UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Orexin modulation of ventral tegmental area dopamine neurons Baimel, Corey 2016

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2016_september_baimel_corey.pdf [ 3.1MB ]
Metadata
JSON: 24-1.0303473.json
JSON-LD: 24-1.0303473-ld.json
RDF/XML (Pretty): 24-1.0303473-rdf.xml
RDF/JSON: 24-1.0303473-rdf.json
Turtle: 24-1.0303473-turtle.txt
N-Triples: 24-1.0303473-rdf-ntriples.txt
Original Record: 24-1.0303473-source.json
Full Text
24-1.0303473-fulltext.txt
Citation
24-1.0303473.ris

Full Text

Orexin modulation of ventral tegmental area dopamine neurons   by   Corey Baimel   B.Sc., Mcgill University, 2009   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF     Doctor of Philosophy    in  THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES     (Pharmacology and Therapeutics )    THE UNIVERSITY OF BRITISH COLUMBIA      (Vancouver)     May 2016      © Corey Baimel, 2016  ii Abstract   Dopamine neurons in the ventral tegmental area (VTA) are critically involved in the expression of motivated behaviour. The activity of dopamine neurons is regulated by intrinsic conductances and by synaptic inputs, both of which are subject to neuromodulatory influences. This thesis explores how orexin signalling alters the synaptic regulation and the activity of VTA dopamine neurons. Chapter 1 describes a role for dopamine in motivated behaviour and highlights how drugs of abuse alter synaptic transmission in the mesocorticolimbic dopamine system to drive compulsive reward seeking. Moreover, it outlines how lateral hypothalamic orexin projections to the VTA alter synaptic transmission onto dopamine neurons to promote motivated behaviour. Chapter 2 examines how orexin signalling gates morphine-induced synaptic plasticity in the VTA. We demonstrate that inhibiting orexin receptor signalling in the VTA blocks morphine-induced increases and decreases in the strength of excitatory and inhibitory synaptic transmission respectively. Orexin neurons coexpress the inhibitory peptide dynorphin and the two are likely coreleased. In chapter 3 we demonstrate that orexin and dynorphin modulate the activity of dopamine neurons in a projection-target specific manner. Orexin preferentially increased the output of dopamine neurons that project to the lateral shell of the nucleus accumbens (NAc), while dynorphin was more effective at inhibiting the activity of dopamine neurons that project to the basolateral amygdala (BLA). Chapter 4 discusses the strength and weaknesses of these experiments and proposes future research to further enhance our understanding of orexin modulation of VTA dopamine neurons.     iii Preface  • Some sections of Chapter 1 of this thesis are modified and updated from sections of published review articles I have written with assistance from S.L. Borgland.  o Baimel C, Bartlett SE, Chiou LC, Lawrence AJ, Muschamp JW, Patkar O, Tung LW, Borgland SL (2015). Orexin/hypocretin role in reward: implications for opioid and other addictions. Br J Pharmacol 172(2): 334-348. o Baimel C, Borgland SL (2012). Hypocretin modulation of drug-induced synaptic plasticity. Prog Brain Res 198:123-131  • A version of Chapter 2 has been published and is reproduced in this thesis: o Baimel C & Borgland SL (2015). Orexin signaling in the VTA gates morphine-induced synaptic plasticity. J Neurosci 35(18): 7295-7303. 
  I worked in collaboration with S.L. Borgland to design the experiments. I collected and analyzed the data and wrote the manuscript with assistance from S.L. Borgland.  
  • A version of Chapter 4 is in preparation for publication: o Baimel C and Borgland SL. Projection target defined effects of orexin and dynorphin on VTA dopamine neurons. In preparation.  I worked in collaboration with S.L. Borgland to design the experiments. I collected and analyzed the data and wrote the manuscript with assistance from S.L. Borgland  • This research was approved by the University of British Columbia and University of Calgary animal care committees: 
  University of British Columbia 
  Certificate: A08-0042 
  Project title: Role of feeding related peptides on brain reward pathways, NSERC 
 
University of Calgary 
   Certificate: AC13-0074 
  Project title: Role of feeding related peptides on brain reward pathways, NSERC      iv Table of Contents Abstract ........................................................................................................................... ii Preface ............................................................................................................................ iii Table of Contents .......................................................................................................... iv List of Figures ............................................................................................................... viii List of Abbreviations ...................................................................................................... ix Acknowledgements ....................................................................................................... xi Dedication ..................................................................................................................... xii Chapter 1: Introduction .................................................................................................. 1 The mesocorticolimbic dopamine system ............................................................... 1 Dopamine and motivated behaviour ......................................................................... 2 Identifying dopamine neurons within a heterogeneous VTA cell population ............ 6 Drugs of abuse target the dopamine system ........................................................... 8 Drugs of abuse hijack synaptic plasticity mechanisms in the VTA ........................... 9 Drugs of abuse alter plasticity at inhibitory synapses onto dopamine neurons ..... 15 Behavioural implication of drug-induced plasticity in the VTA ............................... 17 Drug-induced plasticity in the VTA as a form of metaplasticity .............................. 18 Lateral hypothalamic orexin neurons and motivated behaviour .......................... 22 Neurotransmitter and neuropeptide release from orexin neurons .......................... 25 Drug-induced activation of orexin neurons ............................................................. 27 Drug-induced plasticity at orexin neurons .............................................................. 29 Orexin modulates synaptic transmission in the VTA .............................................. 31 Orexin modulates cocaine-induced plasticity in the VTA ....................................... 33  v Orexin in the VTA modulates motivated behaviour ................................................ 34 Orexin neurons contain the kappa opioid receptor agonist dynorphin ............... 36 Dynorphin signalling in the mesocorticolimbic dopamine system .......................... 39 Orexin and dynorphin have opposing effects on motivated behaviour .................. 42 Summary and objectives .......................................................................................... 43 Chapter 2: Orexin signalling in the VTA gates morphine-induced synaptic plasticity ........................................................................................................................................ 45 Introduction ............................................................................................................... 45 Materials and methods ............................................................................................. 46 Animals ................................................................................................................... 46 Electrophysiology .................................................................................................... 47 Surgical procedures ................................................................................................ 49 Intra-VTA drug infusions ......................................................................................... 50 Tyrosine hydroxylase immunocytochemistry .......................................................... 51 Data analysis ........................................................................................................... 51 Results ....................................................................................................................... 51 OxR1 signalling is required for morphine-induced potentiation of excitatory inputs to VTA dopamine neurons ...................................................................................... 51 Orexin signals locally in the VTA to mediate morphine-induced potentiation of excitatory synaptic transmission ............................................................................ 55 OxR1 signalling is required for a 
 morphine-induced decrease in 
presynaptic GABA release 
 ..................................................................................................................... 58 OxR1 activation in the VTA is required for a morphine-induced decrease in the probability of presynaptic GABA release ................................................................ 60  vi Morphine alters the synaptic excitation/inhibition balance in an OxR1-dependent manner 
 .................................................................................................................... 62 Discussion ................................................................................................................. 64 Orexin and morphine-induced potentiation of AMPAR signalling .......................... 64 Orexin and morphine-induced decreases in inhibitory synaptic transmission ....... 65 Orexin signalling underlies a morphine-induced shift in the synaptic regulation of VTA dopamine neurons .......................................................................................... 66 Morphine-induced activation of orexin neurons ..................................................... 66 Summary and conclusions ..................................................................................... 67 Chapter 3: Projection target defined effects of orexin and dynorphin on VTA dopamine neurons ........................................................................................................ 68 Introduction ............................................................................................................... 68 Materials and methods ............................................................................................. 70 Animals ................................................................................................................... 70 Surgical procedures ................................................................................................ 70 Analysis of action potential firing ............................................................................ 72 Immunohistochemistry and confocal microscopy .................................................. 72 Data analysis ........................................................................................................... 73 Results ....................................................................................................................... 73 NAc lateral shell and BLA projecting VTA dopamine neurons are mostly non-overlapping cell populations with different electrophysiological properties ........... 73 Orexin A preferentially increases the firing activity of NAc lateral shell projecting VTA dopamine neurons .......................................................................................... 79  vii Projection-specific effects of dynorphin A on the firing activity of VTA dopamine neurons ................................................................................................................... 82 Discussion ................................................................................................................. 85 Heterogeneity within the VTA dopamine system .................................................... 85 Projection target specific effects of orexin and dynorphin ..................................... 86 Summary and conclusions ..................................................................................... 90 Chapter 4- General discussion .................................................................................... 91 Orexin signalling and drug-induced synaptic plasticity in the VTA ..................... 92 Endogenous orexin and dynorphin release in the VTA ......................................... 95 Conclusions ............................................................................................................... 96 References .................................................................................................................... 98     viii List of Figures  Figure 1. Projection target defined VTA dopamine neurons ................................................. 21  Figure 2. Systemic administration of the OxR1 antagonist SB 334867 blocks morphine-induced potentiation of excitatory transmission onto VTA dopamine neurons ..................... 54  Figure 3. OxR1 signalling in the VTA is required for morphine-induced plasticity at glutamatergic synapses ......................................................................................................... 57  Figure 4. Morphine decreases the probability of GABA release in an OxR1-dependent manner ................................................................................................................................... 59  Figure 5. OxR1 signalling in the VTA is necessary for a morphine-induced suppression of presynaptic GABA release. ................................................................................................ 61   Figure 6. OxR1 signalling is required for a morphine-induced shift in the balance of excitatory and inhibitory synaptic transmission onto dopamine neurons. ............................ 63  Figure 7. NAc lateral shell and BLA projecting VTA dopamine neurons are two largely non-overlapping cell populations .......................................................................................... 74  Figure 8. NAc lateral shell and BLA projecting VTA dopamine neurons express different Ih ............................................................................................................................................. 76  Figure 9. NAc lateral shell and BLA projecting VTA dopamine neurons have different action potential properties ..................................................................................................... 78  Figure 10. Orexin A preferentially increases the firing activity of NAc lateral shell projecting VTA dopamine neurons. ....................................................................................... 81  Figure 11. Dyn preferentially inhibits the firing activity of BLA projecting VTA dopamine neurons.  ................................................................................................................................ 84   ix List of Abbreviations  AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid  BLA basolateral amygdala  CPA conditioned place aversion  CPP conditioned place preference   CRE Cre recombinase enzyme  DMH dorsomedial hypothalamus   eGFP enhanced green fluorescent protein   EPSC excitatory postsynaptic current   GABA γ-aminobutyric acid  GPCR G-protein coupled receptor   HCN hyperpolarization-activated cyclic nucleotide–gated cation channels  HFS high frequency stimulation   Ih hyperpolarization activated inward current   IPSC inhibitory postsynaptic currents   LDT laterodorsal tegmentum  LH lateral hypothalamus  LTD long-term depression  LTP long-term potentiation   LTPGABA long-term potentiation at GABAergic synapses   MCH melanin-concentrating hormone  mGluR metabotropic glutamate receptor   MOR μ-opioid receptor  MSN medium spiny neuron     x NAc nucleus accumbens   NMDA n-methyl-D-aspartate  norBNI norbinaltorphimine   OxR1 orexin receptor type 1  OxR2 orexin receptor type 2  PFA perifornical area   PFC prefrontal cortex  Pitx3 paired-like homeobox transcription factor   PKC protein kinase C  PLC phospholipase C  RMTG rostromedial tegmental nucleus   SB 334867 N-(2-Methyl-6-benzoxazolyl)-N'-1,5-naphthyridin-4-yl urea  VTA ventral tegmental area        xi Acknowledgements   I would first like to thank my supervisor Dr. Stephanie Borgland for her endless support and guidance throughout my degree. Thank you for your pushing me to challenge myself, while instilling in me the self-confidence needed to do so.   I am grateful to my committee members, Dr. Tony Phillips, Dr. Harley Kurata and Dr. Alasdair Barr, for their time and commitment to the quality of this thesis and for my development as a scientist. Our many discussions have both guided my research and inspired me to broaden my perspective.   Many thanks are owed to the current and former staff members in the department of Anesthesiology, Pharmacology and Therapeutics for their generous favors and support. In particular, I would like to thank Wynne Leung who was invaluable during my time in Vancouver, and perhaps even more after the move to Calgary, and to Andy Jeffries who I am lucky to consider a friend.   Thank you to my funding sources, CIHR, UBC and Brain Canada.   I want to thank all current and former members of the Borgland lab, especially Dr. Gwenaël Labouèbe for teaching me how to patch and for remaining both a resource and a friend. And to Drs. Lindsay Naef, Benjamin Lau, Kimberley Pitman, Jennifer Thompson and Shuai Liu for the much-needed feedback, moral support and the many good times we have shared together; and to Frances Xia, Calvin Ho Ming Li and Dr. Dmitry Mebel for your ongoing friendship.   Thank you to my fellow students and postdocs, both at UBC and at the HBI for making it even more enjoyable to come to the lab nearly everyday, especially Mike Mousseau, Candace Marsters, Phil Colmers, and Mohammed Badran amongst many others.    Special thanks must go to my family: to my brother, Adam, and my cousin, Jordan, for the many memories I will never forget and to my mom, Linda, for her unconditional love and support.  And last but not ever least, thank you to Sami, for everything.       xii Dedication    For my dad 1 Chapter 1: Introduction  The mesocorticolimbic dopamine system   The mesocorticolimbic dopamine system consists of midbrain VTA dopamine neurons and their axonal projections to downstream targets including the NAc, the prefrontal cortex (PFC) and the amygdala (Beier et al., 2015; Swanson, 1982). The VTA is involved in regulating adaptive behaviours related to motivation and consists of dopamine neurons, which are the predominant cell type in the region (~65% of cells), intermingled with both γ-aminobutyric acid (GABA) containing (~30%) and glutamate containing (~5%) neurons. (Hnasko et al., 2012; Margolis et al., 2006a, 2012; Steffensen et al., 1998; Ungless and Grace, 2012; Yamaguchi et al., 2007).   Dopamine neurons fire in either a low frequency single-spike (tonic) or bursting (phasic) pattern, in which action potentials cluster together and fire at high frequencies for several hundred milliseconds (Grace and Bunney, 1984a, 1984b). Burst firing is important for dopamine signalling and results in increased dopamine release per number of spikes as it likely saturates reuptake and degradation mechanisms (Chergui et al., 1994; Floresco et al., 2003; Gonon, 1988; Venton et al., 2003). Because control of dopamine firing modes is critical for motivated behaviour, it is important to consider the processes that regulate these patterns of activity. Firing patterns are driven by intrinsic conductances mediated by inwardly rectifying- and Ca2+- regulated K+ channels, as well as hyperpolarization-activated cyclic nucleotide–gated cation (HCN) channels (Grace, 1991; Hopf et al., 2007; Tateno and Robinson, 2011). Moreover, dopamine neurons receive excitatory glutamatergic and inhibitory GABAergic inputs from many sources, and these inputs coordinate the activity of dopamine neurons. Burst firing is tightly regulated by long range excitatory afferent inputs (Grace and Bunney, 1984b; Grace et  2 al., 2007), primarily through signalling at n-methyl-D-aspartate receptors (NMDARs) (Johnson et al., 1992; Komendantov et al., 2004; Overton and Clark, 1992; Zweifel et al., 2009) and requires Ca2+ entry and the subsequent activation of Ca2+ gated ion channels (Paladini and Roeper, 2014; Paladini and Williams, 2004). Additionally, both pharmacological and optogenetic studies demonstrate that the firing rate and the excitability of dopamine neurons is reduced by activation of GABAA receptors (Johnson and North, 1992a; Polter et al., 2014; Tan et al., 2012; van Zessen et al., 2012). Both the intrinsic conductances and the synaptic inputs that regulate the activity of dopamine neurons are subject to neuromodulatory influences. This thesis presents data supporting a role for lateral hypothalamic orexin neurons in the modulation of dopamine neurons.   Dopamine and motivated behaviour    Most research has been biased towards determining the role of dopamine in the NAc, and this will be the primary focus of this section. However, dopamine has separate and yet related roles in other brain regions, and these functions may be equally important in driving motivated behaviour. For example, dopamine in the PFC has been linked to working memory (Durstewitz and Seamans, 2002; Seamans et al., 1998) and dopamine release in the caudate putamen has important motor functions (Palmiter, 2008).  Nevertheless, despite decades of research, the role of dopamine in the NAc and how it pertains to motivated behaviour is still very much up for debate. Dopamine was initially considered to be the neurochemical substrate of reward, encoding the perception of rewarding stimuli. The dopamine reward hypothesis emerged as an explanation for the finding that rats would work for electrical self-stimulation of the medial forebrain bundle (Bielajew and Shizgal, 1986; Olds and Milner,  3 1954) and was extended to include both natural rewards like food, water and sex. This theory posited that dopamine systems, particularly in the NAc, mediated the primary motivational characteristics of natural stimuli and the subjective pleasure that accompanied them (Wise, 1978). Moreover, it theorized that addictive drugs artificially stimulated this system and that this was critical for the development of addiction (Wise, 1978). However, a substantial body of research has accumulated that does not conform to this view, and so it has been all but abandoned. Several studies, in both humans and animals, demonstrate that blocking dopamine receptors or inhibiting dopamine synthesis does not block the hedonic impact, or the liking, of natural rewards or addictive drugs (Berridge et al., 1989; Brauer and De Wit, 1997; Gawin, 1986; Haney et al., 2001; Leyton et al., 2005; Nann-Vernotica et al., 2001; Venugopalan et al., 2011; Wachtel et al., 2002), and that dopamine concentrations actually drop during reward consumption (Hamid et al., 2016). Moreover, dopamine neurons are activated by noxious, aversion-inducing stimuli (Anstrom and Woodward, 2005; Brischoux et al., 2009), and dopamine release in the NAc and the medial PFC is increased by tail-shock and during social threat (Abercrombie et al., 1989; Kalivas and Duffy, 1995; Tidey and Miczek, 1996).   