwbaan in laag I). Toen we de influx van calcium tijdens dit proces nader onderzochten, bleek dat N-type spanningsafhankelijke calciumkanalen hierbij een belangrijke rol speelden. Tot slot voerden we experimenten uit waarbij we vonden dat dergelijke CB2R activatie de prikkelbaarheid van piramidale neuronen verminderde, vergelijkbaar met de activatie van CB2R met een selectieve agonist in hoofdstuk 3. De in hoofdstuk 4 beschreven data laat zien dat CB2R in de mPFC geactiveerd kunnen worden door endocannabinoïden die worden aangemaakt volgend op fysiologisch relevant vuurgedrag van laag II/III piramidale neuronen. De activatie van CB2R vermindert de prikkelbaarheid van de neuronen. We vermoeden dat CB2R een rol spelen in een feedback mechanisme dat de neuronale activiteit in toom kan houden. Klaarblijkelijk gebeurt dit op een tijdschaal (na enkele minuten) die een stuk langer is dan die van CB1R-gemedieerde effecten op neurotransmitterprocessen (binnen enkele secondes). In hoofdstuk 5 hebben we ons gericht op de rol van CB1R met betrekking tot synaptische transmissie en de balans tussen excitatoire en inhibitoire conductanties in de mPFC. In dit hoofdstuk hebben we laten zien dat CB1R activatie resulteerde in een verminderde afgifte van zowel excitatoire als inhibitoire neurotransmitters. Neurotransmitters als glutamaat en GABA liggen presynaptisch opgeslagen in blaasjes waarvan de inhoud wordt afgegeven als de presynaptische eindiging (elektrisch) actief wordt. De neurotransmitters binden vervolgens aan de postsynaptische receptoren met als gevolg een toename (i.g.v. glutamaat) of een afname (i.g.v. GABA) van de prikkelbaarheid van het postsynaptische neuron. Onze experimenten toonden aan dat CB1R activatie de frequentie van spontane afgifte van neurotransmitterblaasjes reduceerde. Echter, de amplitude van de corresponderende stroompjes in de postsynaptische cel veranderden niet, wat aangeeft dat de inhoud van het blaasje niet


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beïnvloed werd. Deze resultaten ondersteunen een presynaptische lokalisatie van CB1R. Om de presynaptische lokalisatie van CB1R verder te bevestigen, hebben we de expressie van CB1R met immunohistochemische methoden onderzocht. Deze kleuringen kwamen niet alleen overeen met een presynaptische lokalisatie van de receptoren, maar toonden ook aan dat zowel inhibitoire als excitatoire cellen CB1R tot expressie brengen. Vervolgens hebben we voor elektrofysiologie-experimenten gebruik gemaakt van een decompositie methode die ons in staat stelde de totale gemeten synaptische conductantie, in respons op synaptische stimulatie, op te delen in een excitatoire conductantie en een inhibitoire conductantie. Uit de literatuur is bekend het percentage excitatoire en inhibitie conductantie in verschillende corticale gebieden ongeveer 20% en 80%, respectievelijk, bedraagt. Deze verhouding vonden we terug in de laag II/III neuronen van de mPFC. Onze experimenten toonden aan dat activatie van CB1R met WIN leidde tot een afname van alle conductanties. Na toediening van WIN nam de inhibitoire conductantie relatief meer af dan de excitatoire conductantie, waardoor een verschuiving in de balans tussen excitatie en inhibitie optrad ten gunste van excitatie. We vonden dat na CB1R activatie de verhouding van excitatoire en inhibitoire conductantie was veranderd in respectievelijk 25% en 75%. Tot slot hebben we laten zien dat wanneer CB1R in vivo werden geactiveerd (d.m.v. een injectie met een cannabinoïde receptor agonist in de levende rat), een nog grotere verandering in de balans (gemeten in vitro) in dezelfde richting kon worden gemeten. Bij elkaar genomen geven de resultaten van hoofdstuk 5 aan dat CB1R activatie vanaf een presynaptische lokalisatie neurotransmitterafgifte beïnvloedt en dat de activatie van deze receptoren de balans tussen excitatie en inhibitie in de richting van excitatie verschuift. De modulatie van de balans tussen excitatie en inhibitie door CB1R is mogelijk een belangrijk mechanisme voor de regulatie van netwerkactiviteit in de mPFC, een hersengebied wat betrokken is bij hogere cognitieve functies. Verstoring van het endocannabinoïde systeem, voornamelijk tijdens adolescentie wanneer de PFC tot rijping komt, kan blijvende gevolgen hebben voor het gezonde functioneren van de hersenen. Wellicht is de modulatie van de balans tussen excitatie en inhibitie (een van de) onderliggende mechanismen. Het in dit proefschrift beschreven onderzoek heeft aangetoond dat het endocannabinoïde systeem via zowel CB1R als CB2R invloed uitoefent op neuronale activiteit in de mPFC, waarbij de modulatie door CB2R het nieuwste aspect vertegenwoordigt. We hebben als eersten laten zien dat CB2R, vanaf een intracellulaire lokalisatie, betrokken kunnen zijn bij een potentieel belangrijk feedbacksysteem om de neuronale activiteit binnen de perken te houden. Dit feedbacksysteem kan, na een voor hersenprocessen relatief lange tijd (minuten), overmatige neuronale activiteit remmen doordat cellen tijdelijk minder prikkelbaar worden. Ook hebben we aangetoond dat naast de verwachte effecten van CB1R activatie op de neurotransmitterafgifte, de activatie van deze receptoren de mPFC in vitro netwerkactiviteit – via een verschuiving


