Taar1-mediated modulation of presynaptic dopaminergic neurotransmission: Role of D2 dopamine autoreceptors
Introduction
Octopamine (OCT), tyramine (TYR) and β-phenethylamine (β-PEA), as well as several other non-catechol amines, are metabolites of aromatic amino acids and are known as trace amines (TAs). TAs are a family of endogenous compounds with strong structural similarity to the classical monoamine neurotransmitters and are present in mammalian tissues at low (nanomolar) concentrations (Berry, 2004, Grandy, 2007, Lindemann and Hoener, 2005). The endogenous levels of these compounds are at least two orders of magnitude below those of classical monoamine neurotransmitters such as dopamine (DA), noradrenaline (NE) and serotonin (5HT). TAs are found in many species; in invertebrates, tyramine and octopamine are well-characterized neurotransmitters that modulate movement, feeding, metabolism, muscular tone and other functions (Axelrod and Saavedra, 1977, Cooper and Venton, 2009). Trace amines are also produced in bacteria, fungi, and plant cells and can be found in some food products, most notably in chocolate, cheese and red wine (Branchek and Blackburn, 2003). Despite being known for more than a century, the role played by TAs in mammalian, and particularly human, physiology is still enigmatic. However, it has been noted that levels of TAs are altered in a variety of human disorders ranging from schizophrenia, Parkinson's disease, attention deficit hyperactivity disorder (ADHD), and Tourette's syndrome to migraines and drug addiction (Boulton, 1980, Sandler et al., 1980). For decades, TAs were considered to be “false neurotransmitters” that were able to modulate the signaling of monoamines by displacing them from storage vesicles and/or by acting on the plasma membrane transporters in an amphetamine-like manner (Berry, 2004, Parker and Cubeddu, 1988). Interestingly, the rate of synthesis of TAs was found to be comparable with that of classic monoamines, suggesting that the low levels of TAs in brain tissue are most likely determined by the extremely fast rate of metabolism and/or the inability of TAs to be stored in vesicles as classical neurotransmitters (Grandy, 2007, Sotnikova et al., 2009).
However, in 2001, a family of novel mammalian G protein-coupled receptors (GPCRs) were characterized with some members of this family showing a high affinity for TAs (Borowsky et al., 2001). This family of newly discovered receptors was later re-named the Trace Amine-Associated Receptors (TAARs) family (Maguire et al, 2009, Lindemann et al., 2005). The TAAR family includes 6 functional members in humans (TAAR1-9 including 3 members encoded by pseudo-genes) and even more receptors are found in rodents (Borowsky et al., 2001, Bunzow et al., 2001, Lindemann and Hoener, 2005). Interestingly, in the TAAR family only TAAR1 and TAAR4 possess any demonstrable TA responsiveness. TA binding to TAAR1 engages Gαs-type G proteins that activate adenylyl cyclases (Berry, 2004). TAAR1 is the best characterized TAAR member and is found in some areas of the central nervous system and in certain peripheral tissues (Revel et al., 2013). This distribution, which includes components of the limbic system, such as the amygdala, and areas rich in monoaminergic cell bodies, such as the dorsal raphe nucleus and the ventral tegmental area (VTA) (Lindemann et al., 2008), makes TAAR1 a promising target for pharmaceutical treatment of monoamine-related disorders (Revel et al., 2012a, Revel et al., 2013). Because TAs affect multiple targets including TAAR1, TAAR4, and DA transporter (DAT), adrenergic and serotonin receptors, their use in the identification of specific functions of TAAR1 are limited. Only the generation of TAAR1-deficent and -overexpressing mice (TAAR1-KO and TAAR1-OE mice) (Lindemann et al., 2008, Wolinsky et al., 2007, Revel et al., 2012b), the development of selective TAAR1 agonists, such as RO5166017 (Revel et al., 2011), and antagonists, such as ((N-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-trifluoromethyl-benzamide EPPTB) (Bradaia et al., 2009), provided an opportunity to evaluate the specific roles and mechanisms mediated by TAAR1. TAAR1-KO mice appear to be similar to control animals at basal state, but they show enhanced hyperlocomotion and exaggerated striatal release of DA, NE, and 5-HT when challenged with d-amphetamine. Recent studies on TAAR1-KO mice have demonstrated that TAAR1 is able to negatively modulate monoaminergic neurotransmission (Lindemann et al., 2008, Wolinsky et al., 2007). For example, the genetic ablation