Alternative theories have been proposed, which link dopamine to motivation, learning and action, but how dopamine mediates these functions is still somewhat unclear. Because dopamine was associated with the wanting, rather than the liking of rewards (Berridge et al., 1989), the incentive salience hypothesis proposed that dopamine functioned to assign incentive or motivational value to external reward-predicting stimuli or internal representations of reward, therefore making them more likely to command attention and induce approach (Robinson and Berridge, 1993). It reasoned then, that because drugs of abuse increase dopamine transmission, addiction  4 may result from repeated drug use rendering an individual hypersensitive to the act of drug taking and transforming ordinary wanting into excessive craving (Robinson and Berridge, 1993). An alternate theory emphasized a role for dopamine in learning. The reward prediction error hypothesis emerged from the finding that after repeated pairings of a sensory stimulus with a reward, phasic activation of dopamine neurons and downstream dopamine release shifts from the time of reward delivery to the onset of the cue (Day et al., 2007; Mirenowicz and Schultz, 1994; Schultz et al., 1997). Moreover, dopamine neuron activity decreases when predicted rewards are omitted and increases when unpredicted rewards are delivered or predicted rewards are greater than expected (Schultz et al., 1997). Importantly, optogenetic stimulation of dopamine neurons during reward delivery, to mimic a positive prediction error is sufficient to drive cue learning (Steinberg et al., 2013). This suggests that dopamine signals the opportunity for reward and the deviation of the actual reward from that which is predicted, and would represent a mechanism for anticipation and action selection to obtain reward (Collins and Frank, 2015). Furthermore, it is interesting to note that dopamine release to reward predicting cues is paralleled by an increase in the strength of excitatory synapses onto dopamine neurons (Stuber et al., 2008). A third hypothesis suggests that dopamine is important for the behavioural invigoration required to obtain reward, for performing instrumental responses, maintaining effort over time and for allocating behavioral resources based upon cost-benefit analyses (Hernandez et al., 2010; Salamone and Correa, 2002; Salamone et al., 2001).   Although there is a lot of evidence to support these claims, integration of these ideas most appropriately reflects dopamine’s action in the NAc. Recent work demonstrates that after cue associations are acquired, NAc dopamine release is dependent on movement initiation, rather than just reward prediction error alone (Syed  5 et al., 2016). That is, reward-predicting cues induce dopamine release only if and when a correct goal-directed action is initiated, and suggests that increases in dopamine may promote the correct selection and execution of actions that enable the obtaining of reward (Syed et al., 2016). Accordingly, when cues signal a need to inhibit or delay action, phasic dopamine signals are observed only after the delay and with movement initiation to collect the reward, and decreases in dopamine occur when the animal fails to withhold a response (Syed et al., 2016). In this sense, both positive and negative reward prediction errors are observed, but only following action initiation (Syed et al., 2016). Moreover, when rats are required to select between actions that yield different reward probabilities, NAc dopamine levels convey moment-by-moment estimates of available reward and predict task engagement (Hamid et al., 2016). In fact, optogenetic inhibition of dopamine neurons at trial onset decreases the likelihood of the rat engaging in the task (Hamid et al., 2016).  Dopamine also reinforces choice and therefore promotes learning as phasic optogenetic stimulation or inhibition of dopamine neurons following choice increases or decreases the probability of making that choice again (Hamid et al., 2016).   That being said, it is important to consider that dopamine neurons are perhaps equally involved in responding to aversive stimuli. In fear conditioning paradigms, subsets of dopamine neurons show a prediction error like shift in their response (Gore et al., 2014) and inhibiting dopamine neuron activity impairs learning of cues that predict aversive outcomes (Zweifel et al., 2011). Moreover, fear learning is impaired in dopamine deficient mice, and can be restored with dopamine in the NAc and the BLA (Fadok et al., 2010). It will be important to further examine the interplay between dopamine in reward and in aversion.   6 Identifying dopamine neurons within a heterogeneous VTA cell population  To properly understand the function of dopamine, it is critical to have a detailed understanding of the cellular properties and the circuitry of dopamine neurons. Although dopamine neurons are the predominant cell type in the VTA, because they are intermingled with both GABAergic and glutamatergic neurons, proper identification of dopamine neurons within the heterogeneous VTA can be difficult. However, identifying dopamine neurons is critical for understanding how dopamine neurons are engaged during motivated behavior. Both in vivo and in ex vivo slice preparations, dopamine neurons are often identified by indirect electrophysiological markers (Chieng et al., 2011; Margolis et al., 2006a, 2010; Ungless and Grace, 2012). Dopamine neurons are said to have broad action potentials, slow firing rates and often display irregular burst-firing activity (Grace and Bunney, 1980; Grace and Onn, 1989; Guyenet and Aghajanian, 1978); although the latter is rarely observed in slice preparations as afferent inputs are often transected during the slicing procedure (Lodge and Grace, 2006; Ungless and Grace, 2012). Comparatively, non-dopamine neurons in the VTA are considered to have shorter duration action potentials and higher firing rates (Margolis et al., 2012; Steffensen et al., 1998). However, in many cases, assumed dopamine neurons identified with these criteria do not stain positively for tyrosine hydroxylase (Cameron et al., 1997; Jones and Kauer, 1999; Margolis et al., 2003, 2006a), the enzyme responsible for converting L-tyrosine into L-DOPA in the dopamine synthesis pathway, and there is considerable overlap in the characteristics of both dopamine and non-dopamine cells (Chieng et al., 2011; Margolis et al., 2006a; Ungless and Grace, 2012). In slice preparations, dopamine neurons are most commonly identified by the expression of a pronounced hyperpolarization activated inward current (Ih) (Grace and Onn, 1989; Johnson and North, 1992a). There is however a lot of variability between reports in  7 terms of the reliability of Ih and in many cases the expression of Ih does not overlap with tyrosine hydroxylase staining (Margolis et al., 2006a, 2010; Ungless and Grace, 2012; Zhang et al., 2010) or is found in identified GABAergic neurons (Chieng et al., 2011). Species differences may be important here as non-dopamine neurons with Ih are more commonly found in slices from rats, than from mice (Margolis et al., 2006a, 2010; Ungless and Grace, 2012; Zhang et al., 2010). Pharmacological identifiers have also been proposed, in that dopamine neurons were inhibited by signalling at dopamine D2-like autoreceptors (Beckstead et al., 2004; Johnson and North, 1992a), and non-dopamine neurons (presumably GABAergic) inhibited by μ-opioid receptor (MOR) agonists (Johnson and North, 1992b). But not all dopamine neurons are inhibited by the activation of D2-like receptors (Lammel et al., 2008; Margolis et al., 2008) and D2-like and MOR inhibition have both been observed at putative dopamine and non-dopamine neurons (Margolis et al., 2014).  Variability in terms of determining proper markers of dopamine neurons can arise from many factors including the slicing procedure used, which may favor the survival of certain types of neurons over others, experimental bias towards selecting neurons and the length of the experiment, whereby in longer recordings there is increased risk of tyrosine hydroxylase washout, particularly when using a high Cl- internal solution (Margolis et al., 2010; Ungless and Grace, 2012). Therefore, because no neuronal markers clearly differentiate dopamine neurons from other cell populations in the VTA, tyrosine hydroxylase staining is likely the most effective identifier of dopaminergic neurons (Margolis et al., 2010). Alternatively, the use of mouse lines that selectively express fluorescent proteins in dopamine neurons are becoming increasingly popular. Chapter 3 of this thesis makes use of a mouse line in which the enhanced green fluorescent protein (eGFP) reporter gene was inserted into the promoter region of  8 the paired-like homeobox transcription factor 3 (Pitx3) (Hedlund et al., 2008). Pitx3 is expressed in all dopamine neurons (Poulin et al., 2014) from embryonic day 11 and is involved in the maintenance and induction of tyrosine hydroxylase expression at this time and throughout adulthood (Bissonette and Roesch, 2016; Smidt et al., 1997).  In these mice identification of dopamine neurons is immediate and eGFP fluorescence is observed selectively and in nearly 100% of dopamine neurons in the VTA and nearby substantia nigra (Zhao et al., 2004).   Nevertheless, VTA dopamine neurons project to many brain areas (Beier et al., 2015; Swanson, 1982), and recent work suggests that many of these projections are non-overlapping (Beier et al., 2015; Lammel et al., 2012)  and that the intrinsic properties of dopamine neurons segregate according to projection target. Therefore, it is becoming increasingly evident that VTA dopamine neurons consist of several subpopulations of cells that can be somewhat identified by their electrophysiological properties, afferent and efferent projections and their gene expression profiles (Beier et al., 2015; Ford et al., 2006; Lammel et al., 2011, 2012; Margolis et al., 2006b, 2008; Poulin et al., 2014). Chapter 3 of this thesis presents data suggesting NAc lateral shell- and BLA-projecting VTA dopamine neurons have different intrinsic properties and are independently modulated by orexin and dynorphin. Characterizing the diversity within the VTA dopamine system will likely be critical to further understanding how dopamine signalling regulates multiple behavioral phenotypes under different circumstances.   Drugs of abuse target the dopamine system  Addiction is a disease that progresses in a step-wise manner, evolving from irregular, recreational use, to sustained and escalated use that can transition into compulsive drug consumption with disregard for negative consequences (Deroche- 9 Gamonet and Piazza, 2014; Koob and Volkow, 2010). All addictive drugs target the mesocorticolimbic dopamine system and have in common the ability to increase dopamine concentrations in the NAc (Di Chiara and Imperato, 1988), albeit by very different mechanisms. These drugs can be grouped into three categories, those that activate G-protein coupled receptors (GPCRs), those that alter the function of ion channels, which both signal primarily in the VTA, and those that target the monoamine transporters at terminal dopamine release sites (Lüscher and Ungless, 2006). It should however be noted that the effects of this third group, which includes the stimulants cocaine and amphetamine, are still dependent on modulatory mechanisms in the VTA. Specifically cocaine enhances endocannabinoid signalling at presynaptic GABA inputs to increase the firing rate of VTA dopamine neurons through disinhibition (Wang et al., 2015). Drugs of abuse also share the ability to alter synaptic transmission in the mesocorticolimbic dopamine system. Chapter 2 of this thesis focuses on a role for orexin signalling in morphine-induced synaptic plasticity in the VTA.    Drugs of abuse hijack synaptic plasticity mechanisms in the VTA  Addiction is a disorder of neural circuit dysfunction characterized by serial changes in synaptic transmission initiated in the VTA (Lüscher et al., 2015). Synaptic plasticity mechanisms such as long term potentiation (LTP) and long term depression (LTD) are critically involved in learning, and a leading hypothesis suggests that addiction represents a powerful and aberrant form of learning and memory (Hyman et al., 2006; Kalivas and Volkow, 2005; Kauer and Malenka, 2007; Kelley, 2004). In fact, much evidence has accumulated demonstrating that beyond acute increases in dopamine concentration, addictive drugs also have in common the property of inducing synaptic plasticity at synapses in the VTA (Brown et al., 2010; Saal et al., 2003).   10  The study of drug-induced plasticity in the VTA initiated with the finding that a single, in vivo injection of cocaine potentiates excitatory synaptic transmission onto dopamine neurons (Ungless et al., 2001). The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)/NMDAR ratio is a normalized measure of basal synaptic strength for comparing between different cells and slice preparations, which is independent of the positioning of electrodes or the number of synapses activated (Kauer and Malenka, 2007). A single injection of cocaine increases the AMPAR/NMDAR ratio of glutamatergic inputs onto dopamine neurons by way of an NMDAR-dependent increase in postsynaptic AMPAR expression that lasts 5, but not 10 days and which occludes LTP and enhances LTD at these synapses respectively (Ungless et al., 2001). Interestingly, neither the magnitude nor the duration of this plasticity is increased when animals are given 7 days of experimenter-administered drug (Borgland et al., 2004). Together this implies that a single injection of cocaine is perhaps sufficient to saturate potentiation of excitatory inputs onto dopamine neurons. However, others have reported LTP in slices from cocaine-treated rats using different induction protocols (Liu et al., 2005), suggesting that either only a subset of inputs are potentiated by cocaine or that the mechanism for induction of LTP at these synapses was independent of those responsible for the increase in the AMPAR/NMDAR ratio (Liu et al., 2005). Nevertheless, this plasticity is greatly prolonged when animals self-administer cocaine, where increases in the AMPAR/NMDAR ratio are observed up to 3 months after the end of self-administration (Chen et al., 2008). This is in direct contrast to self-administration of natural reinforcers like sucrose and food, where synaptic transmission returns to baseline levels after 21 days (Chen et al., 2008) and highlights the important differences between experimenter-administered and self-administered drugs (Hemby et al., 1997; Kmiotek et al., 2012; O’Connor et al., 2011). Similar drug- 11 induced increases in excitatory synaptic strength have also been demonstrated with single injections of amphetamine (Faleiro et al., 2003; Saal et al., 2003), nicotine, morphine, ethanol (Saal et al., 2003), benzodiazepines (Heikkinen et al., 2009) and delta(9)-tetrahydrocannabinol), the main psychoactive ingredient of marijuana (Good and Lupica, 2010). It remains to be determined if self-administration of these drugs equally prolongs the time-course of drug-induced plasticity in the VTA.   Drug-induced potentiation of AMPAR signalling in the VTA is expressed by a redistribution of AMPAR subunits. AMPARs that contain the GluA2-subunit have a positively charged arginine residue lining the pore which makes the receptors impermeable to Ca2+, and gives them a smaller single channel conductance and a linear current-voltage relationship (Liu and Zukin, 2007). AMPARs containing the GluA2 subunit are the predominant subtype observed at synapses onto dopamine neurons in naïve and saline-treated animals (Brown et al., 2010; Mameli et al., 2011), but are replaced, at least in part, with GluA2-lacking AMPARs following a single injection of cocaine (Bellone and Lüscher, 2006) or other drugs (Brown et al., 2010). After drug exposure, AMPARs containing the GluA2 subunit are internalized and AMPAR excitatory postsynaptic currents (EPSCs) on dopamine neurons show an inwardly-rectifying current-voltage relationship and are sensitive to polyamine block (Bellone and Lüscher, 2006; Brown et al., 2010). Interestingly, after cocaine but not saline exposure, metabotropic glutamate receptor type 1 (mGLUR1) agonists depress AMPAR EPSCs and mGluR activation or mGluR-LTD reverses cocaine-driven AMPAR synaptic plasticity (Bellone and Lüscher, 2006; Mameli et al., 2007). Importantly, this is likely the same reversal mechanism that occurs in vivo as treatment with a dominant negative peptide that prevents mGluR1 signalling, or daily treatment with an mGluR1 antagonist renders cocaine-induced plasticity persistent (Mameli et al., 2009). MGluR1 are  12 expressed largely perisynaptically, and require strong excitatory inputs for their activation (Bellone and Lüscher, 2006; Lujan et al., 1996), it remains unknown which inputs or what stimuli trigger mGluR1 activity in vivo to initiate the reversal of drug-induced plasticity. Finally, although it has received much less attention, it should be noted that a single exposure to cocaine also induces a long-term increase in presynaptic glutamate release (Ungless et al., 2001), which likely further enhances glutamatergic control of postsynaptic dopamine neurons.   An increase in the AMPAR/NMDAR ratio can be induced by either an increase in AMPAR signalling, a decrease in NMDAR signalling, or both. Early work promoted the idea that drugs of abuse induce plasticity only at the level of AMPARs, because no change was observed in response to bath application of NMDA, which targets both extrasynaptic and synaptic NMDARs, following drug exposure (Borgland et al., 2004; Ungless et al., 2001). Nevertheless, recent reports using 2-photon uncaging of glutamate at single synapses demonstrate important changes to NMDAR signalling after cocaine exposure. Relative to saline-treated animals, the amplitude of AMPAR EPSCs is increased and that of NMDAR EPSCs is decreased in cocaine-treated animals when recording from single synapses (Mameli et al., 2011). In fact, a small NMDAR EPSC was predictive of a large AMPAR EPSC that shows inward rectification, indicating that these changes do occur at the same synapse (Mameli et al., 2011). Moreover, the properties of NMDARs are switched following cocaine exposure. Specifically, Ca2+ permeability of NMDARs, Mg2+ block, and sensitivity to zinc are decreased following cocaine exposure while current decay time and sensitivity to ifenprodil, an inhibitor of GluN2B-containing NMDARs is increased (Yuan et al., 2013). Because the properties of NMDARs are subunit dependent, these results are consistent with a switch from GluN1/GluN2A heteromeric NDMARs to GluN1/GluN2B/GluN3A  13 heterotrimeric NMDARs (Yuan et al., 2013). This switch was absent in GluN3A knock out mice, was reversed by stimulation of mGluR1 and was required for cocaine-induced changes in AMPAR subunit composition at these synapses (Yuan et al., 2013).   Taken together, drug-induced switches in both AMPAR and NMDAR subunit composition would drastically alter glutamatergic synaptic transmission and its ability to regulate dopamine neuron activity. The efficacy of synaptic transmission in terms of exciting neurons from rest would be greatly enhanced by way of the increased conductance of AMPARs and decreased Mg2+ block of NMDARs. Moreover, Ca2+ dynamics are reversed following cocaine-exposure, as Ca2+ now preferentially enters the cell at more hyperpolarized potentials. Accordingly, standard spike-timing dependent LTP protocols, which induce Ca2+-dependent plasticity by way of pairing glutamate release with postsynaptic depolarization no longer induce LTP following cocaine, but LTP is induced if glutamate release is paired with hyperpolarization (Mameli et al., 2011). Because glutamatergic inputs are also important for the induction of burst firing (Johnson et al., 1992; Komendantov et al., 2004; Overton and Clark, 1992; Zweifel et al., 2009), plasticity at these synapses may affect the way burst firing is induced during motivationally relevant conditions (Lobb et al., 2011; Paladini and Roeper, 2014).   Dopamine neurons receive glutamatergic synaptic input from many sources and it is unclear which, and how many of these inputs are altered by drug exposure. Because cocaine drives insertion of GluA1 in only a small portion of synapses (Lane et al., 2010), this form of plasticity likely occurs at specific inputs. Although the inputs are unknown, recent findings offer some insight into which subpopulations of dopamine neurons are targeted by addictive drugs. Lammel et al. (2011) demonstrated that a single injection of cocaine increases the AMPAR/NMDAR ratio in dopamine neurons  14 that project to either the medial- or lateral shell of the NAc, but not in those that project to the medial PFC. NAc lateral shell-projecting dopamine neurons are found primarily in the lateral portions of the VTA, express a large Ih current and have a low basal AMPAR/NMDAR ratio (Lammel et al., 2011). These properties and others discussed below are displayed in Figure 1 (page 21).  Because these characteristics were traditionally used to identify dopamine neurons (Chieng et al., 2011; Margolis et al., 2006a, 2010; Ungless and Grace, 2012), many of the earlier findings described here may apply specifically to this subpopulation of dopamine neurons. It should be noted that an aversive hindpaw injection of formalin also potentiates the AMPAR/NMDAR ratio in NAc lateral shell-projecting dopamine neurons, suggesting that plasticity in this population may code for saliency regardless of valence (Lammel et al., 2011). Interestingly, relative to other projection targets, NAc lateral shell-projecting dopamine neurons receive preferential afferent input from the laterodorsal tegmentum (LDT), and optogenetic stimulation of this input is sufficient to induce a conditioned place preference (CPP) (Lammel et al., 2012). Therefore, it is interesting to consider that perhaps there is a link between valence and input specificity in this subpopulation of dopamine neurons in that rewarding stimuli may preferentially alter inputs from the LDT, while other inputs may be altered by exposure to aversive stimuli. Alternatively, because the removal of an aversive stimulus can be considered rewarding (Navratilova et al., 2012; Xie et al., 2014), perhaps it is the cessation of the effects of formalin that induces plasticity at these synapses.   