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in de balans tussen excitatie en inhibitie – aanzienlijk verandert. Hiermee is het duidelijk geworden dat het endocannabinoïde systeem via CB1R en CB2R beschikt over twee zeer verschillende mechanismen om de neuronale activiteit te moduleren. Toekomstig onderzoek zal duidelijk kunnen maken of het endocannabinoïde systeem in andere hersengebieden op vergelijkbare manieren hersenactiviteit kan moduleren en onder welke omstandigheden dit systeem actief is/wordt. Het endocannabinoïde systeem vormt een interessant aangrijppunt voor de ontwikkeling van nieuwe medicijnen voor verslaving en andere hersenaandoeningen. Hierbij is een beter begrip van de functionaliteit van het endocannabinoïde systeem cruciaal. Tot voorheen werd gedacht dat alleen CB1R aanwezig zijn in neuronen, maar het onderzoek beschreven in dit proefschrift heeft aangetoond dat CB2R in laag II/III piramidale neuronen intracellulair aanwezig en functioneel zijn. De psychoactieve effecten van exogene cannabinoïde liganden verlopen voornamelijk door interacties met CB1R. Hierdoor worden (nieuw te ontwikkelen) selectieve CB2R-liganden beschouwd als potentiële geneesmiddelen met relatief weinig psychoactieve bijwerkingen. Dankzij de resultaten beschreven in dit proefschrift hebben we meer inzicht gekregen in het functioneren van het endocannabinoïde systeem in de mPFC en is duidelijk geworden dat bij de ontwikkeling van nieuwe therapeutische stoffen die het cannabinoïde systeem moduleren met beide cannabinoïde receptoren rekening gehouden dient te worden.


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Effects of amisulpride and aripiprazole on progressive-ratio schedule performance: comparison with clozapine and haloperidol. den Boon FS, Body S, Hampson CL, Bradshaw CM, Szabadi E, de Bruin N. Journal of psychopharmacology 2012, 26(9): 1231-43. Excitability of prefrontal cortical pyramidal neurons is modulated by activation of intracellular type-2 cannabinoid receptors. den Boon FS, Chameau P, Schaafsma-Zhao Q, van Aken W, Bari M, Oddi S, Kruse CG, Maccarrone M, Wadman WJ, Werkman TR. PNAS 2012, 109(9): 3534-9. Novel indole and azaindole (pyrrolopyridine) cannabinoid (CB) receptor agonists: design, synthesis, structure-activity relationships, physicochemical properties and biological activity. Blaazer AR, Lange JH, van der Neut MA, Mulder A, den Boon FS, Werkman TR, Kruse CG, Wadman WJ. European journal of medicinal chemistry 2011, 46(10): 5086-98. Comparison of the effects of 2,5-dimethoxy-4-iodoamphetamine and D-amphetamine on the ability of rats to discriminate the durations and intensities of light stimuli. Hampson CL, Body S, den Boon FS, Cheung TH, Bezzina G, Langley RW, Fone KC, Bradshaw CH, Szabadi E. Behavioural pharmacology 2010, 21(1): 11-20.