of TAAR1 induces an increase in the spontaneous firing rate of DA neurons (Lindemann et al., 2008), and similar effects are mediated via application of the selective TAAR1 antagonist EPPTB in control animals (Bradaia et al., 2009), corroborating the idea that TAAR1 normally exerts an inhibitory effect on DA neurons. Although the underlying TAAR1 signaling mechanism remained unclear, Bradaia et al. clearly showed that TAAR1 activates inwardly rectifying K+ channels (Bradaia et al., 2009). They also demonstrated that both the acute application of EPPTB and the constitutive genetic lack of TAAR1 increase the potency of DA at D2 receptors in DA neurons (Bradaia et al., 2009). Studies in vitro and in vivo offered further indication of a physical and functional interaction between TAAR1 and D2 receptors (Espinoza et al., 2011), whereas others have suggested that TAAR1 may directly alter DAT function (Miller et al., 2005). To complement the information about TAAR1 function, a line of transgenic mice that overexpresses TAAR1 in the brain has been recently generated (Revel et al., 2012b). This model is hyposensitive to amphetamine and it shows constitutive hyperactivity of monoaminergic nuclei (Revel et al., 2012b). Overall, the growing body of evidence suggests a modulatory role of TAAR1 on monoaminergic activity (Reese et al., 2014, Cichero et al., 2013), particularly on presynaptic function. To determine whether deletion of the Taar1 gene or application of TAAR1 ligands perturbs the functional presynaptic activity of DA neurons at the level of axon terminals, we investigated extracellular DA dynamics using fast scan cyclic voltammetry (FSCV) and in vivo microdialysis techniques in the dorsal striatum (DStr) and nucleus accumbens (NAcc) of wild type (WT) and TAAR1-KO mice. Furthermore, we applied FSCV to evaluate the evoked DA release and clearance rates in these brain regions of WT and TAAR1-KO mice in the presence of the selective TAAR1 agonist (RO5166017) and a TAAR1 antagonist ((N-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-trifluoromethyl-benzamide EPPTB).
We found that DA release evoked by a single stimulus was higher in NAcc, and the basal extracellular level of DA was significantly higher in this brain region in TAAR1-KO mice. The TAAR1 agonist RO5166017 decreases DA release in WT mice but not in TAAR1-KO animals, and application of the TAAR1 antagonist EPPTB prevented the reduction in the evoked DA release induced by the TAAR1 agonist in WT animals. We further gained functional evidence suggesting that these presynaptic effects could be mediated by an interaction between TAAR1 and D2 DA autoreceptors.
Section snippets
Animals
All experiments were conducted in compliance with the Italian Ministry of Health (DL 116/92; DL 111/94-B) and European Community (86/609/EEC) directives regulating animal research. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques, if available. Animals were housed under a 12 h light/12 h dark cycle with ad libitum access to food and water. TAAR1-KO mice of mixed backgrounds (C57BL/6J × 129Sv/J
Lack of TAAR1 leads to an increased DA release predominantly in the nucleus accumbens
Previous studies employing conventional microdialysis have revealed similar basal levels of dialyzate DA in the NAcc and striatum (Di Cara et al., 2011) of TAAR1-KO and WT mice, revealing a difference in monoaminergic transmission only following amphetamine challenge (Lindemann et al., 2008). To evaluate the mechanisms of TAAR1-dependent modulation of DA and 5-HT transmission, we first analyzed the total tissue content of monoamines and their metabolites in different brain areas (Fig. 1). HPLC
Discussion
The aim of this study was to understand the mechanism of the modulatory action of TAAR1 on presynaptic DA transmission. Our data support the previous reports showing a close interaction between TAAR1 and the dopaminergic system (Lindemann et al., 2008, Revel et al., 2011, Bradaia et al., 2009) and specifically highlights an interaction between the D2 class of autoreceptors and TAAR1 receptors. While several recent studies have been performed on understanding the role of TAAR1 in the modulation
Acknowledgments
This work was supported in part by research awards to RRG from F. Hoffmann-La Roche Ltd. (Basel, Switzerland) and Fondazione Compagnia di San Paolo (Torino, Italy). We are grateful to Lundbeck A/G and Lundbeck USA for generously providing the TAAR1 knockout mice. We thank Dr. M. Morini, D. Cantatore and F. Piccardi for their excellent technical assistance.
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