NAc medial shell-projecting neurons on the other hand express a relatively small Ih current and have a high basal AMPAR/NMDAR ratio (Lammel et al., 2011). Because of this, these neurons would likely have been excluded in early reports, and yet it was in this subpopulation of dopamine neurons that cocaine had the largest effect in terms of  15 both the magnitude and the duration of the increase in the AMPAR/NMDAR ratio (Lammel et al., 2011). In this population, the effect persisted for 3 weeks after a single injection (Lammel et al., 2011), compared to the transient nature of the effect as previously described (Borgland et al., 2004). Interestingly, relative to other parts of the dorsal and ventral striatum, the NAc medial shell is also the primary site of behaviourally relevant dopamine release to unconditioned rewarding stimuli (Aragona et al., 2008; Bassareo et al., 2002; Stuber et al., 2005).   Drugs of abuse alter plasticity at inhibitory synapses onto dopamine neurons  Although the activity of VTA dopamine neurons is also regulated by inhibitory synaptic inputs (Johnson and North, 1992a; Lobb et al., 2011; Tan et al., 2012; van Zessen et al., 2012), fewer studies have examined drug-induced plasticity at inhibitory synapses onto dopamine neurons. Repeated experimenter-administered cocaine decreases the amplitude of GABAA miniature inhibitory postsynaptic currents (mIPSCs) in dopamine neurons (Liu et al., 2005). This facilitates LTP induction at excitatory synapses, in that an LTP protocol that is insufficient in saline-treated animals, induces LTP in cocaine-treated animals (Liu et al., 2005). Moreover, LTP could be induced in control animals when recorded in the presence of the GABAA antagonists bicuculine or picrotoxin (Liu et al., 2005). D1-expressing GABAergic medium spiny neurons (MSNs) in the NAc project to the VTA where they synapse preferentially on VTA GABA neurons, and optogenetic stimulation of axonal terminals of these neurons inhibits the activity of VTA GABA neurons and disinhibits dopamine neurons (Bocklisch et al., 2013). Repeated cocaine administration increases the frequency of spontaneous IPSCs and occludes high frequency stimulation induced LTP (HFS-LTP) at D1-MSN to VTA GABA synapses (Bocklisch et al., 2013). This in turn decreases spontaneous IPSC frequency  16 at synapses onto VTA dopamine neurons and increases both basal firing rate and burst firing of these neurons (Bocklisch et al., 2013). This increase in firing is not observed after a single injection of cocaine, where excitatory synaptic transmission is enhanced, suggesting that increased excitation alone is not sufficient to alter the output of dopamine neurons and highlights the importance of inhibitory signalling onto dopamine neurons (Bocklisch et al., 2013).   Moreover, a single injection of morphine blocks LTP at GABAergic synapses  (LTPGABA) onto dopamine neurons (Niehaus et al., 2010; Nugent et al., 2007). Similar to changes in glutamatergic synaptic transmission, opioid-induced inhibition of LTPGABA is transient and reverses 5 days after exposure through an unknown mechanism. GABAergic synapses onto dopamine neurons also undergo an NMDAR-independent postsynaptic LTD which is absent and presumably occluded in slices from morphine-treated animals (Dacher and Nugent, 2011). Therefore, addictive drugs simultaneously increase the strength of excitatory synapses while decreasing that of inhibitory synapses, creating an imbalance in the synaptic regulation of dopamine neurons.  Because dopamine neurons were identified by the presence of a large Ih in these studies, it is tempting to speculate that this inhibition of LTP and occlusion of LTD at GABAergic synapses occurs at dopamine neurons that project to the lateral shell of the NAc (Lammel et al., 2011). NAc lateral shell projecting-dopamine neurons receive inhibitory inputs from the rostromedial tegmental nucleus (RMTG) (Lammel et al., 2012). Interestingly, opioids disinhibit VTA dopamine neurons through inhibition of the projection from the RMTG (Jalabert et al., 2011; Lecca et al., 2012; Matsui and Williams, 2011; Matsui et al., 2014) and so it is possible that these mechanisms represent a long-term adaptation at these synapses.  17 Behavioural implication of drug-induced plasticity in the VTA  The behavioural significance of drug-induced plasticity of glutamatergic signalling in the VTA is somewhat contentious. Behavioural sensitization reflects a long-lasting enhanced motor response that occurs with repeated drug exposure (Vanderschuren and Kalivas, 2000). Behavioural sensitization is initiated in the VTA and has been linked to dopamine receptor dependent cellular and molecular changes (Cornish and Kalivas, 2001; Perugini and Vezina, 1994; Pierce et al., 1996; Vezina, 1993; Vezina and Stewart, 1989), but changes in glutamatergic transmission are also likely involved. Sensitization is blocked with intra-VTA NMDAR antagonists (Kalivas and Alesdatter, 1993; Vezina and Queen, 2000; Wolf and Jeziorski, 1993) or AMPAR antagonists (Carlezon et al., 1999). Moreover, drug exposure renders dopamine neurons more responsive to glutamatergic input (White et al., 1995; Zhang et al., 1997), and enhanced transmission at these synapses may be responsible for attaching incentive value to stimuli associated with reward (Borgland et al., 2004). Therefore, more efficient excitatory synaptic transmission onto dopamine neurons could underlie sensitization. Further supporting this notion is the finding that tetanic stimulation of the medial PFC, which sends glutamatergic afferents to dopamine neurons in the VTA, can induce sensitization in drug-naïve rats (Schenk and Snow, 1994) and that GluA1 overexpression is sufficient to elicit sensitization (Carlezon et al., 1997). Nevertheless, although locomotor activity strongly correlates with synaptic potentiation after a single dose of drug, the correlation is lost following a sensitizing regimen (Borgland et al., 2004). Moreover, (Engblom et al., 2008) showed a clear dissociation between synaptic potentiation and locomotor sensitization with GluA1 knock out in DAT-CRE (Cre recombinase enzyme) mice. This reflects the fact that although sensitization is initiated  18 in the VTA, it is likely expressed by adaptations in downstream target structures like the NAc (Vanderschuren and Kalivas, 2000; Wolf, 1998).  Similar controversy exists when looking at CPP tasks. On the one hand, GluA1 overexpression potentiates reward in a morphine CPP task (Carlezon et al., 1997), and context-dependent associative components of CPP are lost in global GluA1 knock out mice. In other cases, CPP was observed in global GluA1 knockout mice and in mice that lack GluA1 specifically in VTA dopamine neurons (Engblom et al., 2008; Mead et al., 2005). Therefore, future research will need to further examine the link between synaptic plasticity at dopamine neurons and the promotion of reward seeking behaviour.   Drug-induced plasticity in the VTA as a form of metaplasticity   Although the behavioural significance of drug-induced plasticity in the VTA is ambiguous, the functional significance from a neural circuit perspective is not. Drug-induced synaptic plasticity in the VTA represents both a critical and a permissive step in the induction of downstream plasticity within the mesocorticolimbic system (Creed and Lüscher, 2013; Mameli et al., 2009). Although a single injection of cocaine enhances excitatory synaptic transmission in the VTA (Borgland et al., 2004; Brown et al., 2010; Saal et al., 2003; Ungless et al., 2001), it does not alter synaptic transmission in the NAc. However, cocaine does induce bidirectional plasticity in the NAc with repeated exposure to the drug. Five injections of cocaine followed by 10-14 days of withdrawal induces an increase in the AMPAR/NMDAR ratio of excitatory synapses onto NAc MSNs (Kourrich et al., 2007), but is reversed to a depression of the AMPAR/NMDAR, which occludes low frequency stimulation-induced LTD in the NAc, with a single re-exposure to cocaine (Thomas et al., 2001). Interestingly, when animals are treated with  19 an mGluR1 antagonist to block the internalization of GluA2-lacking AMPARs, making VTA plasticity persistent, a single injection of cocaine is sufficient to depress synaptic transmission in the NAc (Mameli et al., 2009). The same occurs with a local infusion of a dominant negative peptide that interferes with mGluR1 function (Mameli et al., 2009). Conversely, if repeated injections of cocaine are given with a positive allosteric modulator of mGluR1, cocaine-induced plasticity in the NAc is abolished (Mameli et al., 2009). Therefore, drug-induced synaptic plasticity in the VTA is often described as metaplasticity, such that these drug-induced changes in synaptic transmission set the stage for further plasticity in regions like the NAc, rather than coding for specific events or behaviours itself (Creed and Lüscher, 2013). Moreover, it suggests that inhibiting, or reversing plasticity in the VTA may be a means to interrupt the development of addiction early on.   Persistent plasticity in the VTA likely increases the activity of dopamine neurons that project to the NAc, where dopamine release modulates glutamatergic synaptic activity (Nicola et al., 2000; Wolf et al., 2004). Excitatory inputs to the NAc from regions like the hippocampus, the BLA and the PFC provide information about contextual cues in the environment (Britt et al., 2012; Loweth et al., 2014; Sesack and Grace, 2010) and so dopamine neuron activity and downstream dopamine release in the NAc might alter excitatory synaptic transmission onto MSNs, to attribute salience to specific cues related to the drug or reward experience.  Importantly many drug-induced phenotypes are observed with selective stimulation of VTA dopamine neurons. Brown et al. (2010) demonstrated that a 2 hour intermittent optogenetic stimulation protocol, which mimics the time course of cocaine-induced increases in DA concentration (Frank et al., 2008) is sufficient to induce the insertion of GluA2-lacking AMPARs at synapses onto VTA dopamine neurons in a D1R-dependent manner. Moreover, withdrawal from VTA  20 dopamine neuron self-stimulation is sufficient to induce the insertion of GluA2-lacking AMPARs and to increase the AMPAR/NMDAR ratio at synapses onto D1-MSNs in the NAc (Pascoli et al., 2015). Finally, VTA dopamine neuron self-stimulation is sufficient to induce hyper-excitability of pyramidal neurons in the orbitofrontal cortex, which drives self-stimulation and compulsive-like cocaine self-administration in foot-shock resistant mice (Pascoli et al., 2015). Together, this suggests the ability of drugs to target the mesocorticolimbic dopamine system is fundamental to their ability to drive the development of addiction.  All that being said, it is unclear how drugs of abuse, a pharmacologically diverse group, all converge to alter excitatory and inhibitory synaptic transmission in the VTA. It has been postulated that all drugs may act on a common substrate that in turn influences synaptic strength and maintains the effects over time (Niehaus et al., 2010). Chapter 2 of this thesis presents data indicating that the lateral hypothalamic orexin system may fulfill this role.     21     Figure 1: Input and output neurocircuits of projection target defined VTA dopamine neurons   This schematic depicts a subset of the afferent inputs and the efferent outputs of VTA dopamine neurons. It also illustrates the specific properties of these neurons as revealed by electrophysiological recordings in retrograde labelled cells, pharmacological analysis and by selective optogenetic stimulation.    22 Lateral hypothalamic orexin neurons and motivated behaviour   The lateral hypothalamus (LH) has long been studied for its role in motivated behaviour. Electrical stimulation of the LH can evoke behaviours including locomotion and goal-directed activities like feeding, drinking, and copulation (Valenstein et al., 1970). Because both rodents and humans will perform operant tasks for electrical stimulation of the LH (Heath, 1963; Olds and Milner, 1954), the LH is now recognized as a critical part of the complex circuitry that regulates reward and reinforcement (Baimel et al., 2015). Accordingly, addictive drugs modulate operant responding for self-stimulation of the LH (Adams et al., 1972; Carey and Goodal, 1975), and animals will perform operant tasks for direct administration of drugs into the LH (Cazala et al., 1987).      The orexin neuropeptides, also known as hypocretins, were independently discovered by two groups nearly two decades ago (de Lecea et al., 1998; Sakurai et al., 1998). Orexins consist of 2 peptides, orexin A and orexin B, both cleaved from the same pre-pro-orexin precursor molecule (de Lecea et al., 1998; Sakurai et al., 1998). Orexin neurons reside in the portions of the LH that support self-stimulation (Hollander et al., 2008; Peyron et al., 1998; Valenstein et al., 1970), and the functional effects of orexin receptor activation mimic those of LH stimulation. Central orexin administration evokes feeding (Dube et al., 1999; Haynes et al., 1999; Sakurai et al., 1998), drinking (Kunii et al., 1999), copulation (Gulia et al., 2003) and locomotion (Kotz et al., 2002). Together this suggests that orexin neurons may be one of the critical substrates in the LH involved in the regulation of motivated behaviour. Much literature supports this view and proposes that the orexin system is critical to the integration of both external cues and internal states to coordinate arousal levels and drive motivational action (Baimel et al., 2015; Mahler et al., 2014). These behaviours are partly engaged through orexin  23 projections to reward-responsive dopamine neurons in the VTA (Baimel et al., 2015; Balcita-Pedicino and Sesack, 2007; Fadel and Deutch, 2002).   Orexins bind and activate two GPCRs:  orexin receptor type 1 (OxR1) and orexin receptor type 2 (OxR2) (Sakurai et al., 1998). Although most of the functional roles of orexin receptor activation have been linked to the Gq pathway (Borgland et al., 2006, 2008; Uramura et al., 2001; Yang et al., 2003), both receptors have been shown to interact with Gq, Gs and Gi/o proteins (Kukkonen and Leonard, 2014). A lack of direct methods to measure G-protein activation has prevented a definitive evaluation of orexin receptor G-protein coupling (Baimel et al., 2015; Kukkonen and Leonard, 2014). Despite being small in number, the orexin cell population sends dense projections throughout the brain, including a dense projection to the VTA (Baldo et al., 2003; Peyron et al., 1998). OxR1 and OxR2 are similarly widely distributed within the brain (Trivedi et al., 1998), but their expression patterns are not uniform. For example, there is higher expression of OxR1 in cortical regions, the bed nucleus of the stria terminalis and the locus coeruleus, whereas OxR2 density is enriched in the NAc and in subregions of the thalamus and hypothalamus; the are relatively similar levels of expression of both receptors in the VTA (Baimel et al., 2015; Sakurai et al., 1998; Trivedi et al., 1998).   Orexin peptides were discovered for their role in regulating feeding and arousal (Adamantidis et al., 2007; Chemelli et al., 1999; Lin et al., 1999; Peyron et al., 2000; Sakurai et al., 1998; Thannickal et al., 2000). Accordingly, the loss of orexin neurons results in the neurological syndrome of narcolepsy, characterized by excessive daytime sleepiness (Burgess and Scammell, 2012; Chemelli et al., 1999; Lin et al., 1999). Interestingly, narcoleptic patients have a lower prevalence of drug abuse (Akimoto et al., 1960; Guilleminault et al., 1974; Nishino and Mignot, 1997), although it is unclear if and how narcolepsy alters reward processing  in narcoleptic patients (Dimitrova et al.,  24 2011). Orexin peptides are released in a circadian rhythm and are elevated in the cerebrospinal fluid during active phases and are low during the resting phase (Estabrooke et al., 2001, Blouin et al., 2013). These fluctuations in peptide levels are consistent with the level of activity of orexin neurons which show a similar pattern ((Lee et al., 2005; Mileykovskiy et al., 2005). Orexin signalling has been linked to many different behavioural phenotypes, but a recent hypothesis suggests that across all domains orexin plays a critical role in driving motivational activation (Mahler et al., 2014). Orexin neurons are activated by internal, homeostatic or external, motivationally relevant signals and coordinate both psychological and physiological processes to facilitate adaptive behaviours (Mahler et al., 2014).     Orexin neurons are so named by way of the fact that they are the only known source of orexin A and orexin B. That being said, this label is potentially misleading due to the fact that these cells also contain dynorphin (Chou et al., 2001; Muschamp et al., 2014) and release glutamate (Rosin et al., 2003; Schöne et al., 2012, 2014). Although it has been largely overlooked, potential simultaneous or corelease of these transmitters may have important functional consequences. Moreover, although orexin neurons are not thought to be GABAergic (Rosin et al., 2003), recent evidence suggests that orexin neurons may be capable of releasing GABA at certain sites (Apergis-Schoute et al., 2015). Optogenetic stimulation of orexin neurons evokes currents in melanin-concentrating-hormone (MCH) cells that persist in the presence of CNQX and APV, blockers of AMPAR and NMDARs, and which decrease the firing of postsynaptic MCH neurons in a GABAA dependent manner (Apergis-Schoute et al., 2015). Together, this suggests that orexin neurons are capable of very complex regulation of downstream targets.     25 Neurotransmitter and neuropeptide release from orexin neurons  Because orexin neurons synthesize and release multiple transmitters and peptides (Apergis-Schoute et al., 2015; Chou et al., 2001; Muschamp et al., 2014; Rosin et al., 2003; Schöne et al., 2012, 2014), it is important to consider the conditions under which each would be released. Although there is substantial overlap in the processes that regulate neurotransmitter and neuropeptide release (Südhof, 2012a), the differences confer important functional considerations. Small molecule amino acid neurotransmitters and neuropeptides are stored in separate pools of vesicles, clear synaptic vesicles and dense-core vesicles respectively. Interestingly, both orexin and glutamate containing vesicles are found in the same terminals (Balcita-Pedicino and Sesack, 2007; de Lecea et al., 1998). Nevertheless, amino acid neurotransmitters are released by Ca2+ dependent exocytosis at specialized sites in the presynaptic “active zone” (Couteaux and Pécot-Dechavassine, 1970), where docked and primed vesicles are stored in close proximity to voltage-gated Ca2+ channels to allow fast synchronous excitation-release coupling (Südhof, 2012a). Neuropeptides, on the other hand, are less restrictive and are released from many sites void of synaptic specializations away from the active zone (van den Pol, 2012). The dispersed nature of peptide release sites creates an interesting paradox in release requirements. Although dense-core vesicles have a lower Ca2+threshold for vesicle fusion (~1 μM compared to 10-100 μM for synaptic vesicles) (Südhof, 2012b), they require a greater Ca2+ signal to be released, such that cytosolic Ca2+ concentrations need to be high enough to permit diffusion of Ca2+ to the extrasynaptic sites (van den Pol, 2012; Südhof, 2012b). Therefore, neuropeptide release likely requires high frequency trains of action potentials. In line with this is the observation that optogenetic stimulation of orexin terminals in the tuberomammillary nucleus at 1, 5, 10 and 20 Hz induces glutamate receptor currents in  26 postsynaptic histamine neurons, but orexin release, measured by an increase in orexin receptor dependent histamine neuronal firing rate is only observed with 20 Hz stimulations (Schöne et al., 2014). Neuropeptide release form extrasynaptic sites does not imply that neuropeptides cannot act on cells immediately postsynaptic to the axon, but rather that neuropeptide release and activity is likely much less confined in space (van den Pol, 2012). In fact, the diffusion of amino acid neurotransmitters is somewhat limited by the structure of the synapse itself and so they only diffuse small distances in the tens of nanometers, before they undergo rapid degradation or reuptake (van den Pol, 2012). Neuropeptides, on the other hand, often lack specific reuptake mechanisms and rather are subject to degradation by peptidases that are present in the extracellular space (van den Pol, 2012). Because of this, peptides often have longer extracellular half-lives, which increases the time available for diffusion and are said to be released by volume transmission (Agnati et al., 1986, 1995, 2010).  The extrasynaptic nature of peptide release is of particular relevance for the orexin input to the VTA. Although orexin fibres are present throughout the VTA, only 15% of orexin axons make appositional contacts in the VTA and even less (5%) show identifiable synaptic specializations (Balcita-Pedicino and Sesack, 2007). However, most axons stain heavily for dense-core vesicles suggesting that orexin release in this region is largely extrasynaptic (Balcita-Pedicino and Sesack, 2007). Given that neuropeptide release can occur at sites void of synaptic specializations, the low synaptic incidence of orexin neurons in the VTA likely does not impede its neuromodulatory influence over VTA dopaminergic and GABAergic neurons (Baimel et al., 2015). It is also important to consider that peptides are generated through protein synthesis in the endoplasmic reticulum and are processed in the Golgi apparatus before  27 being packaged in dense-core vesicles and transported to presynaptic terminals through axonal transport (Tallent, 2008; van den Pol, 2012). Because of the large energy expenditure required for this process, neuropeptides may be released in smaller number of molecules, although this can be compensated for by amplification at GPCRs and increased potency at its receptors (van den Pol, 2012). That being said, the time scale required to replenish pools of dense-core vesicles is much greater than that of synaptic vesicles, which are regulated within the terminals (van den Pol, 2012).  Because of this, orexin release may occur in intermittent bouts.    