Attenuation of the effects of d-amphetamine on interval timing behavior by central 5-hydroxytryptamine depletion. Body S, Cheung TH, Hampson CL, den Boon FS, Bezzina G, Fone KC, Bradshaw CM, Szabadi E. Psychopharmacology 2009, 203(3): 547-59.

Endocannabinoids produced upon action potential firing evoke a Cl- current via type-2 cannabinoid receptors in the medial prefrontal cortex. den Boon FS, Chameau P, Houthuijs K, Mastrangelo N, Kruse CG, Maccarrone M, Wadman WJ, Werkman TR. submitted Modulation of the balance between excitation and inhibition in the rat medial prefrontal cortex by type-1 cannabinoid receptors. den Boon FS, Werkman TR, Schaafsma-Zhao Q, Houthuijs K, Vitalis T, Kruse CG, Wadman WJ, Chameau P. submitted

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Curriculum Vitae Femke Susanne den Boon was born on November 26, 1983 in Amsterdam, the Netherlands. After finishing her secondary school education at the Vossius Gymnasium in Amsterdam, she started her bachelor’s degree Psychobiology at the University of Amsterdam. She continued her education with a master’s degree Neurobiology (with distinction) at the same university. During this master’s degree she carried out two research projects. Her first research project was performed at the Department of Pharmacology and Pharmacotherapy of the Amsterdam Medical Centre under the supervision of Dr. Astrid Alewijnse and Prof. Martin Michel. Here, she focused on the signalling cascade following sphingolipid receptor activation. For her second research project, she spent just over a year at the Psychopharmacology Section of the University of Nottingham, United Kingdom. Under the supervision of Prof. Chris Bradshaw she investigated the effects of antipsychotics on rat behaviour. In July 2008 she started her PhD project in the group of Prof. Wytse Wadman at the Swammerdam Institute for Life Sciences at the University of Amsterdam. She focused on the function of endocannabinoid receptors in the rodent medial prefrontal cortex and the results of this research are presented in this thesis.

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Dankwoord

En dan nu het dankwoord; het allerlaatste schrijfwerk, maar wel altijd het meest gelezen stuk van een proefschrift! Hoewel er soms geen einde aan leek te komen, gingen die (ruim) vier jaar toch ineens een stuk harder dan ik dacht. Voor je het weet ben je je dankwoord aan het schrijven! Mijn promotieonderzoek heeft me een heleboel plezier en leuke ervaringen opgeleverd, maar uiteraard ook stress en moeilijke momenten. Hieronder een lijst mensen die ik wil bedanken voor hulp bij het tot stand komen van dit boekje.

Ik wil ten eerste mijn promotor Wytse Wadman bedanken. Tijdens mijn master heb ik dankzij jou een stageplek in Engeland gekregen, waar ik voor Solvay (onderhand Abbott) aan de slag kon bij de universiteit van Nottingham. Daarna kon ik meteen in jouw groep beginnen met mijn eigen promotie-onderzoek. Ik heb de laatste jaren erg veel van je geleerd, wetenschap is heel wat meer dan alleen experimenten doen! Bedankt daarvoor.

Dan mijn dagelijks begeleider, Taco. Ik denk dat ik met jou als begeleider ontzettend veel geluk heb gehad! Je stond werkelijk altijd klaar om me te helpen met praktische en niet-praktische zaken. Ik heb het heel prettig gevonden dat ik altijd bij je kon binnen lopen om je data te laten zien en te overleggen! Heel fijn dat jij altijd positief bleef, ook als ik het even niet zag zitten.

Dan mijn andere, onofficiële dagelijks begeleider, Pascal. Pascal, you got involved in my project during my second year and you turned out to be a very essential ingredient! I cannot thank you enough for all your help with experiments, hypothesizing, analysis and, importantly, being a great person to talk to about science and everything else! My time in the lab would certainly not have been the same without you.

Een speciaal bedankje ook voor mijn copromotor Chris. We hebben elkaar in mijn laatste jaar vaak gesproken en ik heb veel aan je adviezen gehad bij het schrijven. Bedankt daarvoor!

Ik wil ook de andere leden van de Wadmangroep bedanken, Hans, Natalie, Jan en Erwin. Ik heb veel gehad aan jullie feedback op presentaties en natuurlijk gezellige momenten bij Polder tussendoor!