Drug-induced activation of orexin neurons  In terms of anatomical connectivity, orexin neurons are well situated to mediate reward and motivation. Many studies have examined the role of orexin neurons in drug-seeking behaviour. In CPP tasks, orexin neurons in the LH, but not the dorsomedial hypothalamus (DMH) or the perifornical area (PFA), and that project to the VTA but not the locus coeruleus (Richardson and Aston-Jones, 2012), are activated by cues associated with both drug (cocaine and morphine) and food rewards (Harris et al., 2005). Moreover, the preference for the drug-paired environment positively correlates with expression levels of the immediate early gene cFOS in orexin neurons (Harris et al., 2005), a surrogate marker of neuronal activation. Interestingly, following extinction training, electrical stimulation of the LH or microinfusion of orexin A into the VTA reinstates CPP, both of which are inhibited by administration of the OxR1 antagonist N-(2-Methyl-6-benzoxazolyl)-N'-1,5-naphthyridin-4-yl urea (SB 334867) (Harris et al., 2005). Conversely, stressful stimuli like foot shock, which can also reinstate drug seeking, do not activate LH orexin neurons but rather activate those in the DMH and PFA (Baimel et al., 2015; Harris et al., 2005). Together, this suggested that there may be  28 a functional dichotomy of orexin neurons in terms of their response to pharmacological and environmental stimuli and how they in turn organize behaviour (Harris and Aston-Jones, 2006). It was proposed that DMH and PFA orexin neurons were involved in promoting arousal (Harris and Aston-Jones, 2006; Sharf et al., 2010), whereas LH orexin neurons stimulated reward seeking (Harris and Aston-Jones, 2006; Willie et al., 2003). This dichotomy was thought to result from discrete targeting of orexin projections within each sub-region; such that DMH and PFA orexin neurons projected to regions like locus coeruleus to induce arousal; and LH orexin neurons likely influenced reward through projections to the reward circuit including, but not limited to, the VTA (Baimel et al., 2015; Harris and Aston-Jones, 2006). However, orexin neurons that project to both the locus coeruleus and the VTA are present and spread throughout the orexin field, and VTA-projecting orexin neurons outnumber locus coeruleus-projecting neurons in medial areas (González et al., 2012). In addition, a sensitizing regimen of amphetamine induces cFos orexin double-labeling throughout the orexin field (McPherson et al., 2007), as does naloxone-induced morphine withdrawal (Georgescu et al., 2003; Laorden et al., 2012). Therefore, an alternative hypothesis is that differential recruitment of orexin neurons results from the activation of distinct upstream afferent projections to these neurons rather than anatomically defined segregation within the hypothalamus. Accordingly, orexin neurons in the medial regions receive preferential innervation from hypothalamic neurons while those in the LH are targeted by inputs from the brain stem and reward-related regions (Yoshida et al., 2006). Moreover, neurons in the rostral lateral septum, which are activated by addictive drugs and cues in the environment linked to the drug taking experience (Shoji et al., 1997, 1998), send projections to the LH, but not to the DMH or PFA (Baimel et al., 2015; Sartor and Aston-Jones, 2012).   29 Drug-induced plasticity at orexin neurons  Orexin neurons are somewhat unique in terms of their regulation by synaptic input (Gao and Hermes, 2015). In most long projecting neurons, inhibitory synapses are clustered around the cell body and excitatory inputs are found on more distal dendrites (Douglas and Martin, 2004; Horvath and Gao, 2005). However, orexin neurons have more asymmetric, presumably excitatory, synapses than symmetric inhibitory synapses on their cell bodies (Horvath and Gao, 2005). Accordingly, the frequency of excitatory mEPSCs is 10-fold higher than that of mIPSCs in these neurons (Horvath and Gao, 2005). Furthermore, blocking glutamatergic transmission onto orexin neurons significantly inhibits firing of these neurons while inhibition of GABAA receptors has little effect (Li et al., 2002; Xie et al., 2006). Interestingly under basal conditions, orexin neurons express GluA2-lacking AMPARs and the amplitude of AMPAR EPSCs are far greater than that of NMDAR EPSCs, giving them a high AMPAR/NMDAR ratio (Gao and Hermes, 2015; Rao et al., 2007, 2008, 2013). Together this suggests that orexin neurons are likely highly responsive to presynaptic glutamate input and that Ca2+ regulation of synaptic plasticity in these cells may be dependent on AMPARs, rather than NMDARs (Gao and Hermes, 2015). Moreover, efficient glutamatergic transmission onto orexin neuron may facilitate excitation of these neurons in response to motivationally relevant sensory input (Gao and Hermes, 2015).   The critical role of drug-induced plasticity within the mesocorticolimbic dopamine system in the development and the expression of addiction-like behaviours has been well characterized (Creed and Lüscher, 2013; Kauer and Malenka, 2007; Lüscher et al., 2015), but drug-induced modifications in synaptic transmission are not limited to these brain regions. In fact, given the unique synaptic regulation of orexin neurons, and their connections to the mesocorticolimbic dopamine system, drug- 30 induced alterations in excitatory synaptic transmission onto orexin neurons may have important functional consequences towards motivated behaviour.   In vivo repeated cocaine administration induces synaptic plasticity at excitatory synapses onto orexin neurons. In mice that express a CPP for cocaine, both the AMPAR/NMDAR ratio and the amplitude of AMPAR mEPSCs are increased relative to saline control mice (Rao et al., 2013). This plasticity was not induced with a single injection of cocaine, nor was it dependent on OxR1 signalling and was observed 5, but not 10 days after exposure (Rao et al., 2013). Interestingly, this up regulation of postsynaptic AMPARs did not occlude, but rather facilitated the induction of high frequency stimulation induced LTP (Rao et al., 2013). This suggests that cocaine-induced plasticity at these synapses, similar to that at synapses in the VTA, may serve as a type of metaplasticity lowering the threshold for plasticity induced by other synaptic stimulations (Gao and Hermes, 2015).  In contrast, others have reported presynaptic cocaine-induced plasticity including an increase in the frequency of mEPSCs, paired pulse-depression, and an increase in the number of vesicular glutamate transporter 2 (VGLUT-2) positive puncta onto orexin neurons (Yeoh et al., 2012). Nevertheless, it should be noted that the behavioural implication of cocaine-induced plasticity at orexin neurons has not been examined, and it is unclear whether or not synaptic plasticity at these synapses is involved in the development of drug seeking behaviours. In fact, the OxR1 antagonist SB 334867 blocks cocaine CPP despite the persistence of cocaine-induced plasticity at synapses onto orexin neurons (Rao et al., 2013). Therefore, downstream release of orexin peptides is likely the critical factor driving motivated behaviour and plasticity at orexin neurons may therefore contribute to motivated behaviour by increasing the excitability of these neurons in order to enhance the likelihood of orexin release (Baimel et al., 2015).   31  Given the role of orexin receptor signalling in morphine CPP (Harris et al., 2005), it is surprising that application of MOR agonists decrease the firing rate and the frequency of mEPSCs onto orexin neurons (Baimel et al., 2015; Li and van den Pol, 2008). That being said, the effect of in vivo morphine exposure on the activity of orexin neurons has not been determined. Moreover, because orexin neurons are a functionally segregated cell population (Harris and Aston-Jones, 2006) and only half of orexin cells express MORs (Georgescu et al., 2003), it is possible that the population of cells inhibited by morphine do not project to reward-related brain areas but may mediate the sedative effects of the drug through projections to arousal related regions. The acute effects of other addictive drugs on the activity of orexin neurons have not been examined.    Orexin modulates synaptic transmission in the VTA   In whole-cell patch clamp recordings from VTA dopamine neurons, orexin A induces a dose-dependent transient increase in NMDAR EPSCs (Borgland et al., 2006). This potentiation is due to the activation of protein kinase C (PKC)/ phospholipase C (PLC) intracellular signalling cascades and results in increased trafficking of NMDARs containing the GluN2A subunit to the membrane (Borgland et al., 2006). Additionally, NMDAR mEPSC amplitude is increased 15 minutes, but not 3-4 hours after orexin A application, further indicating the short-term nature of this potentiation (Borgland et al., 2006). One of the major roles of NMDARs in dopamine neurons is the promotion of burst firing (Overton and Clark, 1992; Suaud-Chagny et al., 1992; Tong et al., 1996) a phenomenon associated with more efficient dopamine release in downstream target structures. Therefore, orexin A-induced potentiation of NMDAR may result in increased dopamine neuron activity and increased dopamine release in projecting regions. Orexin  32 A application to the VTA increases the firing rate of dopamine neurons and in some cases induces burst firing (Korotkova et al., 2003; Muschamp et al., 2007), although it is still unknown if this increase in the firing rate is dependent on potentiation of glutamatergic transmission. Nevertheless, intra-VTA infusions of orexin A increase dopamine release in the both the NAc (Narita et al., 2006) and the PFC (Vittoz and Berridge, 2006; Vittoz et al., 2008) and can induce CPP, which is blocked with PKC and PLC inhibitors (Narita et al., 2007).   NMDARs are involved in the induction of certain forms of LTP. It is therefore possible that orexin A-induced potentiation of NMDAR EPSCs facilitates the induction of synaptic plasticity in dopamine neurons (Baimel and Borgland, 2015; Borgland et al., 2006). Interestingly, there are no acute effects of orexin A application on AMPAR EPSCs (Borgland et al., 2006). However, orexin A application increases the AMPAR/NMDAR ratio when measured 3-4 hours later, an effect that is associated with an increase in both the frequency and the amplitude of AMPAR mEPSCs (Borgland et al., 2006). The increase in mEPSC amplitude, but not the frequency, was inhibited with the NMDAR antagonist APV, suggesting that AMPAR EPSCs were potentiated through an NMDAR-dependent postsynaptic mechanism as well as a NMDAR-independent increase in glutamate release (Borgland et al., 2006).   Orexin B is presumably coreleased with orexin A and increases the firing rate of dopamine neurons (Korotkova et al., 2003), as well as potentiating NMDAR and AMPAR EPSCs (Borgland et al., 2008). Orexin B in the VTA signals primarily through OxR2 and potentiates NMDAR EPSCs via a PKC-dependent mechanism (Borgland et al., 2008). Additionally, orexin B induces a presynaptic increase in glutamate release, which surprisingly is not blocked by antagonists for either OxR1 nor OxR2 suggesting that the enhanced presynaptic glutamate release may be due to orexin B or one of its  33 metabolites acting at an unidentified PKC-coupled receptor (Borgland et al., 2008). Interestingly, although orexin B infusions in the VTA induce a place preference and increase dopamine release in the NAc (Narita et al., 2007), inhibition of OxR2 does not alter reward-seeking behaviours (Prince et al., 2015; Smith et al., 2009; Wang et al., 2009). Therefore, orexin B likely mediates reward seeking by acting on OxR1, while OxR2 in the VTA may primarily mediates arousal-related functions (Aston-Jones et al., 2010).  Although there are multiple reports demonstrating the ability of orexin to increase the firing rate of dopamine neurons, the effect is quite variable in that  many dopamine neurons do not respond to orexin application (~50%) (Korotkova et al., 2003). Given the newfound heterogeneity of the dopamine system, both in terms of cellular properties and circuit level connections (Lammel et al., 2008; 2011; 2012; Margolis et al., 2008), the inconsistent response to orexin application within the VTA might suggest that specific subsets of dopamine neurons are sensitive to the activating effects of orexin (Baimel et al., 2015). Accordingly, orexin A preferentially induces cFOS in VTA dopamine neurons that project to the medial PFC and the NAc shell (Vittoz et al., 2008). Chapter 3 of this thesis presents data suggesting that orexin and dynorphin independently target different subpopulations of dopamine neurons.    Orexin modulates cocaine-induced plasticity in the VTA   Although no one has directly measured the acute effects of in vivo drug exposure on the activity of orexin neurons, indirect evidence suggests that drugs of abuse activate orexin neurons. Orexin neurons show high levels of cFos expression in drug CPP paradigms (Harris et al., 2005) and both systemic or intra-VTA SB 334867 attenuates cocaine- or amphetamine-induced increases in dopamine concentration in  34 the NAc (España et al., 2010a; Quarta et al., 2010). Moreover, intra-VTA infusion of orexin A potentiates cocaine-induced increases in dopamine concentration and the efficacy of cocaine-mediated dopamine reuptake inhibition (España et al., 2011a). Because orexin neurons are likely activated by addictive drugs, and orexin potentiates NMDAR EPSCs in the VTA (Borgland et al., 2006), signalling at OxR1 may be involved in the NMDAR-dependent increase in excitatory transmission induced by addictive drugs (Ungless et al., 2001; Saal et al., 2003; Borgland et al., 2004; Brown et al., 2010). Interestingly, when animals are pretreated with the OxR1 antagonist SB 334867, cocaine-induced potentiation of excitatory synaptic transmission is blocked (Borgland et al., 2006). Moreover, intra-VTA SB 334867 blocks the development, but not the expression of cocaine sensitization (Borgland et al., 2006), as well as sensitization induced by morphine (Narita et al., 2006) and amphetamine (Quarta et al., 2010).   Interestingly, self-administration of drugs or other highly salient reinforcers enhances the functional circuit between orexin and dopamine neurons. Following cocaine or high fat food pellet self-administration, the magnitude of orexin-induced potentiation of NMDAR EPSCs is greater than that in naïve animals, or animals that self-administered regular food (Borgland et al., 2009). Moreover, in these animals orexin potentiates NMDAR EPSCs at doses that are subthreshold in control animals (Borgland et al., 2009). Therefore, exposure to salient reinforcers may facilitate the ability of orexin neurons to alter the output of VTA dopamine neurons.   Orexin in the VTA modulates motivated behaviour   Orexins modulate many aspects of reward seeking, but several lines of evidence point to a particular role for orexin in motivated drug seeking. In self-administration studies, OxR1 antagonists reduce cocaine self-administration under progressive ratio  35 schedules of reinforcement, where animals must exert progressively greater effort to obtain the same cocaine infusion (Borgland et al., 2009; España et al., 2010b), but not on low fixed ratio schedules of reinforcement when drug infusions are easily obtained (Smith et al., 2009; España et al., 2010b). Similarly, in an experiment where the dose of cocaine progressively declines within a session, such that the animal must progressively increase responding to maintain preferred levels of cocaine, SB 334867 has little effect in the early portion of the session when the response requirement is low, but attenuates responding later in the session as the response requirement increases (España et al., 2010b). Moreover, local infusion of orexin A in the VTA increases the breakpoint for cocaine self-administration on progressive ratio schedules of reinforcement (España et al., 2011b), suggesting that VTA orexin A increases effortful responding for cocaine. In contrast, OxR1 antagonists do reduce heroin self-administration on low effort fixed-ratio 1 schedules of reinforcement (Smith and Aston-Jones, 2012), suggesting that the role of orexin in reinforcement may differ across drugs or perhaps that the motivational aspect of heroin self-administration may differ from that of cocaine. Interestingly, in a behavioural economics task, SB 334867 only reduces cocaine demand when cocaine-associated cues are present (Bentzley and Aston-Jones, 2015),  further supporting the idea that orexin neurons integrate sensory stimuli to drive motivated behaviour (Mahler et al., 2014).   Relapse to drug seeking is a common characteristic of addiction that can be modeled in rodents. Because cues in the environment hold salient motivational properties (Robinson and Berridge, 1993), they are capable of reinstating drug-seeking. OxR1 antagonists block cue-induced reinstatement of drug seeking for cocaine (Smith et al., 2009) and heroin (Smith and Aston-Jones, 2012). Interestingly, cue-induced reinstatement of cocaine seeking requires both OxR1 and AMPAR signalling in the VTA  36 (Mahler et al., 2013). Treatment with a positive allosteric modulator of AMPAR reverses SB 334867 inhibition of reinstatement of drug seeking (Mahler et al., 2013). Moreover, intracerebroventricular orexin administration is sufficient to reinstate cocaine seeking (Boutrel et al., 2005). Although intra-VTA infusions of an OxR1 antagonist reduces cocaine-induced increases in dopamine concentration (España et al., 2010b), it does not block drug-primed reinstatement of cocaine-seeking (Smith et al., 2009). Together, this suggests that orexin in the VTA promotes motivated behaviour when the effort requirement is high and drives reinstatement of drug seeking by modulating signalling at AMPARs.  Orexin neurons contain the kappa opioid receptor agonist dynorphin  The neuropeptide dynorphin is widely distributed in the brain and is the endogenous ligand for the kappa opioid receptor (KOR), but also binds with low affinity to both MORs and delta opioid receptors (Zhang et al., 1998). Dynorphin is cleaved from the precursor prodynorphin (Chavkin et al., 1982; Corbett et al., 1982) and is widely known to mediate negative emotional states. KOR agonists induce place aversions, depression-like behaviour, and dysphoria in both humans and animals (Mucha and Herz, 1985; Pfeiffer et al., 1986; Shippenberg et al., 2007).  In the LH, dynorphin is almost exclusively expressed in orexin neurons, and nearly all orexin neurons contain dynorphin at both the mRNA and the protein level (Chou et al., 2001). Although dynorphin release from orexin neurons has been largely overlooked, it likely has important functional consequences. Both orexin and dynorphin levels in the LH increase at night, when animals are awake (Przewłocki et al., 1983; Fujiki et al., 2001), suggesting that both peptides share a circadian rhythm. Moreover, human narcoleptic patients show a marked reduction in both orexin and dynorphin in  37 the hypothalamus (Crocker et al., 2005), and ablating orexin neurons yields a phenotype very similar to that of human narcolepsy (Hara et al., 2001), while this phenotype is only mildly evident in orexin peptide knockout mice (Chemelli et al., 1999; Willie et al., 2001). Together this suggests that orexin and dynorphin may act together to regulate arousal and other behaviours, and highlights the importance of taking into account the full peptide and neurotransmitter profile of a neuron when examining function.  Orexin receptors (Sakurai et al., 1998) and KORs (DePaoli et al., 1994) are expressed widely throughout the brain and have opposing actions on cellular excitability. Orexin binds to excitatory, primarily Gq coupled receptors, and increases the firing rate of postsynaptic neurons, while dynorphin binds to Gi/Go-coupled kappa-opioid receptors (Chavkin et al., 1982) and inhibits neural activity by activating GIRK channels . Nevertheless, recent evidence suggests that orexin and dynorphin are co-released (Muschamp et al., 2014). Orexin and dynorphin colocalize in 94% of neurons in the LH and are found together within the Golgi apparatus, in axonal processes, and importantly in axonal terminals at asymmetric synapses, where both peptides are located in the same large vesicles (Muschamp et al., 2014).   There are many ways in which corelease of orexin and dynorphin could modulate the activity of downstream target neurons. Because orexin and dynorphin have opposing effects on cellular excitability, the obvious notion is that they would counteract each other, but this is not necessarily the case. In the arcuate nucleus, orexin and dynorphin have synergistic effects in that orexin directly excites neuropeptide Y containing cells and dynorphin inhibits GABA release onto these neurons (Li and van den Pol, 2006). The same is true of orexin and dynorphin release onto histaminergic tuberomammillary neurons (Eriksson et al., 2004). Both of these examples highlight the fact that the effect of corelease of orexin and dynorphin is very  38 much dependent on the receptor expression of the target neurons (Li and van den Pol, 2006). It is possible that at terminal release sites, cells may only express the receptor for orexin or dynorphin, and therefore would only respond to one of the peptides (Li and van den Pol, 2006). Chapter 3 of this thesis presents data suggesting that this is one way that orexin neurons coordinate the output of the VTA. That being, said, even if target cells express both receptors, receptor activity may be differentially regulated. For example, MCH neurons in the hypothalamus are more responsive to dynorphin than they are to orexin, therefore coapplication of these peptides induces a hyperpolarizing current (Li and van den Pol, 2006). However, with repeated application of these peptides, the current becomes depolarizing and dominated by orexin signalling due to more rapid desensitization of KORs in these cells (Li and van den Pol, 2006). Therefore, in some cases the level and the pattern of release may determine the response. Alternatively, the peptides may act with different time courses due to latencies in signalling or in duration of action. In this scenario, one peptide may limit the effect of the other over time (Li and van den Pol, 2006). Moreover, the activation of one receptor may alter the signalling of the other. For example, in cells heterologously expressing both OxR1s and KORs, OxR1 activation promotes preferential β-arrestin/p38 signalling over Gi of KORs (Robinson and McDonald, 2015). Finally, and perhaps the most intriguing notion, is that although these peptides are copackaged in vesicles, they are encoded by different genes whose transcription is likely regulated by different factors (Li and van den Pol, 2006; Li et al., 2014; Muschamp et al., 2014). Therefore, it is possible that under certain circumstances or physiological states, orexin and dynorphin may be differentially synthesized. This may drastically alter the modulatory effect of orexin neuron activity on downstream structures like the VTA.  39  Dynorphin signalling in the mesocorticolimbic dopamine system    The behavioural profile of KOR activation differs greatly from that of other opioid receptor agonists. MOR agonists like morphine induce a CPP (Shippenberg and Herz, 1986) by indirectly increasing the activity of VTA dopamine neurons and subsequent  downstream dopamine release in target structures (Latimer et al., 1987; Leone et al., 1991; Spanagel et al., 1992; Devine et al., 1993). Accordingly, direct infusion of MOR agonists into the VTA, but not in the NAc or the medial PFC, is sufficient to induce a place preference (Bals-Kubik et al., 1993). KOR activation on the other hand induces a conditioned place aversion (CPA) (Shippenberg and Herz, 1986), which is lost in selective dopamine neuron KOR knockout mice and is mediated in part by agonist-induced decreases in NAc dopamine release (Chefer et al., 2013). Interestingly, although CPA and KOR-induced decreases in NAc dopamine levels are restored with KOR expression in the VTA (Chefer et al., 2013), DA concentrations in the NAc are not decreased by direct  administration of KOR agonists into the VTA, but rather by local signalling within the NAc (Spanagel et al., 1990; Thompson et al., 2000). This suggests that KORs translated in the VTA are likely transported to axonal terminals in the NAc to mediate KOR-induced CPA. Accordingly, in the NAc KOR immunoreactivity is found on axons that express DAT, as well as on DAT-negative axons that make contacts onto pre-synaptic DAT-positive terminals (Svingos et al., 2001).  Taken together it is not surprising to note that KOR agonist administration into the NAc induces a CPA (Bals-Kubik et al., 1993). That being said, place aversions are also observed with microinjections of KOR agonists into the medial PFC, the LH and the VTA (Bals-Kubik et al., 1993), suggesting that multiple KOR pools may be involved, or that KOR  40 signalling in different brain areas may activate different phenotypes but all of negative valence and therefore all capable of inducing place aversion.   Because direct injection of KOR agonists into the VTA induces a CPA, Margolis et al. (2003) aimed to determine if this was due to inhibition of dopamine neurons in this region. Dopamine neurons in the VTA express KOR (Speciale et al., 1993; Arvidsson et al., 1995; Mansour et al., 1996) and multiple dynorphin expressing neurons project to the VTA, including neurons in the hypothalamus (Fallon et al., 1985). KOR agonists inhibited a subset (about 50%) of tyrosine hydroxylase positive neuron that expressed an Ih current via postsynaptic activation of GIRK channels (Margolis et al., 2003). Moreover, KOR agonists induced a small decrease in the probability of presynaptic glutamate release onto these neurons (Margolis et al., 2005). Because these effects were only observed on a subset of dopamine neurons, a few groups attempted to identify which neurons were responsive to KOR agonist administration. Interestingly, the effects of KOR agonists on VTA dopamine neurons are dependent on projection target. Margolis et al. (2006, 2010) determined that KOR agonists inhibit both amygdala- and medial PFC-projecting dopamine neurons, but not neurons that project to the medial portions of the NAc. Accordingly, intra-VTA infusion of KOR agonists in vivo reduces mPFC, but not NAc, dopamine concentration (Margolis et al., 2006a). This suggests that KOR-induced aversion could be regulated at the level of the VTA for neurons projecting to the medial PFC and the amygdala, and at dopamine terminals in the NAc (Spanagel et al., 1990; Bals-Kubik et al., 1993; Thompson et al., 2000). However, intra-VTA microinjections of KOR agonists decrease haloperidol-induced elevations of dopamine (Leyton et al., 1992). Furthermore, only 4 of the NAc-projecting neurons tested in this study were confirmed to be dopaminergic (Margolis et al., 2006a), suggesting that non-dopaminergic neurons may account for this discrepancy.   41 Moreover, others have reported that 94% of NAc medial shell-projecting putative dopamine neurons are responsive to KOR agonist application, compared to only 13% of neurons that project to the BLA (Ford et al., 2006) and GABAA, GABAB, and D2 IPSCS are differentially inhibited by KOR agonists in these populations (Ford et al., 2006). Therefore, there remains a lot of uncertainty regarding the ability of KOR signalling to modulate the output of subpopulations of dopamine neuron. Chapter 3 of this thesis examines the effects of dynorphin on projection-target specific subpopulations of dopamine neurons.    Importantly, not all aversive events decrease dopamine neuronal activity (Horvitz, 2000). Similarly, inhibition of dopamine release may not be the only mechanism by which KOR agonists mediate their aversive effects (Ehrich et al., 2015). For example, KOR agonist induced CPA requires KOR activation of beta-arrestin dependent p38 MAPK signalling in VTA dopamine neurons. Interestingly, inhibiting p38 MAPK blocks KOR agonist induced CPA without changing KOR-induced decreases in dopamine release in the NAc (Ehrich et al., 2015). Therefore, it is important to consider all factors when examining neuromodulation through signalling at GPCRs.    KOR agonists have inhibitory effects on the rewarding aspects of addictive drugs and dose-dependently decrease morphine (Glick et al., 1995; Kuzmin et al., 1997), ethanol (Nestby et al., 1999; Lindholm et al., 2001; Logrip et al., 2009), cocaine (Glick et al., 1995; Negus et al., 1997; Mello and Negus, 1998; Schenk et al., 1999, 2001), nicotine (Galeote et al., 2009), and cannabis (Mendizábal et al., 2006) self-administration. On the other hand, KOR signalling is critically involved in the reinstatement of drug seeking and has an important interaction with stress. Acute stress promotes escalation of addictive drug consumption and stimulates relapse in abstinent individuals (Brady and Sinha, 2005; Mantsch et al., 2016). KOR antagonists  42 block stress-induced but not drug-primed reinstatement of cocaine self-administration (Beardsley et al., 2005) and CPP (Aldrich et al., 2009), and stress-induced reinstatement is lost in KOR or prodynorphin knock out mice (Redila and Chavkin, 2008). Therefore, while KOR signalling has an inhibitory effect on the acute reinforcing properties of addictive drugs, it drives consumption during stress. Interestingly, KOR signalling underlies acute stress-induced inhibition of LTP at GABAergic synapses in the VTA, but does not alter stress-induced plasticity at excitatory synapses (Graziane et al., 2013). This imbalance in synaptic regulation of dopamine neurons after stress may make them more responsive to motivationally relevant cues.   Recent evidence suggests that there are populations of dynorphin positive neurons that induce a rewarding phenotype.  Optogenetic stimulation of neurons in the dorsal NAc shell is rewarding and drives a place preference (Al-Hasani et al., 2015). This suggests that there is likely a lot of complexity in the endogenous dynorphin system, and that activating specific populations of neurons will be required to properly understand the manner in which each subpopulation alters motivated behaviour.   Orexin and dynorphin have opposing effects on motivated behaviour   Recently, the interplay between orexin and dynorphin on motivated behaviour was examined for the first time. It was proposed that a key function of orexin is to negate the antireward effects of dynorphin (Muschamp et al., 2014). Intra-VTA administration of an OxR1 antagonist increases reward thresholds for electrical self-stimulation of the LH, decreases impulsive responding in the five choice serial reaction time task and decreases cocaine self-administration of a fixed ratio 5 schedule of reinforcement (Muschamp et al., 2014). Interestingly, all of these effects are blocked by pretreatment with norbinaltorphimine (norBNI), which suggests that in the absence of  43 orexin signalling, the inhibitory effects of dynorphin on motivated behaviour are unopposed (Muschamp et al., 2014). However, the effect is not reciprocal because unopposed orexin signalling does not increase reward-seeking behaviour (Muschamp et al., 2014); the reason for this remains to be determined. Nevertheless, these effects appear to be due to equal but opposing actions of orexin and dynorphin on the firing rate of VTA dopamine neurons, although other structures may also be involved (Muschamp et al., 2014). Although this work suggests that orexin and dynorphin signalling may oppose one another, this may be behaviour specific. For example, both SB 334867 and norBNI block stress-induced reinstatement of cocaine seeking (Boutrel et al., 2005; Graziane et al., 2013). Moreover, morphine withdrawal increases cFos levels in orexin neurons (Georgescu et al., 2003; Laorden et al., 2012). It is interesting to consider that under these conditions orexin and dynorphin may work synergistically to promote drug seeking, with orexin motivating drug seeking to alleviate some of the negative affect induced by dynorphin.   Summary and objectives  Dopamine signalling is linked to motivated behaviour and dopamine release is dependent on both the level and the pattern of activity of VTA dopamine neurons. These patterns of activity are determined by intrinsic conductances and afferent synaptic input, both of which are subject to neuromodulatory influences. Orexin neurons project to the VTA and promote motivated behaviour through OxR1 dependent increases in firing and changes in synaptic transmission.   Drugs of abuse target the mesocorticolimbic dopamine system and induce synaptic plasticity at both excitatory and inhibitory synapses onto these neurons. Despite much research, it remains unclear how this pharmacologically diverse group of  44 drugs all converge to induce synaptic plasticity in the VTA. Orexin in the VTA is required for both cocaine-induced potentiation of excitatory transmission and the development of behavioural sensitization (Borgland et al., 2006). In chapter 2, we aim to determine if orexin signalling is involved in morphine-induced plasticity in the VTA.   Recent evidence highlights the complexity of the VTA dopamine system. There exists distinct subpopulations of dopamine neurons that differ in their intrinsic electrophysiological properties and their afferent and efferent connections (Lammel et al., 2008, 2011; Beier et al., 2015). Moreover, these different subpopulations may be subject to separate neuromodulatory influences (Ford et al., 2006; Margolis et al., 2006a, 2008; Vittoz et al., 2008). Because orexin neurons contain both excitatory orexin and inhibitory dynorphin not only in the same cell (Chou et al., 2001), but also within the same vesicles (Muschamp et al., 2014), potential corelease of these opposing peptides may have distinct effects on different subpopulations of dopamine neurons. In chapter 3, we examine the neuromodulatory effects of orexin and dynorphin on projection-target defined subpopulations of VTA dopamine neurons.     45 Chapter 2: Orexin signalling in the VTA gates morphine-induced synaptic plasticity  Introduction The mesolimbic dopamine system is a well-recognized target of addictive drugs, and plasticity within this neural circuitry is involved in the development and maintenance of addiction (Hyman et al., 2006; Kauer and Malenka, 2007). The activity and output of VTA dopamine neurons are tightly regulated by excitatory glutamate and inhibitory GABA synaptic inputs, and drug-induced plasticity at these synapses occurs with the very first drug exposure. A single in vivo injection of multiple addictive drugs induces an LTP of excitatory synaptic transmission in the VTA (Ungless et al., 2001; Saal et al., 2003; Brown et al., 2010). This drug-evoked plasticity is thought to increase the incentive properties of the drug, to mediate early behavioural responses to drug exposure, and to trigger long-term neural adaptations in regions that receive dopamine input (Kauer and Malenka, 2007). Moreover, drug treatment inhibits inhibitory control mechanisms that increase GABA transmission to the VTA (LTPGABA) (Nugent et al., 2007; Niehaus et al., 2010; Graziane et al., 2013), likely further increasing the incentive properties of the drug.  Orexin neurons have emerged as an important mediator of drug-seeking behaviour and alter synaptic transmission in the VTA. Orexins are neuropeptides synthesized in the LH that contribute to arousal, feeding, and reward seeking (de Lecea et al., 1998; Sakurai et al., 1998; Harris et al., 2005; Adamantidis et al., 2007). Orexin A and B signal at OxR1 and OxR2, which are widely expressed in the brain, including in the VTA (Korotkova et al., 2003; Narita et al., 2006). Orexin neurons are activated by drug cues (Harris et al., 2005), and drug administration enhances excitatory drive at  46 glutamate synapses onto orexin neurons (Yeoh et al., 2012; Rao et al., 2013). Importantly, orexins mediate drug seeking through their projections to the VTA (Harris et al., 2005; Borgland et al., 2006; Narita et al., 2006). Orexins increase the firing rate of dopamine neurons and can enhance dopamine release in downstream target structures (Korotkova et al., 2003; Narita et al., 2006, 2007; Vittoz and Berridge, 2006; Vittoz et al., 2008; España et al., 2010a). Short-term administration of orexin A increases NMDAR current amplitudes in dopamine neurons (Borgland et al., 2006), an effect that is enhanced by long-term drug self-administration (Borgland et al., 2009), and promotes a long-term increase in AMPAR signalling (Borgland et al., 2006).  Drugs of abuse differ greatly in terms of their behavioural profiles and molecular targets. Although the mechanisms by which addictive drugs increase phasic dopamine release have been well characterized (Lüscher and Ungless, 2006), it is unclear how these drugs all converge to induce synaptic plasticity in the VTA. Previously, we found that systemic administration of an OxR1 antagonist blocks both cocaine-induced synaptic plasticity at excitatory synapses in the VTA and the development of behavioural sensitization (Borgland et al., 2006). However, it is unknown whether orexin contributes to plasticity in the VTA induced by other drugs with different mechanisms of action. Here, we used whole-cell patch-clamp electrophysiology to elucidate the contribution of orexin signalling to morphine-induced synaptic plasticity of VTA dopamine neurons.   Materials and methods  Animals   All protocols were performed in accordance with the ethical guidelines established by the Canadian Council for Animal Care, and were approved by the  47 University of British Columbia and the University of Calgary Animal Care Committees. Sprague Dawley rats were obtained from the University of British Columbia breeding facility or Charles River Laboratories (Montreal, Qc) and were housed in the facility for 3 days before use. Rats were kept in groups of two to six, except following surgery, at which time rats were single housed. Rats were maintained on a 12-hour light/dark schedule, and were given food and water ad libitum. All experiments were performed during the animals’ light cycle.  Electrophysiology  All electrophysiological recordings were performed in slice preparations from male Sprague Dawley rats (postnatal day 19 to 30). Briefly, rats were anesthetized with isoflurane and decapitated, and their brains were extracted into ice-cold sucrose solution containing the following (in mM): 75 sucrose, 87 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 7 MgCl2, and 0.95 CaCl2. Horizontal sections (250 μm) containing the VTA were cut on a vibratome (Leica), and incubated in a holding chamber for at least 45 min before being transferred to a recording chamber and superfused with bicarbonate-buffered solution (artificial CSF) containing the following (in mM): 126 NaCl, 1.6 KCl, 1.1 NaH2PO4, 1.4 MgCl2, 2.4 CaCl2, 26 NaHCO3, and 11 glucose (32oC–34oC), and saturated with 95%-O2/5%-CO2. Cells were visualized on an upright microscope using “Dodt-type” gradient contrast infrared optics (Dodt et al., 2002) and whole-cell voltage-clamp recordings were made using a MultiClamp 700B Amplifier (Molecular Devices). Recording electrodes (3–5 MΩ) were filled with the following (in mM): 117 cesium methanesulfonate, 20 HEPES, 0.4 EGTA, 2.8 NaCl, 5 TEA-Cl, 2.5 Mg-ATP, and 0.25 Na-GTP, pH 7.2–7.3 and 270 –285 mOsm to record EPSCs; and 0.1 mM spermine was added only when examining the rectification index. For IPSCs, electrodes were filled  48 with the following (in mM): 125 KCl, 2.8 NaCl, 2 MgCl2, 10 HEPES, 0.6 EGTA, 2.5 Mg-ATP, and 0.25 Na-GTP, pH 7.2–7.3 and 270–285 mOsm. Series resistance (10 –25 MΩ) and input resistance were monitored on-line with a 10 mV depolarizing step (400 ms) given before every afferent stimulus. Dopamine neurons, which are the most abundant cell type in the VTA, were located medial to the medial nucleus of the optic tract and were identified by the presence of a large Ih current, and in a subset of cells by tyrosine hydroxylase staining. A bipolar stimulating electrode was placed 100–300 μm rostral to the recording electrode and was used to stimulate either excitatory or inhibitory afferents at 0.1 Hz. Neurons were voltage clamped at -70 mV to record AMPAR EPSCs. EPSCs were recorded in the presence of picrotoxin (100 μM) to block GABAA receptor IPSCs; and IPSCs were recorded with strychnine (1 μM) to block potential glycine IPSCs (Zheng and Johnson, 2001); with AP5 (50 μM) and DNQX (10 μM) to block excitatory synaptic transmission; and with sulpiride (200 nM) to block dopamine D2 receptor IPSCs (Gantz et al., 2013) in the bath solution.   The AMPAR/NMDAR ratio was recorded at +40 mV. To calculate the AMPAR/NMDAR ratio, an average of 12 EPSCs at +40 mV was computed before and after application of the NMDAR blocker AP5 (50 μM). NMDAR responses were calculated by subtracting the average response in the presence of AP5 (AMPAR only) from that seen in its absence, and the peak of the AMPAR EPSC was divided by the peak of the NMDAR EPSC to yield an AMPAR/ NMDAR ratio. For the rectification index, AMPAR EPSCs were evoked at -70, 0, and +40 mV in the presence of AP5 (50 μM) and the polyamine spermine (via pipette, 0.1 mM). The rectification index was calculated by dividing the gradient of the slope at negative potentials by the gradient of the slope at positive potentials.   49  mEPSCs and mIPSCs were recorded at -70 mV in the presence of tetrodotoxin (500 nM) to block action potential driven spontaneous events. AMPAR mEPSCs were selected based on their amplitude (>12 pA), decay time (<3 ms), and rise time (<1 ms) using the MiniAnalysis program (Synaptosoft). Similarly, GABAA mIPSCs were selected for amplitude (>12 pA), rise time (<4 ms), and decay time (<10 ms). For both mEPSC and mIPSC experiments, recordings were 3 minutes long and a maximum of 300 events were counted per cell.  The excitatory/inhibitory balance was recorded with an internal solution containing the following (in mM): 132 cesium methanesulfonate, 8 CsCl, 10 HEPES, 0.6 EGTA, 4 Mg-ATP, 0.3 Na-GTP, and 10 Na-phosphocreatine. EPSCs were recorded at the reversal potential for GABAA IPSCs (-67 mV), and IPSCs were recorded at the reversal potential for EPSCs (+8 mV). The Ge/Gi ratio was calculated by converting the average peak current amplitude of the EPSC and the IPSC into a conductance.  Systemic drug treatment  Rats were removed from their home cage and placed individually in a clean cage. Rats were given either SB 334867 (10 mg/kg, intraperitoneal ; Tocris Bioscience) or vehicle (10% DMSO, 20% β-hydroxycyclodextrin in saline), or were not given an injection (naïve rats). Fifteen minutes later, rats were treated with morphine HCl (10 mg/kg, intraperitoneal) or an equivalent volume of saline (0.9% NaCl). Following injection procedures, rats were returned to their home cage and were left for 24 h.  Surgical procedures   Sprague Dawley rats (50–60g at the start of the experiment) were housed individually following surgical procedures. Animals were anesthetized with isoflurane  50 and placed in a stereotaxic frame (Kopf) and bilateral cannulas (26 gauge; Plastics One) were lowered into the VTA (coordinates in mm: anteroposterior, -4.6 to -5.2; mediolateral, ±0.5; dorsoventral, -7.0). As a control, some animals were implanted with cannulas that only reached dorsoventral -5.0 mm to test infusions outside of the VTA. Cannulas were anchored to the skull surface with dental cement and were occluded with a dummy cannula of the same length. Rats were treated post-surgically with ketoprofen (5 mg/kg, subcutaneous), and weights were monitored daily during the recovery period.  Intra-VTA drug infusions   During the recovery period, rats were familiarized with the infusion procedures with three habituation sessions. During these sessions, rats were transferred from their home cage to a clean cage and were left for 30 min. Afterward, rats were handled for 5–10 min and the dummy cannula was replaced with a microinjector, cut above the length of the cannula to prevent tissue damage to the VTA. The microinjector was attached to a Hamilton syringe with tubing, and a mock infusion was performed with a microinfusion pump. The microinjector was left in place for 5 min before being replaced with the dummy cannula, and rats were left in the injection cage for 15 min before being returned to their home cage. On injection day, microinfusions were conducted using 33 gauge microinjectors that protruded 0.5 mm below the base of the guide cannula to a final dorsoventral coordinate of -7.5 mm. SB 334867 (0.3 or 0.03 nmol/0.3 l) or vehicle (50% saline, 50% DMSO) were infused bilaterally into the VTA (0.3 l/side at 0.1 l/min) with the microinfusion pump. Microinjectors were left in place for 5 min following the injection, at which point rats were injected systemically with morphine (10 mg/kg, i.p.) or saline. Rats were then returned to their home cage and were left for 24 h.   51 Tyrosine hydroxylase immunocytochemistry   Brain slices from patch-clamp recordings were fixed overnight in cold 4% paraformaldehyde, rinsed in phosphate-buffered saline, blocked in 10% normal donkey serum, incubated with monoclonal mouse anti-tyrosine hydroxylase antibody (Sigma, 1:1000) for 48 hours at 4oC. DyLight 594 conjugated streptavidin (Jackson Labs; 1:200) was applied overnight at 4oC. Secondary donkey anti-mouse fluorescein isothiocyanate (FITC) antibody (1:50) was applied for two hours at 4oC and slices were mounted using Fluoromount (Sigma, St. Louis).  Data analysis   All values are expressed as the mean ± SEM. Statistical significance was assessed using two-way or one-way ANOVA with Bonferroni post-tests. In all experiments, the sample size is expressed as N/n where N refers to the number of cells recorded from n animals. Prism 5 software (GraphPad Software) was used to perform the statistical analysis. Figures were generated using Illustrator CS2 software (Adobe Systems). The levels of significance are indicated as follows: ***p<0.001, **p<0.01, *p<0.05.   