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Ook al mijn collega AIOs bedankt! Qiluan, Marlies, Linda, Xin, Laura, Fleur, Yoav, Grazia, Lana en Sicco. Qiluan, wij zaten samen op het endocannabinoïde project. Jij bent wat eerder begonnen dan ik, maar algauw deden we samen experimenten en dat heb ik altijd heel gezellig gevonden! Ook zaten we in het nieuwe gebouw naast/tegenover elkaar waardoor er af en toe meer koffie werd gedronken en gekletst dan gewerkt. En natuurlijk heel leuk dat je mijn paranimf wil zijn!

Marlies, met jou heb ik vooral in de laatste anderhalf jaar veel lief en leed gedeeld. Erg prettig om dichtbij iemand te zitten die telkens door dezelfde fases gaat en precies dezelfde frustraties kent! Goed om af en toe even stoom af te blazen, toch? Ook heb ik met jou een geweldige tijd gehad in de VS, eerst op congres, daarna roadtrippen: Elvis lives! En ik kom gauw naar Edinburgh om whiskey met je te proeven, ik geloof dat jij nu expert bent!

Ik wil ook mijn drie stagestudenten bedanken, Willem, Ton en Kas. Jullie hebben alle drie veel experimenteel werk gedaan in het lab en bij alle drie leidde dat tot (bijna) publicaties, bedankt voor jullie inzet!

Voor alle hulp bij het spenen van muizen (= popcorn) wil ik Chris bedanken. Voor o.a. alle normal goat serum die ik op het laatste moment zelf niet bleek te hebben, maar mocht lenen wil ik Els bedanken. Also thanks to my Italian collaborators, without whom some of the projects would not have been as successful; Mauro, Monica, Sergio and Nicolina.

I spent two years in the SILS PhD PD council where I learned a lot and had a lot of fun! Nice to get out of the lab and organize things with such a fun group of people, thanks Clemens, Fleur, Oskar, Alex, Martijn and Jeroen.

Uiteraard heb ik ook onderwijs gedaan in mijn AIO tijd en daarbij Judith en Etienne leren kennen, waarmee het altijd heel gezellig was!

Los van mijn collegas zijn mijn vrienden en familie heel belangrijk voor me geweest! Met veel vrienden heb ik mijn frustraties en ook succesmomenten kunnen delen, thanks guys! Drie mensen die ik speciaal wil noemen zijn Daphne, Kobus en Mila. Daphne, als PhD student political science weet jij als geen ander hoe je statistiek moet gebruiken. Wij neurowetenschappers vertrouwen veel te vaak op simpele t-testen en ANOVAs! Ook heb ik vaak inhoudelijk dingen met je besproken en was jouw blik vanuit een ander perspectief meer dan nuttig! Kobus, jij bent altijd mijn maatje geweest tijdens

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de bachelor en master. Ik vond het super toen jij ook je PhD begon op SILS, één verdieping beneden iemand die altijd zin had in een koffie of een biertje, heel gezellig! Ik weet zeker dat je het fantastisch gaat doen, ik wens je heel veel succes in je project. Nu moeten we elkaar maar wat meer buiten werk zien! Mila, we kennen elkaar al heel wat jaartjes en jij bent een van mijn paranimfen. Erg handig trouwens dat je vlakbij Science Park woont, waardoor ik recht uit het lab kon aanschuiven!

Een hele belangrijke drijfveer voor mij tijdens mijn AIO periode waren mijn ouders. Jullie hebben me altijd gesteund en me zelfvertrouwen gegeven. Al zolang ik me kan herinneren hebben jullie me gestimuleerd om te doen wat ik leuk vind. Daarbij kon ik altijd bij jullie terecht als ik er wat minder positief in stond, of juist wat meer! Dank jullie lieve papa en mama!!!! Jan, ook jij bedankt voor je steun. Ik bof ontzettend met zo’n lieve broer!

Dan als laatste, mijn aller-allerliefste Enda. The most amazing husband in the world, of course! You have always lifted my spirits when I needed that and gotten me through the rough patches. There is no way I could have made it through these years without your endless love, support and sense of humour. Tá mé i ngrá leat.

pure.uva.nl · the term ‘endocannabinoid (ecb) system’ refers to a group of endogenous...

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