Results  OxR1 signalling is required for morphine-induced potentiation of excitatory inputs to VTA dopamine neurons   Morphine, like other addictive drugs, induces an LTP-like potentiation of excitatory inputs to VTA dopamine neurons (Ungless et al., 2001; Saal et al., 2003; Borgland et al., 2004; Brown et al., 2010). To examine the role for orexin signalling in morphine-induced plasticity, we measured the relative contribution of AMPAR and  52 NMDAR EPSCs 24 h after a single injection of morphine (10 mg/kg) or saline (0.9% NaCl), with or without pretreatment with the OxR1 antagonist SB 334867 (10 mg/kg). Consistent with previous observations (Saal et al., 2003), a single injection of morphine significantly potentiated the AMPAR/NMDAR ratio in both naive (saline, 0.4 ± 0.03; morphine, 0.6 ± 0.04; p<0.01) and vehicle-treated rats [saline, 0.4 ± 0.04; morphine, 0.6 ± 0.06; p<0.05; Fig. 2 A, B; two-way-ANOVA: drug (morphine vs. saline) pretreatment (naive, vehicle, SB 334867) interaction, F(2,33)=3.870, p=0.031; drug, F(1,33)=10.30, p=0.003; pretreatment, F(2,33)=5.558, p=0.008]. This potentiation was blocked in rats pretreated with SB 334867 15 min before morphine exposure (saline, 0.4 ± 0.05; morphine, 0.4 ± 0.06; p<0.05; Fig. 2 A, B).   To further investigate the effect of morphine on AMPARs at excitatory synapses of dopamine neurons, we recorded AMPAR mEPSCs to determine the locus of synaptic change. Morphine induced a long-lasting increase in the frequency of AMPAR mEPSCs in naive rats (saline, 0.6 ± 0.1 Hz; morphine, 1.6 ± 0.3 Hz; p<0.01) and vehicle-treated rats (saline, 0.4 ± 0.1 Hz; morphine, 1.2 ± 0.3; p<0.05), but not in SB 334867-treated rats (saline, 0.6 ± 0.2 Hz; morphine, 0.6 ± 0.1 Hz; p>0.05; Fig. 2C,D; two-way ANOVA: interaction, F(2,54)=2.488, p=0.093; drug, F(1,54)=8.861, p=0.004; pretreatment, F(2,54)=2.252, p=0.115). This increase in the probability of presynaptic glutamate release was confirmed with the paired-pulse ratio. Morphine induced a paired-pulse depression in naive (saline: 0.9 ± 0.07; morphine: 0.6 ± 0.03, p<0.01) and vehicle-treated rats (saline: 0.9 ± 0.06; morphine: 0.6 ± 0.05, p<0.01; Fig. 2F). Morphine-induced paired-pulse depression was inhibited by SB 334867 (saline, 0.9 ± 0.05; morphine, 0.9 ± 0.06; p<0.05; Fig. 2F; two-way ANOVA: interaction, F(2,38)=3.371, p=0.045; drug, F(1,38)=20.37, p<0.0001; pretreatment, F(2,38)=4.468, p=0.018).   53  Orexin signalling also modulated post-synaptic AMPAR effects induced by morphine exposure. Morphine treatment increased the amplitude of AMPAR mEPSCs in VTA neurons of control rats (naive: saline, 17.4 ± 0.6 pA; morphine, 19.4 ± 0.6 pA; p<0.05; vehicle: saline, 16.2 ± 0.5 pA; morphine, 18.1 ± 0.5 pA; p<0.05), an effect that was blocked by pretreatment with SB 334867 (saline, 16.9 ± 0.6 pA; morphine, 17.1 ± 0.5 pA; p>0.05; Fig. 2C,E; two-way ANOVA: interaction, F(2,54)=1.583, p=0.214; drug, F(1,54)=9.102, p=0.004; pretreatment, F(2,54)=4.193, p=0.020). Because morphine treatment is associated with a change in the subunit composition of AMPARs in the VTA (Brown et al., 2010), we measured the rectification index following morphine treatment. Morphine increased the rectification index in naive rats (saline, 1.1 ± 0.06; morphine, 1.5 ± 0.1; p<0.05) and vehicle-treated rats (saline, 1.1 ± 0.08; morphine, 1.5 ± 0.1; p<0.05), but not in those that were treated with SB 334867 (saline, 1.2 ± 0.06; morphine, 1.2 ± 0.07; p>0.05; Fig. 2G; two-way ANOVA: interaction, F(2,28)=2.549, p=0.088; drug, F(1,54)=8.876, p=0.005; pretreatment, [F(2,48)=1.520, p=0.230). Together, these data suggest that orexin signalling is necessary for morphine-induced potentiation of presynaptic and postsynaptic efficacy of glutamatergic synapses.           54   55 Figure 2: Systemic administration of the OxR1 antagonist SB 334867 blocks morphine-induced potentiation of excitatory transmission onto VTA dopamine neurons.  A. Example recordings of evoked NMDAR (light) and AMPAR (dark) EPSCs at +40 mV, from VTA dopamine neurons of rats 24 h after exposure to morphine or saline in naïve (upper), vehicle- (middle) and SB 334867- (10 mg/kg; lower) treated rats. Scale bars, 50 pA, 20 ms. B. Morphine (10 mg/kg, filled bars), but not saline (open bars) treatment increased the AMPAR/NMDAR ratio in naïve and vehicle-treated, but not SB 334867- treated rats (p<0.05, 2-way ANOVA). C. Example traces of AMPAR mEPSCs recorded at -70 mV 24 h after morphine or saline treatment in naïve (left), vehicle- (centre) and SB 334867- (right) treated rats. Scale bars, 50 pA, 100 ms. D. Left panel, AMPAR mEPSC frequency was increased in morphine- compared to saline-treated rats in naïve and vehicle- but not SB 334867-treated rats (p<0.05, 2-way ANOVA). Right panel, cumulative probability plots (averaged across all cells) comparing morphine or saline exposure on mEPSCs for naïve, vehicle- and SB 334867-treated animals. E. Left panel, morphine increased AMPAR mEPSC amplitude compared to saline in naïve and vehicle-treated, but not SB 334867-treated rats (p<0.05, 2-way ANOVA). Right panel, cumulative probability plots (averaged across all cells) comparing morphine or saline exposure on mEPSC amplitude for naïve, vehicle- and SB 334867-treated rats. F. Morphine (filled bars) induced a paired pulse depression of evoked AMPAR EPSCs in naïve and vehicle-treated, but not SB 334867-treated rats (p<0.05, 2-way ANOVA). Inset, Sample traces of evoked AMPAR EPSC paired pulses recorded at -70 mV. Scale bars, 50 pA, 20 ms. G. Pretreatment with SB 334867 blocked a morphine-induced increase in the rectification index (p<0.05, 2-way ANOVA). Inset, Sample traces of AMPAR EPSCs recorded at -70, 0 and +40 mV with spermine in the pipette solution. Current voltage relationship of AMPAR EPSCs for morphine and saline in vehicle- and SB 334867-treated rats. Scale bars, 50 pA, 10 ms. n/N = cells/rats. Bars represent mean + s.e.m, * p < 0.05, ** p < 0.01   Orexin signals locally in the VTA to mediate morphine-induced potentiation of excitatory synaptic transmission   OxR1 is expressed widely throughout the brain (Trivedi et al., 1998; Marcus et al., 2001). Therefore, systemic administration of SB 334867 precluded identification of the site of action of orexin in modulating morphine-induced plasticity. Because microinfusion of SB 334867 into the VTA inhibits the reinstatement of morphine-induced CPP (Narita et al., 2006), we hypothesized that orexin was signalling locally in the VTA to mediate morphine-induced potentiation of glutamatergic synaptic transmission. To test this, we implanted rats with a bilateral cannula aimed at the VTA and microinfused SB 334867 locally. In rats that received vehicle infusions into the VTA,  56 systemic morphine administration increased the AMPAR/NMDAR ratio compared with saline (saline, 0.4 ± 0.06; morphine, 0.8 ± 0.1; p<0.01; Fig. 3 A, B). This effect of morphine was blocked by an intra-VTA infusion of SB 334867 (0.3 nmol/0.3 μl; saline, 0.4 ± 0.05; morphine, 0.5 ± 0.03; p>0.05; Fig. 3 A, B; two-way ANOVA: interaction, F(1,25)5.122, p=0.033; drug, F(1,25)=12.89, p=0.001; pretreatment, F(1,25)=5.103, p=0.033). However, SB 334867 did not block a morphine-induced increase in the AMPAR/NMDAR ratio when infused outside the VTA (morphine, 0.8 ± 0.07) or when the dose was 10-fold lower (0.03 nmol/0.3 μl; morphine, 0.8 ± 0.05; one-way ANOVA: F(3,25)=10.85, p<0.0001).   To test whether intra-VTA orexin signalling modulates morphine-induced presynaptic or postsynaptic plasticity at excitatory synapses, we recorded AMPAR mEPSCs. Morphine increased the frequency of AMPAR mEPSCs compared with saline in intra-VTA vehicle-treated (saline, 0.4 ± 0.1 Hz; morphine, 1.5 ± 0.3 Hz; p<0.001), but not SB 334867-treated rats (0.3 nmol/0.3 μl; saline, 0.4 ± 0.08 Hz; morphine, 0.5 ± 0.2 Hz; p>0.05; Fig. 3C,D; two-way ANOVA: interaction, F(1,32)=6.551, p=0.015; drug, F(1,32)=12.84, p=0.001; pretreatment, F(1,32)=8.289, p=0.007). Similarly, morphine increased AMPAR mEPSC amplitude in an orexin-dependent manner (two-way ANOVA: interaction, F(1,32)=3.685, p=0.064; drug, F(1,32)=4.894, p=0.034; pretreatment, F(1,32)=10.26, p=0.003). In morphine-exposed rats, mEPSC amplitude was increased in intra-VTA vehicle-treated (saline, 15.9 ± 0.4 pA; morphine, 17.7 ± 0.5 pA; p<0.05), but not in intra-VTA SB 334867-treated rats (0.3 nmol/ 0.3 μl; saline, 15.3 ± 0.4 pA; morphine, 15.4 ± 0.4 pA; p>0.05; Fig. 3C,E). Together, these results suggest that orexin signalling in the VTA is necessary for morphine-induced presynaptic and postsynaptic potentiation at excitatory synapses onto dopamine neurons.   57     58  Figure 3: OxR1 signalling in the VTA is required for morphine-induced plasticity at glutamatergic synapses.  A. Sample traces of evoked AMPAR (dark) and NMDAR (light) EPSCs recorded at +40 mV, 24 h after exposure to morphine or saline in rats microinfused with intra-VTA vehicle or SB 334867 rats. Scale bars, 50 pA, 20 ms. B. Morphine (filled bars) potentiated the AMPAR/NMDAR ratio compared to saline (open bars) in intra-VTA vehicle-, but not intra-VTA SB 334867-treated animals (p<0.05, 2-way ANOVA). SB 334867 was ineffective when infused outside the VTA or when the dose was lowered to 0.03 nmoles/0.3 μl. C. Sample traces of AMPAR mEPSCs recorded at-70 mV, 24 h after morphine or saline exposure in intra-VTA vehicle- and intra-VTA SB 334867-treated rats. Scale bars, 50 pA, 100 ms. D. Left panel, morphine increased the frequency of AMPAR mEPSC relative to saline in intra-VTA vehicle-, but not intra-VTA SB-334867 treated rats (2-way ANOVA, p<0.05).  Right panel, cumulative probability plot (averaged across all cells) comparing morphine and saline exposure on mEPSCs for intra-VTA vehicle or SB 334867 treated rats.  E. Left panel, intra-VTA SB 334867 inhibited a morphine-induced increase in the amplitude of AMPAR mEPSCs in VTA dopamine neurons (2-way ANOVA, p<0.05). Right panel, cumulative probability plot (averaged across all cells) comparing morphine and saline exposure on mEPSC amplitude for intra-VTA vehicle or SB 334867 treated rats. n/N = cells/rats. Bars represent mean + s.e.m. *p < 0.05, ** p < 0.01.   OxR1 signalling is required for a 
 morphine-induced decrease in 
presynaptic GABA release 
  Short-term morphine administration in 
creases VTA dopamine neuronal activity 
by way of disinhibition resulting from 
 μ-opioid receptor activation on GABAergic 
inputs to dopamine neurons (Di Chiara and Imperato, 1988; Johnson and North, 1992b; Jalabert et al., 2011). Moreover, 
morphine has long-term effects on GABA 
transmission in the VTA (Nugent et al., 2007; Dacher and Nugent, 2011). Therefore, we recorded GABAA mIPSCs in VTA 
 dopamine neurons 24 h after a single 
 morphine exposure to assess whether 
 orexin signalling had a role in morphine- 
induced plasticity at GABAergic synapses 
in the VTA. Morphine treatment decreased GABAA mIPSC frequency on to 
dopamine neurons of control rats (naive 
 rats: saline, 2.2 ± 0.4 Hz; morphine, 1.1 ± 
0.2 Hz; p<0.05; vehicle-treated rats: sa 
line, 2.1 ± 0.3 Hz; morphine, 0.9 ± 0.08 
Hz; p<0.05; Fig. 4A-C; two-way 
 ANOVA: interaction, F(2,62)=3.262, p  59 =0.045; drug, F(1,62)=11.19, p 0.001]; 
pretreatment, [F(2,62)=7.566, p<0.001). 
However, SB 334867 inhibited morphine- 
induced suppression of mIPSC frequency 
 (saline, 2.5 ± 0.3 Hz; morphine, 2.5 ± 0.3 
Hz; p>0.05; Fig. 4A-C). There was no effect of morphine on GABAA mIPSC amplitude (two-way ANOVA: interaction, [F(2,62)=1.493, p=0.232; drug, F(1,62)=3.011, p=0.088; pretreatment, F(2,62)=2.537, p=0.088; Fig. 4 A, D, E).     60 Figure 4: Morphine decreases the probability of GABA release in an OxR1-dependent manner.  A. Example recordings of GABAA mIPSCs recorded at -70 mV, 24 h after exposure to morphine or saline in naïve, vehicle- or SB 334867-treated rats. Scale bars, 50 pA, 200 ms. B. Left panel, morphine (filled bars) decreased the frequency of GABAA mIPSCs relative to saline (open bars) in naïve and vehicle-, but not SB 334867-treated rats (p<0.05, 2-way ANOVA). Right panel, cumulative probability plots (averaged across all cells) comparing morphine or saline exposure on mIPSC for naïve, vehicle- and SB 334867-treated animals. C. Left panel, Morphine exposure did not alter GABAA mIPSC amplitude compared to saline exposure in naïve, vehicle- or SB 334867-treated animals (p>0.05, 2-way ANOVA). Right panel, cumulative probability plots (averaged across all cells) comparing morphine or saline exposure on mIPSC amplitude for naïve, vehicle- and SB 334867-treated animals. n/N = cells/rats. Bars represent mean + s.e.m. *p < 0.05.  OxR1 activation in the VTA is required for a morphine-induced decrease in the probability of presynaptic GABA release  
We next examined whether the morphine-induced decrease in the probability of GABA release was mediated by OxR1 activation within the VTA. Morphine decreased GABAA mIPSC frequency in intra-VTA vehicle-treated rats (saline, 2.3 ± 0.4 Hz; morphine, 1.2 ± 0.1; p<0.05), but not in those that received intra-VTA SB 334867 (0.3 nmol/0.3 μl; saline, 2.0±0.3 Hz; morphine, 2.1 ± 0.3; p>0.05; Fig. 5 A, B; two-way ANOVA: interaction, F(1,52)=5.845, p=0.019; drug, F(1,52)=4.118, p=0.047; pretreatment, F(1,52)=1.951, p=0.169). There was no effect of morphine treatment on GABAA mIPSC amplitude in either group (two-way ANOVA: interaction, F(1,52)=1.316, p=0.256; drug, F(1,52)=0.588, p=0.447; pretreatment, F(1,52)=0.955, p=0.333; Fig. 5 A, C).       61     Figure 5: OxR1 signalling in the VTA is necessary for a morphine-induced suppression of presynaptic GABA release.  A. Sample recordings of GABAA mIPSCs at -70 mV, from rats that were exposed to morphine or saline after pretreatment with intra-VTA vehicle or intra-VTA SB 334867. B. Left panel, morphine (filled bars) decreased GABAA mIPSC frequency relative to saline (open bars) in intra-VTA vehicle-, but not intra-VTA SB 334867-treated rats (2-way ANOVA, p<0.05). Right panel, cumulative probability plot comparing morphine and saline exposure on mIPSC frequency for intra-VTA vehicle or SB 334867 treated rats. C. Left panel, there was no effect of morphine on the amplitude of GABAA mIPSCs (2-way ANOVA, p>0.05). Right panel, cumulative probability plots comparing morphine and saline exposure on mIPSC amplitude for intra-VTA vehicle- or SB 334867-treated rats. n/N = cells/rats. Bars represent mean + s.e.m. ** p < 0.01.  62 Morphine alters the synaptic excitation/inhibition balance in an OxR1-dependent manner 
  Our results demonstrate that a single morphine exposure induces a simultaneous OxR1-dependent increase in glutamate transmission and a decrease in GABA transmission on to VTA dopamine neurons. By acting at both excitatory and inhibitory synapses, orexin may drive a morphine-induced shift in the excitatory/ inhibitory (Ge/Gi) balance of synaptic inputs on to dopamine neurons. To verify this, we recorded locally evoked EPSCs and IPSCS onto the same cell. EPSCs were recorded at the IPSC reversal potential (-67 mV), and IPSCs were recorded at the EPSC reversal potential (+8 mV). Morphine induced a shift in the Ge/Gi balance onto dopamine neurons from naive rats (saline, 0.08 ± 0.01; morphine, 0.4±0.1; p<0.05) and vehicle-treated rats (saline, 0.09 ± 0.01; morphine, 0.3 ± 0.07; p<0.05; Fig. 6 A, B), but not in those that were treated with SB 334867 (saline, 0.1 ± 0.02; morphine, 0.1 ± 0.03; p>0.05; Fig. 6 A, B; two-way ANOVA: interaction, F(2,56)=3.184, p=0.049; drug, F(1,56)=15.37, p=0.0002; pretreatment, F(2,56)=2.338, p=0.106). Together, these data suggest that orexin signalling is necessary for a morphine-induced shift in the balance of inhibitory and excitatory control of dopamine neurons.         63    Figure 6: OxR1 signalling is required for a morphine-induced shift in the balance of excitatory and inhibitory synaptic transmission onto dopamine neurons.  A. Example recordings of EPSCs (dark) at -67 mV, and IPSCs (light) at +8 mV from VTA dopamine neurons in naïve, vehicle- and SB 334867-treated animals. Scale bars, 200 pA, 20 ms. B. Morphine (filled), compared to saline (open), induced a switch in the ratio of excitatory and inhibitory conductances onto dopamine neurons from naïve or vehicle-treated animals. This shift was inhibited by systemic SB 334867 (2-way ANOVA, p<0.05) C. SB 334867 inhibited morphine-induced increased Ge/Gi ratio in 7/9 identified TH-positive neurons. TH was labeled with anti-tyrosine hydroxylase antibodies and FITC. Biocytin was labeled with streptavidin-conjugated Texas Red. n/N = cells/rats. Bars represent mean + s.e.m. *p < 0.05,  *** p < 0.001.  64 Discussion   Here, we establish a critical role for orexin signalling in morphine-induced synaptic plasticity in the VTA. Both systemic and intra-VTA administration of the OxR1 antagonist SB 334867 inhibited morphine-induced potentiation of excitatory synaptic transmission. Additionally, SB 334867 blocked a long-lasting decrease in the probability of presynaptic GABA release at inhibitory synapses onto dopamine neurons. Last, OxR1 signalling was required for a morphine-induced shift in the balance of excitatory and inhibitory inputs to dopamine neurons. These data provide further evidence that orexin signalling in the VTA plays a critical role in drug-induced plasticity of dopamine neurons.  Orexin and morphine-induced potentiation of AMPAR signalling   Previous work (Saal et al., 2003; Brown et al., 2010) has demonstrated that a single morphine exposure increases the AMPAR/NMDAR ratio and promotes the insertion of GluA2-lacking AMPARs to the post-synaptic site, as revealed by an increase in the rectification index. Not only have we replicated these results, but we have demonstrated that morphine also increases the probability of presynaptic glutamate release, and that both presynaptic and postsynaptic changes induced by morphine to enhance synaptic efficacy onto dopamine neurons require OxR1 signalling in the VTA. Because overexpression of GluA1 subunits in the VTA potentiates morphine reward in a CPP task (Carlezon et al., 1997), orexin-dependent morphine-induced plasticity at excitatory synapses likely contributes to learning the association between context and the drug experience. Consistent with this, intra-VTA administration of an orexin receptor antagonist inhibits morphine CPP (Narita et al., 2006; Harris et al., 2007). Drug-induced plasticity and changes in AMPAR subunit composition have  65 previously been attributed to activation of dopamine D5 receptors and the cAMP-dependent protein kinase A signalling pathway (Argilli et al., 2008; Brown et al., 2010). Because the effects of orexin in the VTA are dependent on PKC signalling (Borgland et al., 2006; Narita et al., 2007), OxR1 likely represents an alternative mechanism for drug-induced synaptic plasticity. Interestingly, both OxR1 and D5 receptor signalling can potentiate NMDAR receptor currents in the VTA, albeit through different mechanisms (Borgland et al., 2006, 2009; Schilström et al., 2006). Because NMDAR activation is required for drug-induced plasticity (Ungless et al., 2001; Argilli et al., 2008; Engblom et al., 2008), OxR1 and D5 receptors may act cooperatively to enhance NMDAR signalling and to promote drug-induced synaptic changes in the VTA. Exposure to morphine also induced an OxR1-dependent increase in the probability of presynaptic glutamate release. Orexin-induced increases in glutamate release have previously been observed in multiple brain regions, including the VTA (van den Pol et al., 1998; Burlet et al., 2002; Li et al., 2002; Smith et al., 2002; Lambe and Aghajanian, 2003; Borgland et al., 2006, 2009). Increased glutamate release probability may reflect a mechanism additional to postsynaptic trafficking of AMPARs to further enhance synaptic efficacy cis- or trans-synaptically.  Orexin and morphine-induced decreases in inhibitory synaptic transmission  In addition to morphine action at excitatory synapses, we observed that morphine induces an OxR1-dependent decrease in the probability of GABA release. Previous work has demonstrated that morphine modulates GABAergic synapses onto VTA dopamine neurons. A single exposure to morphine blocks the induction of LTPGABA, and likely occludes LTDGABA at synapses onto dopamine neurons (Nugent et al., 2007; Dacher and Nugent, 2011; Graziane et al., 2013). LTDGABA requires the  66 activation of postsynaptic dopamine D2 receptors (Dacher and Nugent, 2011). Here, we propose a novel mechanism by which orexin signalling in the VTA facilitates a morphine-induced inhibition of GABA release. Future work will need to determine the mechanism by which orexin signalling can modulate GABAergic synapses, although preliminary studies (work done by the group of Lih-Chu CHiou, published in Baimel et al., 2015) have indicated that orexin induces an endocannabinoid-mediated inhibition of GABA release onto dopamine neurons.  Orexin signalling underlies a morphine-induced shift in the synaptic regulation of VTA dopamine neurons  Rapid modulation of the Ge/Gi is important for optimal information processing (Sarti et al., 2013). Here we demonstrate that orexin facilitates the morphine-induced shift from inhibitory to excitatory influence on dopaminergic neurons. Because the balance of excitatory and inhibitory inputs of dopamine neurons is one of the key regulators of dopamine neuron activity (Floresco et al., 2003), this shift likely reflects a mechanism by which orexin can modulate the output of dopamine neurons in response to reward-predictive cues (Harris et al., 2005). The ability of orexin to increase glutamate transmission in parallel with a decrease in GABA transmission suggests an important role for orexin in gating the output of dopamine neurons.  Morphine-induced activation of orexin neurons  How morphine exposure induces orexin release remains to be determined. Orexin neurons express MORs (Georgescu et al., 2003) and are cFos activated with morphine administration (Harris et al., 2005, 2007; Richardson and Aston-Jones, 2012) or withdrawal (Georgescu et al., 2003). Orexin neurons that project to the VTA, but not those that project to the locus coeruleus, are activated by in vivo exposure to morphine  67 in proportion to the level of CPP (Richardson and Aston-Jones, 2012). Furthermore, OxR1 antagonists reduce heroin self-administration and cue-induced reinstatement of opioid seeking (Smith and Aston-Jones, 2012), suggesting that increased orexin in the VTA is required for opioid seeking. Consistent with this, orexin A in the VTA is sufficient to reinstate drug seeking (Harris et al., 2005).   In contrast to these studies, a short-term (30 s) morphine application to LH slices has direct inhibitory effects on orexin neuronal activity (Li and van den Pol, 2008). Because orexin neurons are a heterogeneous cell population, and only 50% of orexin cells express high levels of MOR immunoreactivity (Georgescu et al., 2003), opioids like morphine may differentially regulate orexin neurons that project to different brain areas. For example, opioid-induced inhibition of orexin neurons may be restricted to those neurons that project to arousal-related brain areas and thus mediate the sedating effects of morphine exposure. Alternatively, orexin neurons may be activated upon removal of morphine inhibition, potentially leading to increased orexin release in the VTA. Further research is required to test these possibilities.  Summary and conclusions  In summary, we propose a novel role for orexin signalling in morphine-induced plasticity at both glutamate and GABA synapses in the VTA. Morphine enhances excitatory synaptic efficacy onto VTA dopamine neurons by way of an orexin-dependent increase in presynaptic glutamate release, and a postsynaptic increase in AMPAR function. Moreover, orexin facilitated a long-term decrease in presynaptic GABA release to dopamine neurons. Finally, orexin signalling was required for a shift in the balance of excitatory and inhibitory control of dopamine neurons. Together, these findings provide novel insights into how orexin gates drug-induced plasticity in the VTA.   68 Chapter 3: Projection target defined effects of orexin and dynorphin on VTA dopamine neurons   Introduction   Dopamine neurons of the VTA attribute salience to environmental cues that predict motivationally relevant information (Robinson and Berridge, 1993) and provide the primary source of dopamine to target structures such as the BLA and the NAc. Recent evidence has established that distinct subpopulations of dopamine neurons exist within the VTA, with cells differing in their anatomical location, their electrophysiological properties and their afferent and efferent connections. Thus, different subpopulations of VTA dopamine neurons likely subserve different functions to drive motivated behaviour. Phasic dopamine in the NAc increases effort to obtain reinforcers (Correa et al., 2002; Salamone and Correa, 2002). Alternatively, BLA dopamine modulates synaptic transmission and associative plasticity (Bissière et al., 2003; Marowsky et al., 2005; Chu et al., 2012) to promote the learning and recall of fear and reward related memories and to drive cue-induced reinstatement of reward seeking (Fuchs and See, 2002; Berglind et al., 2006; Gremel and Cunningham, 2009; Fadok et al., 2010; Lintas et al., 2011; Heath et al., 2015). However, little is known about the afferent neuromodulatory control of VTA dopamine neurons and the circuits in which they are embedded.   Orexin neurons in the LH respond to reward predicting cues (Harris et al., 2005) and drive motivated reward-seeking behaviour through their projections to the VTA (España et al., 2010a, 2011a; Richardson and Aston-Jones, 2012; Mahler et al., 2013). Orexin A activates Gq-coupled OxR1s on VTA dopamine neurons to potentiate excitatory synaptic transmission (Borgland et al., 2006, 2009) and to increase neuronal firing (Korotkova et al., 2003; Muschamp et al., 2007, 2014; Moorman and Aston-Jones,  69 2010). Orexin is co-expressed with dynorphin (Chou et al., 2001), the endogenous ligand of Gi-coupled KORs, in dense core vesicles and the two are presumably coreleased (Li and van den Pol, 2006; Muschamp et al., 2014). Unlike orexin, activation of KORs in the VTA is aversive (Bals-Kubik et al., 1993; Ehrich et al., 2015) and reduces reward-seeking behaviour (Zhang et al., 2004; Shippenberg et al., 2007; Muschamp et al., 2014). Interestingly, orexin and dynorphin exert balanced but opposing effects on the firing activity of VTA dopamine neurons and orexin signalling in the VTA facilitates reward seeking by attenuating the antireward effects dynorphin (Muschamp et al., 2014). However, the specific circuits underlying the modulatory effects of these peptides remain unknown. Projection specific effects of orexin and dynorphin have been previously reported, albeit with contrasting results. Orexin infusion in the VTA preferentially induces cFos immunoreactivity in dopamine neurons that project to the medial PFC and the shell, but not the core, of the NAc and increases dopamine efflux in these regions (Narita et al., 2006; Vittoz and Berridge, 2006; Vittoz et al., 2008). Nevertheless, direct infusion of the OxR1 antagonist SB 334867 into the VTA attenuates cocaine-induced increases in dopamine concentration in the NAc core (España et al., 2010a). Similarly, there have been reports of mixed effects of KOR agonists on dopamine neurons that project to the medial PFC, the NAc and the amygdala (Ford et al., 2006; Margolis et al., 2006a, 2008). Here we test the hypothesis that orexin and dynorphin modulate separate circuits within the VTA. We examined the ability of orexin and dynorphin to alter the firing activity of NAc lateral shell- and BLA-projecting VTA dopamine neurons.     70 Materials and methods Animals All protocols were in accordance with the ethical guidelines established by the Canadian Council for Animal Care and were approved by the University of Calgary Animal Care Committees. C57BL/6J mice were obtained from Charles River Laboratories and Pitx3-GFP mice were obtained from the University of Calgary breeding facility; both were housed in groups of 2-5. Mice were maintained on a 12-hour light:dark schedule and were given food and water ad libitum. All experiments were performed during the animal’s light cycle.     Surgical procedures   Male and female mice were 8-10 weeks old at the time of surgery. Animals were anaesthetized with isoflurane and placed in a stereotaxic frame (Kopf; Tujunga, CA). Red Retrobeads (max excitation at 530 nm/max emission at 590 nm) (200 nL; Lumafluor Inc., Naples, FL) were infused bilaterally into the lateral shell of the NAc (from bregma: anteroposterior +1.425 mm; mediolateral, ± 1.75 mm; dorsoventral, −4.25 mm) or the BLA (from bregma: anteroposterior -1.0 mm; mediolateral, ± 3.1 mm; dorsoventral, -5.3 mm). Mice were treated post-surgically with ketoprofen (10 mg/kg, subcutaneous) and were left for a minimum of 2 weeks to allow adequate time for retrograde transport of the Retrobeads. Injection sites were confirmed in all animals by preparing coronal section of the lateral shell of the NAc and in horizontal sections of the BLA. Electrophysiology All electrophysiological recordings were performed in slice preparations from adult (minimum 10-weeks old) Pitx3-GFP mice. Briefly, mice were anaesthetized with isoflurane and transcardially perfused with an ice-cold sucrose solution containing (in  71 mM): 50 sucrose, 26.2 NaHCO3, 1.25 glucose, 4.9 MgCl2, 3 kynurenic acid, 0.1 CaCl2 and 1.32 ascorbic acid in bicarbonate-buffered solution (aCSF, described below). Mice were then decapitated and brains were extracted. Horizontal sections (180 µm) containing the VTA were cut on a vibratome (Leica, Nussloch, Germany) and incubated in a holding chamber for at least 45 minutes before being transferred to a recording chamber and superfused with aCSF containing (in mM): 126 NaCl, 1.6 KCl, 1.1 NaH2PO4, 1.4 MgCl2, 2.4 CaCl2, 26 NaHCO3, 11 glucose (32-34oC), and saturated with 95% O2/5% CO2. Cells were visualized on an upright microscope using “Dodt-type” gradient contrast infrared optics (Dodt et al., 2002) and whole-cell recordings were made using a MultiClamp 700B amplifier (Axon Instruments, Union City, CA).  Recording electrodes (3-5 MΩ) were filled with (in mM): 136 potassium-D-gluconate, 4 MgCl2, 1.1 HEPES, 5, EGTA, 10 sodium creatine phosphate, 3.4 Mg-ATP and 0.1 Na2GTP. After breaking into the cell, Ih currents were recorded in voltage-clamp mode by applying a series of hyperpolarizing steps (+10 to -60 mV) to dopamine neurons voltage-clamped at -70 mV. Cells were then switched into current-clamp mode and spontaneous firing activity was recorded. Orexin A (Tocris Bioscience) and dynorphin A (1-17, American Peptide) were both dissolved in distilled water and were bath applied for 5 minutes. Dynorphin was dissolved along with DL-Thiorphan (Sigma-Aldrich) and bestatin hydrochloride (Sigma-Aldrich), an enkephalinase and aminopeptidase inhibitor respectively. Because the vast majority of dopamine neurons ceased firing within 5 minutes of recording, current-step induced firing was used for all experiments, except dose-response analysis. For current-step experiments, the membrane potential for each neuron was set to -60 mV by DC injection via the patch amplifier and a series of 5 current pulses (400 ms in duration, 5-25 pA apart, adjusted for each cell) were applied every 45 seconds, where the minimum current amplitude was set for each cell so that  72 the first pulse was subthreshold and did not yield firing.  Analysis of action potential firing  Firing data for all neurons was analyzed with the MiniAnalysis program (Synaptosoft) using the same criteria. Drug-induced changes in firing are expressed as a percentage of baseline. For a given cell, a current step was selected that yielded 3-4 action potentials during the baseline period (minutes 5-10). This same current step was then analyzed following drug administration (minutes 20-25). Immunohistochemistry and confocal microscopy    Brain slices from patch-clamp recordings in Pitx3-GFP mice were fixed overnight in cold 4% paraformaldehyde and then stored in phosphate-buffered saline until processing. To enhance intrinsic GFP in dopamine neurons, slices were blocked in 3% normal goat serum, and incubated with monoclonal chicken anti-GFP antibody (1:10 000) overnight at room temperature. Goat anti-chicken Alexa Fluor 488 (1:1000) was then applied for 4 hours along with Dylight 405-conjugated streptavidin to identify neurons tagged with biocytin through the patch-pipette. Slices were then mounted with Fluoromount. For the slices used for the dual retrograde labeling experiment, brains were perfused and stored overnight in paraformaldehyde, switched to sucrose and coronal sections were cut on a cryostat. Sections were then blocked in 10% normal donkey serum and incubated with mouse anti-tyrosine hydroxylase (1:1000) for 48 hours at 4oC. Donkey anti mouse AMCA secondary antibody (1:50) was applied for 4 hours at room temperature and slices were mounted with Fluoromount. All images were obtained on a Nikon Eclipse C1si confocal microscope with a motorized stage (Nikon Canada Inc. Ontario, Canada). The objectives used were 20× Plan Apo DIC (NA 0.75), 40× oil immersion DIC (NA 1.30), and 60× Plan Apo water immersion DIC (NA 1.20).      73 Data analysis  All data is expressed as mean ± SEM. For each data set, distribution was assessed with a Shapiro-Wilk normality test and parametric or non-parametric statistical tests were used accordingly. Data was analyzed with two-tailed Student’s t-test or the Mann Whitney test, the Wilcoxon matched-pairs signed rank test, and the Kruskal-Wallis test for unpaired, paired and multiple group comparisons respectively. In all experiments, sample size is expressed as n/N where “n” refers to the number of cells recorded rom “N” animals. Prism 5 software (GraphPad Software, Inc., La Jolla, CA) was used to perform statistical analysis. Figures were generated using Prism 5 and Illustrator CS6 software (Adobe Systems Incorporated). The levels of significance are indicated as follows: ***P < 0.001, **P < 0.01, *P < 0.05.  Results NAc lateral shell- and BLA-projecting VTA dopamine neurons are mostly non-overlapping cell populations with different electrophysiological properties  Although VTA dopamine neurons were once considered a homogeneous cell population, recent evidence suggests a far more complex midbrain dopaminergic system with distinct neuronal subtypes differing in their topographical organization, their afferent and efferent connections and their basal electrophysiological properties (Ford et al., 2006; Margolis et al., 2006a, 2008; Lammel et al., 2008, 2011, 2012; Beier et al., 2015). Here, we examined the properties of dopamine neurons that project to the lateral shell of the NAc or the BLA. To confirm that these represent two distinct populations of dopamine neurons, we performed a double-tracing experiment in which we injected red Retrobeads into the lateral shell of the NAc and green Retrobeads into the BLA of adult C57BL/6J mice (Fig.7 A,D injection sites shown in Fig.7 B,C).  74 Retrograde bead infusions into the NAc lateral shell or the BLA yielded a similar number of labeled neurons in the VTA (NAc: 38.8 ± 5.6 neurons per slice, n=6/6; BLA: 31.0 ± 6.4 neurons per slice, n=5/5; t(9)=0.93, p=0.377; Fig.7 E). Importantly, we observed very few co-localized retrograde labels in VTA neurons (1.2 ±0.4 neurons per slice, n=5/5, Fig.7 E). Additionally, within slices, NAc lateral shell-projecting dopamine neurons were located in more ventral regions of the VTA, while BLA-projecting neurons resided in the more dorsal ventral portions with only minor regions of overlap between the two cell populations  (Fig.7 D). The majority of retrograde labeled neurons were immunoreactive for TH (NAc: 85.5 ± 2.2% of cells, n=6/6; BLA: 80.5 ± 4.2% of cells, n=5/5; Fig.7 F), with no difference in the degree of TH colocalization between the projection targets (t(9)=1.181, p=0.29). Taken together, these results suggest that BLA- and lateral shell- projecting VTA dopamine neurons are non-overlapping cell populations.  75 Figure 7: NAc lateral shell- and BLA-projecting VTA dopamine neurons are two largely non-overlapping cell populations. A. To determine that NAc lateral shell- and BLA-projecting VTA dopamine neurons are non-overlapping cell populations, we injected red Retrobeads into the NAc lateral shell and green Retrobeads into the BLA of adult C57Bl6J mice. B, C. 4x image of sample injection sites in the lateral shell of the NAc or the BLA. D. 10x and 40x (inset) image of a coronal section of the VTA where tyrosine hydroxylase positive neurons are shown in blue, along with retrograde labeled neurons from the NAc lateral shell (red) or the BLA (green). E. Retrograde bead infusions into the NAc lateral shell or the BLA yielded a similar number of retrograde labeled neuron in the VTA, with little to no overlap between the two projection targets. F. The majority of retrograde labeled neurons from both sites were immunoreactive for tyrosine hydroxylase. Bars represent mean + s.e.m.     Because NAc lateral shell- and BLA-projecting VTA dopamine neurons represent two separate cell populations, we examined whether or not they could be differentiated based on their intrinsic electrophysiological properties. To do this, we performed whole-cell patch-clamp electrophysiology from retrograde labeled VTA dopamine neurons in 10-12 week old Pitx3-GFP mice (Fig. 8 A,B,C). In these mice, GFP is expressed exclusively in dopamine neurons (Zhao et al., 2004; Hedlund et al., 2008), which permitted immediate identification of each recorded neuron as dopaminergic. Dopamine neurons have often been identified by the presence of a large HCN channel mediated Ih current (Lacey et al., 1989; Johnson and North, 1992a), although the reliability of this method has repeatedly been questioned (Margolis et al., 2010; Ungless and Grace, 2012). Nevertheless, recent reports suggest that the presence or lack of an Ih current in VTA dopamine neurons segregates on the basis of projection-target (Ford et al., 2006; Margolis et al., 2006a; Lammel et al., 2008, 2011). To activate Ih, we applied a series of hyperpolarizing steps to dopamine neurons voltage-clamped at -70 mV. Similar to previous reports (Lammel et al., 2011), NAc lateral shell-projecting dopamine neurons displayed a large Ih current (-67.0 ± 7.8 pA, n=51/29 with a step to -130 mV, Fig. 8 D,E) relative to BLA-projecting dopamine neurons, which expressed little to no Ih  76 (-12.5 ± 4.6 pA, n=25/17 with a step to -130 mV, Fig. 8 D,E) (Mann-Whitney test: U=460.0, p<0.0001). However, the presence of an Ih current was not a unique identifier of NAc lateral shell-projecting dopamine neurons as a subpopulation of BLA-projecting dopamine neurons also express the current (10 of 25 neurons (40%) had an Ih > -15 pA), and the lack of an Ih was not specific to BLA-projecting neurons (6 of 51 (12%) NAc lateral shell-projecting had an Ih <-15 pA). Moreover, inward leak currents mediated by background conductances were greater in NAc lateral shell-projecting relative to BLA-projecting dopamine neurons (NAc: -215.9 ± 18.5 pA, n=51/29; BLA: -84.6 ± 15.6 pA; n=25/17; Mann-Whitney test: U=227.0, p<0.0001, Fig. 8 D,F).     77 Figure 8: NAc lateral shell- and BLA-projecting VTA dopamine neurons express different Ih. A, B. 20X (left) and 60x (right) confocal images of horizontal sections of the VTA from Pitx3-GFP mouse two weeks after infusion of retrograde beads in the lateral shell of the NAc (red) (A) or the BLA (red, shown in green) (B). GFP expressing dopamine neurons are shown in blue. C. Confocal images (60X) of a biocytin labeled (magenta), GFP positive (blue), retrograde labeled neuron from the NAc lateral shell. D. Sample Ih recordings from NAc lateral shell- (red) and BLA-projecting (green) VTA dopamine neurons. E, F. Relative to BLA-projecting dopamine neurons, NAc lateral shell-projecting dopamine neurons express a significantly larger Ih current, which is associated with increased activation of leak conductances (F). Bars represent mean + s.e.m, *** p < 0.001.     Because previous reports have suggested that projection target is a determinant of the properties of action potentials in dopaminergic neurons, we recorded the spontaneous firing of projection target defined dopamine neurons in current clamp. Figure 9A displays single action potentials recorded from NAc lateral shell- or BLA-projecting VTA dopamine neurons. These subpopulations of dopamine neurons had a similar threshold voltage (NAc: -30.5 ± 0.4 mV, n=21/13, BLA: -31.0 ± 0.6 mV, n=12/10; t(31)=0.67, p=0.511; Fig. 9 B), but differed in spike width (measured at half maximal amplitude) (NAc: 1.7 ± 0.1 ms, n=21/13, BLA: 2.2 ± 02 ms, n=12/10; t(31)=3.72, p=0.0034; Fig. 9 C) and in the properties of the AHP. NAc lateral shell-projecting dopamine neurons had a greater spike height (measured from the peak to the minimum voltage of AHP) (NAc: 100.6 ± 1.6 mV, n=21/13, BLA: 88.9 ± 3.1 mV, n=12/10; t(31)=3.72, p=0.0008; Fig. 9 D), a greater minimum voltage of the AHP (measured as the peak negative voltage reached in the trough of the action potential) (NAc:-64.9 ± 0.8 mV, n=21/13, BLA:-58.4 ± 2.1 mV, n=12/10; t(31)=3.44, p=0.0017; Fig. 9 E) and a longer AHP width (measured as the period of time during which the membrane voltage was below pre-spike level in the trough of the action potential) (NAc: 0.53 ± 0.05 mV, n=21/13, BLA: 0.29 ± 0.39 mV, n=12/10; t(31)=3.44, p=0.0017; Fig. 9 F). Together this suggests that NAc lateral shell- and BLA-projecting VTA dopamine neurons represent  78 two distinct subpopulations of neurons with distinct electrophysiological characteristics.         Figure 9: NAc lateral shell- and BLA-projecting VTA dopamine neurons have different action potential properties. A. Sample traces of an individual spontaneous action potential recorded in current clamp mode from NAc lateral shell- (red) and BLA-projecting (green) VTA dopamine neurons. Action potentials from NAc lateral shell- projecting VTA dopamine neurons had a similar threshold voltage (B), but a shorter width (C), greater height (D) and more hyperpolarized (E) and wider (F) afterhyperpolarization potentials relative to BLA-projecting VTA dopamine neurons. Scatter plots display individual data points with mean + s.e.m, ** p < 0.01.    79 Orexin A preferentially increases the firing activity of NAc lateral shell-projecting VTA dopamine neurons  Although previous reports have established that orexin A increases the firing rate of dopamine neurons, the effects are variable in that many neurons show no response to application of orexin A (Korotkova et al., 2003; Muschamp et al., 2014). To determine if differential responses of orexin A segregate on the basis of projection target, we examined the effect of orexin A application on the firing activity of projection target defined VTA dopamine neurons. In non-retrograde labeled dopamine neurons, we confirmed that orexin A increases the spontaneous firing rate in a concentration-dependent manner to a maximum of 174.1 ± 10.6% (EC50=56.3 nM; Fig. 10 A,B,C). Depolarization block occurred in several neurons after application of 300 nM orexin A, leading to significant variability in firing rate at this concentration. We then examined the effect of a saturating concentration of orexin A (100 nM) on action potential firing of projection-specific VTA dopamine neurons during depolarizing currents steps, where a series of five 400 ms depolarizing steps was applied every 45 seconds. In NAc lateral shell-projecting VTA dopamine neurons, orexin A increased the firing (baseline: 102.9 ± 2.0% vs. Orexin A 148.6 ± 17.7%; n=10/7; Fig. 10 D,E,F). Although orexin A potentiates glutamatergic synaptic transmission at synapses onto VTA dopamine neurons (Borgland et al., 2006, 2009), this increase in firing was independent of changes in synaptic transmission as the increase was equally observed when APV (50 μM), DNQX (10 μM) and picrotoxin (100 μM), antagonists of AMPARs, NMDARs and GABAARs respectively, were included in the bath (baseline: 103.0 ± 2.6% vs. Orexin A 149.9 ± 12.0%; n=6/4, Fig. 10 D,E,F). The effect was dependent on OxR1 signalling and was inhibited when recordings were made with the OxR1 antagonist SB 334867 (1 μM) in the bath solution (baseline: 99.8 ± 2.6% vs. Orexin A 105.1 ± 6.8%; n=10/6, Fig. 10  80 D,E,F) (Kruskal-Wallis test: H(2)=8.576, p=0.0137; Fig. 10 F). Overall, orexin A increased the firing of 11 of 16 (69%, pooled aCSF and aCSF + synaptic blockers, Fig. 10 G) NAc lateral shell-projecting VTA dopamine neurons. Conversely, orexin A had no effect on the firing rate of BLA-projecting VTA dopamine neurons (baseline: 102.1 ± 1.2% vs. Orexin A 106.0 ± 5.0%; n=9/7, p=0.734. W=-7.0 pairs=9; Fig. 10 H,I,J), with only 1 out of 9 (11%) neurons showing an increase in firing greater than 15%. This suggests that orexin release in the VTA modulates dopamine firing in a subpopulation specific manner.                81   82  Figure 10: Orexin A preferentially increases the firing activity of NAc lateral shell-projecting VTA dopamine neurons. A, Sample recordings of spontaneous action potential firing of dopamine neurons before and after bath application of 100 nM orexin A. B, Example time course and C, dose-response curve (n=3-5 per concentration) for orexin A-induced changes in firing rate in VTA dopamine neurons recorded in Pitx3-GFP mice. D, Sample recordings of current-step induced firing before and after application of 100 nM orexin A in dopamine neurons that project to the lateral shell of the NAc. E, F, 100 nM orexin A increases the firing activity of NAc lateral shell-projecting dopamine neurons (blue), This effect persists in the presence of blockers of AMPARs, NMDARs, and GABAARs (orange) and is dependent on OxR1 signalling (grey). G, In the absence of an antagonist (pooled aCSF and aCSF + synaptic blockers) orexin A increased the firing rate of 69% (11 of 16) of recorded neurons. H, 100 nM orexin A did not increase the firing activity of VTA dopamine neurons that project to the BLA. I,J, Sample traces and summary of all recordings. K, Only 11% (1 of 9) of BLA-projecting dopamine neurons showed an increase in firing in the presence of orexin A. Bars represent mean + s.e.m, * p < 0.05.   Projection-specific effects of dynorphin A on the firing activity of VTA dopamine neurons   Orexin neurons contain the excitatory orexin peptides and the inhibitory peptide dynorphin in the same dense core vesicles (Muschamp et al., 2014). Therefore, given projection target defined effects of orexin on VTA dopamine neurons, potential co-release of dynorphin represents an important additional regulatory mechanism for orexin neurons to modulate the activity of VTA dopamine neurons. Dynorphin signalling could counteract the effects of orexin to temper the level of activity of dopamine neurons, or might signal within different subcircuits of the VTA to further promote orexin-driven reward seeking. Here we examined how dynorphin A alters the activity of NAc lateral shell- and BLA-projecting VTA dopamine neurons. Dynorphin A decreased the spontaneous firing rate of non-retrograde labeled dopamine neurons in a dose-dependent manner to a minimum of 58.99 ± 10.07% (IC50=25.6 nM, Fig. 11 A,B,C). We then examined the effect of dynorphin A (200 nM) on current step evoked firing of  83 projection defined VTA dopamine neurons. Interestingly, we observed mixed effects of dynorphin on the firing activity of NAc lateral shell-projecting dopamine neurons. Although dynorphin A did not induce a significant depression of the firing activity of NAc lateral shell-projecting dopamine neurons when all cells were averaged together (baseline: 98.5 ± 1.9% vs. dynorphin 81.3 ± 8.8%; n=13/10; Wilcoxon matched-pairs signed rank test: W=33.00, p=0.105, pairs=13; Fig. 11 D,E,F), there was a clear dichotomy in the response to dynorphin. While the majority of cells were non-responsive (9 of 13 cells (64%), baseline: 100.4 ± 2.14% vs. dynorphin 100.2 ± 2.60%, Fig. 11 D,E,F,G), the firing activity of a subset of neurons was significantly decreased in response to application of dynorphin A (4 of 13 cells (36%), baseline: 94.39 ± 3.65% vs. dynorphin 38.75 ± 9.76%, Fig. 11 D,E,F,G) (responders vs. non-responders: Mann-Whitney test: U=0.0, p=0.0028). In contrast to NAc lateral shell-projecting neurons, the majority of BLA-projecting dopamine neurons were inhibited by dynorphin A application (baseline: 102.6 ± 2.2% vs. dynorphin 66.5 ± 10.7%; n=11/9, Wilcoxon matched-pairs signed rank test: W=56.00, p=0.0098, pairs=11; Fig. 11 H,I,J).  64%  (7 of 11) of neurons tested were inhibited by dynorphin and the remainders were non-responders (Fig.11 K). This inhibition of firing was blocked with the KOR antagonist norBNI (baseline: 99.4 ± 0.66% vs. dyn 93.0 ± 18.45%; N/n=4/3 , Wilcoxon matched-pairs signed rank test W=0.0, p=1.0 pairs=4; Fig. 11 J). Taken together, dynorphin inhibits the majority of BLA-projecting dopamine neurons, but only modulates a small population of NAc lateral shell projecting dopamine neurons.      84     85  Figure 11: Dyn preferentially inhibits the firing activity of BLA-projecting VTA dopamine neurons. A, Sample recordings of spontaneous action potential firing of dopamine neurons before and after bath application of 100 nM Dyn. B, Example time course and C, dose-response curve (n=3-5 per concentration) for Dyn-induced changes in firing rate in VTA dopamine neurons recorded in Pitx3-GFP mice. D, Sample recordings of current-step induced firing before and after application of 200 nM Dyn in dopamine neurons that project to the lateral shell of the NAc. E, F, G, The majority of dopamine neurons that project to the lateral shell of the NAc were unaffected by application of 200 nM Dyn (purple, 9 of 13 cells, 64%). But, Dyn significantly decreased firing in a subset of NAc lateral shell-projecting neurons (red, 4 of 13, 36%). H, Sample traces of current-step evoked firing in BLA-projecting VTA dopamine neurons before and after bath application of 200 nM Dyn in aCSF (red) or with norBNI in the bath (grey). I,J,K, Dyn decreased the firing activity of BLA-projecting VTA dopamine neurons (red). This decreases was blocked in the presence of the KOR antagonist norBNI (grey).   Discussion   Here we report that orexin and dynorphin have distinct modulatory effects on the activity of projection-target defined VTA dopamine neurons. Dopamine neurons that project to the NAc lateral shell or the BLA represent two distinct cell populations with largely non-overlapping axonal projections and divergent electrophysiological properties. Orexin selectively increased the firing activity of NAc lateral shell-projecting dopamine neurons, while dynorphin primarily inhibited firing in BLA-projecting dopamine neurons. Thus, presumed corelease of orexin and dynorphin in the VTA targets independent dopaminergic circuits to mediate its net effects.  Heterogeneity within the VTA dopamine system  Previous work has established a complex dopaminergic system whereby intrinsic properties and neural circuit connections are dependent on projection target (Ford et al., 2006; Margolis et al., 2006a, 2008; Lammel et al., 2008, 2011, 2012; Beier et al., 2015). Consistent with previous reports (Lammel et al., 2008; Beier et al., 2015), we confirm that NAc lateral shell-projecting and BLA-projecting VTA dopamine neurons  86 are two largely discrete cell populations. We observed little to no dual labelling in a double retrograde tracing experiment. Accordingly, a recent report demonstrated that NAc lateral shell-projecting VTA dopamine neurons send very few axonal arbors to the amygdala (Beier et al., 2015) and NAc lateral shell- and BLA-projecting dopamine neurons were differentially distributed within the VTA (Lammel et al., 2008). NAc lateral shell- and BLA-projecting dopamine neurons also differed in their intrinsic electrophysiological properties. We replicate the observations that relative to BLA-projecting dopamine neurons, NAc lateral shell-projecting neurons express a greater Ih current, and have shorter action potentials with a more pronounced AHP (Lammel et al., 2008, 2011). Given that NAc lateral shell-projecting neurons had a longer and more hyperpolarized AHPs, they likely fire in a lower frequency range than do dopamine neurons that project to the BLA. Correspondingly, NAc lateral shell-projecting dopamine neurons have lower maximal firing rates in response to ramp depolarization protocols (Lammel et al., 2008). Moreover, differences in the AHP indicate differential recruitment of ion channels during spike firing, which may be subject to distinct neuromodulatory influences.  Projection target specific effects of orexin and dynorphin   Orexin and dynorphin had projection target specific effects on the firing activity of VTA dopamine neurons. Orexin preferentially increased firing in NAc lateral shell-projecting dopamine neurons while dynorphin was more effective at inhibiting firing in BLA-projecting dopamine neurons. Importantly, we measured the effect on dopamine neuronal firing using saturating steady-state concentrations of either peptide. Thus, it is likely that non-responding neurons did not express sufficient receptor to initiate a signaling response.  Although previous reports have demonstrated that orexin increases  87 firing in VTA dopamine neurons (Korotkova et al., 2003; Muschamp et al., 2007, 2014; Moorman and Aston-Jones, 2010), our results indicate that these effects are restricted to specific subpopulations within the VTA. Orexin had no effect on the firing activity of BLA-projecting dopamine neurons. The orexin-mediated increase in firing was independent of orexin induced changes in synaptic transmission, which occurred with a similar time course and required OxR1 signalling (Borgland et al., 2006, 2009). OxR1 signalling has been linked to the closing of K+ channels, the activation of the electrogenic Na+/Ca2+ exchanger, and the activation of non-selective cation channels (Leonard and Kukkonen, 2014). Because all of these mechanisms can contribute to an orexin-induced increase in firing, future experiments should aim to identify the specific mechanism by which OxA excites NAc lateral shell-projecting dopamine neurons. Moreover, because NAc lateral shell-projecting dopamine neurons express Ih, and enhancing Ih can increase firing of dopamine neurons (Wanat et al., 2008), OxA may target HCN channels to enhance neuronal firing. Taken together, orexin increases firing of NAc shell, but not BLA-projecting dopamine neurons.    Dynorphin strongly inhibited the majority of BLA-projecting dopamine neurons, as well as a small population of NAc lateral shell-projecting dopamine neurons. Projection target specific effects of KOR agonists have been previously examined, albeit with contrasting results. KOR agonists did not hyperpolarize the membrane potential of NAc medial shell-projecting dopamine neurons, nor did they reduce dopamine concentration in the NAc in a rat (Margolis et al., 2006a), but did induce large whole-cell K+ currents in this population of neurons in mice (Ford et al., 2006). The discrepancy between these two studies may be due to a difference in species. Combined with our results here, there may be important subregional differences for projections within the NAc as we observed both non-responders to dynorphin as well  88 as a population that was strongly inhibited by dynorphin signaling in NAc lateral shell-projecting dopamine neurons. Future work will need to determine if there are further characteristics that differentiate dynorphin sensitive neurons from dynorphin insensitive neurons within this projection target. Nevertheless, it is important to note that orexin and dynorphin modulation of dopamine neurons is not restricted to acute changes in neuronal firing and so we cannot rule out other modulatory effects of these peptides on these target neurons. Both orexin and dynorphin alter synaptic transmission at synapses onto VTA dopamine neurons (Borgland et al., 2006, 2009; Ford et al., 2006) and can induce signalling cascades that are independent of changes in neuronal activity (Ehrich et al., 2015).   Although we report here that NAc lateral shell- and BLA-projecting dopamine neurons were largely sensitive to orexin or dynorphin, there exists a subset of dopamine neurons that are sensitive to both peptides (Muschamp et al., 2014). Application of orexin and dynorphin to VTA dopamine neurons that respond individually to both peptides had no net effect on firing (Muschamp et al., 2014), suggesting that orexin cancels the inhibitory effect of dynorphin. While the projection target of the population that respond to both peptides was not defined in this study, it is possible that they project to the NAc lateral shell as we found that neurons in this population of dopamine neurons can respond to either orexin or to a lesser extent, dynorphin. Furthermore, given that orexin and dynorphin can alter downstream release in target structures like the medial PFC (Margolis et al., 2006a; Narita et al., 2006; Vittoz and Berridge, 2006), it will be important to further characterize the mechanisms by which orexin and dynorphin interact to influence dopamine neurons in a projection target specific manner.   Together these results suggest that corelease of orexin and dynorphin in the VTA alter the activity and likely the downstream release of dopamine in a projection  89 target specific manner. Given the different roles of dopamine in the NAc and the BLA, coordinated regulation of the VTA by orexin neurons may have important functional considerations. Here we speculate that influence of orexin projections to the VTA would likely reduce dopamine concentration in the BLA. Dopaminergic regulation of the amygdala is complex with certain reports demonstrating that dopamine drives plasticity to promote learning and memory retention (Bissière et al., 2003; Marowsky et al., 2005; Chu et al., 2012) while  others demonstrate that dopamine receptor activation enhances memory extinction (Abraham et al., 2016) or acts as a filter to discriminate and prevent less salient experiences from forming persistent memories (Kwon et al., 2015; Lee and Kim, 2016). Both orexin and dopamine overlap in their regulation of fear and threat learning (de Oliveira et al., 2011; de Souza Caetano et al., 2013; Sears et al., 2013; Heath et al., 2015). However, because threat learning is associated with orexin signalling in the locus coeruleus (Sears et al., 2013; Soya et al., 2013) or in the BLA itself (Flores et al., 2014), and LC- and VTA-projecting orexin neurons differ in their activation profiles, it is conceivable that this effect of orexin is independent of its projections to the VTA. Conversely, because NAc lateral shell-projecting dopamine neurons were preferentially excited by orexin, recruitment of orexin projections to the VTA would likely drive dopamine release in the NAc. Given that orexin neurons are recruited primarily during highly motivated reward seeking, when effort requirements are high (Borgland et al., 2009; Smith et al., 2009; España et al., 2010a, 2011a; Muschamp et al., 2014), orexin and dynorphin signalling in the VTA may act to coordinate the output of the VTA to bias activity and energy resources towards projection targets like the NAc, which are critical for effort driven reward seeking (Correa et al., 2002) while dampening activity in neurons projecting to the BLA, which  90 are not essential to reward seeking in high effort demand tasks (McGregor and Roberts, 1993; McGregor et al., 1994; Wassum and Izquierdo, 2015).  Summary and conclusions  In summary, we demonstrated that NAc lateral shell- and BLA-projecting VTA dopamine neurons represent two distinct cell populations within the VTA that are subject to differential neuromodulatory influence from lateral hypothalamic orexin neurons. We propose that through corelease of orexin and dynorphin, orexin projections coordinate the activity of VTA dopamine neurons to drive motivated reward seeking behaviour.    91 Chapter 4-General discussion   The activity of dopamine neurons is tightly regulated by intrinsic conductances, synaptic inputs and neuromodulatory influences including that of lateral hypothalamic orexin neurons. This thesis examined two mechanisms by which orexin neurons regulate the output of VTA dopamine neurons. The mesocorticolimbic dopamine system is a direct target of addictive drugs and synaptic plasticity within this circuitry underlies some of the core features of addiction. Drug-induced plasticity at excitatory glutamatergic synapses onto VTA dopamine neurons represents a critical permissive step in the development of addiction-like behaviour. In chapter 2, we determined that orexin signalling at OxR1 locally within the VTA gates morphine-induced plasticity at excitatory and inhibitory synapses onto dopamine neurons. A single exposure to morphine potentiated excitatory synaptic transmission by both an OxR1 dependent switch in the subunit composition of postsynaptic AMPARs and an increase in the probability of presynaptic glutamate release. Moreover, morphine induced an OxR1 dependent decrease in the probability of presynaptic GABA release at inhibitory synapses onto dopamine neurons, which contributed to a shift in the balance of excitatory and inhibitory control of dopamine neurons.   Recent evidence highlights the heterogeneity of VTA dopamine neurons, which differ in their afferent and efferent connectivity and their intrinsic electrophysiological properties. Chapter 3 of this thesis demonstrates projection target specific neuromodulatory effects of orexin and dynorphin on VTA dopamine neurons. Orexin preferentially increased the firing activity of VTA dopamine neurons that project to the lateral shell of the NAc, while dynorphin was more effective at inhibiting firing in dopamine neurons that project to the BLA. Taken together, activation of orexin projections to the VTA likely has both immediate and long-term consequences on the  92 regulation of the activity of dopamine neurons, which is critically linked to the promotion of motivated behaviour.  Orexin signalling and drug-induced synaptic plasticity in the VTA  A leading theory hypothesizes that drug addiction arises from neural circuit dysfunction initiated in the VTA (Kauer and Malenka, 2007; Lüscher et al., 2015). Drug-induced plasticity at synapses onto VTA dopamine neurons occurs with the very first exposure (Ungless et al., 2001; Saal et al., 2003; Nugent et al., 2007; Brown et al., 2010) and represents a metaplastic signal for further modifications within the mesocorticolimbic system (Mameli et al., 2009; Creed and Lüscher, 2013; Pascoli et al., 2015). Nevertheless, it remains unknown how drugs with different molecular targets and different mechanisms of action converge to induce plasticity in the VTA. We have now established that orexin signalling in the VTA underlies drug-induced plasticity for both cocaine (Borgland et al., 2006) and morphine (Baimel and Borgland, 2015, Chapter 2). Given that orexin signalling has been implicated in drug-seeking behaviours across the spectrum of addictive drugs, orexin may represent the unifying substrate responsible for this shared drug-induced phenotype. For the experiments described in chapter 2 with morphine, and in previous work with cocaine (Borgland et al., 2006), dopamine neurons were identified by the expression of a large Ih current. Given that we have confirmed here that a large Ih current is a marker of dopamine neurons that project to the lateral shell of the NAc (Lammel et al., 2011), it is conceivable that this subpopulation of neurons is highly represented in these experiments. Further supporting this is the fact that the majority of NAc lateral shell-projecting dopamine neurons tested in chapter 3 were sensitive to the excitatory effects of orexin A. It will be important to determine if orexin is equally effective in gating footshock-induced  93 plasticity in these neurons (Lammel et al., 2011). Moreover, because dopamine neurons were identified by large Ih currents, this suggests that dopamine neurons that lack Ih, such as those that project to the BLA (chapter 3), the NAc medial shell and the medial PFC (Lammel et al., 2008, 2011) were likely omitted from these studies. Therefore, it will be equally important to characterize the role of orexin signalling in plasticity in these populations of neurons. Because these subpopulations of dopamine neurons receive different afferent inputs (Lammel et al., 2012; Beier et al., 2015), and we demonstrate that orexin signalling underlies morphine-induced increases in presynaptic glutamate release, orexin may act specifically on a subset of glutamatergic terminals in the VTA. Given that the laterodorsal tegmentum preferentially targets dopamine neurons that project to the NAc lateral shell (Lammel et al., 2012), this represents a strong candidate for the presynaptic effects of orexin.   The specific aspect of OxR1 signalling that is responsible for gating drug-induced plasticity in the VTA remains unknown. Cocaine induces NMDAR dependent potentiation of AMPAR signalling in dopamine neurons (Ungless et al., 2001; Borgland et al., 2004; Brown et al., 2010). Similarly, orexin A potentiates NMDAR currents in VTA dopamine neurons, which in turn promotes increased AMPAR signalling (Borgland et al., 2006). Together this suggests that drug-induced activation of orexin terminals in the VTA may drive orexin release and lead to increased NMDAR currents and the induction of AMPAR plasticity. However, selective optogenetic stimulation of dopamine neurons is sufficient to induce drug-like changes in AMPAR expression in the VTA (Brown et al., 2010) and bath application of cocaine potentiates NMDARs through signalling at dopamine receptor D5 (Schilström et al., 2006). This promotes the idea that somatodendritic dopamine release within the VTA may also contribute to drug-induced plasticity at glutamatergic synapses. Given that orexin increases the firing activity of  94 cocaine sensitive NAc lateral shell-projecting dopamine neurons (Lammel et al., 2011), orexin induced increases in dopamine transmission may also be involved in driving plasticity at synapses onto these neurons.   It is also important to consider what role corelease of dynorphin may have under these conditions. Although the majority of NAc lateral shell-projecting dopamine neurons recorded here were insensitive to dynorphin, others have reported KOR agonist induced acute decreases in GABAA, GABAB and dopamine receptor 2 IPSCs (Ford et al., 2006). Dynorphin induced disinhibition paired with simultaneous orexin-induced activation of dopamine neurons would likely further enhance excitability of these neurons. Furthermore, although KOR signalling has no effect on stress-induced plasticity at excitatory synapses onto VTA dopamine neurons, nor is it involved in morphine-induced inhibition of LTPGABA (Graziane et al., 2013), KOR antagonists do block stress-induced inhibition of LTPGABA in the VTA and stress-induced reinstatement of cocaine seeking (Graziane et al., 2013). It is interesting to consider that exposure to stress might cause orexin neurons to release both orexin and dynorphin in the VTA to drive synaptic plasticity at excitatory and inhibitory synapses respectively. Nevertheless, because morphine and footshock activate different subpopulations of orexin neurons in the LH (Harris et al., 2005), which are thought to differ in their projection targets, this may not be the case. Alternatively, stress may induce selective release of dynorphin in the VTA to drive these effects, although this may not necessarily involve orexin neurons, as there are many other dynorphin producing cells in the brain that project to the VTA, including those from the BNST, the NAc and the amygdala (Fallon et al., 1985; Meredith, 1999; Dong and Swanson, 2004; Poulin et al., 2009; Graziane et al., 2013). Moreover, because exposure to highly salient reinforcers enhances the effects of OxR1 signalling in the VTA (Borgland et al., 2009), it is  95 interesting to consider that repeated drug exposure might also alter KOR signalling in certain subpopulations of dopamine neurons.   Endogenous orexin and dynorphin release in the VTA  Given the recently established heterogeneity of dopamine neurons (Ford et al., 2006; Margolis et al., 2006a; Lammel et al., 2011, 2012; Beier et al., 2015) and the diverse cellular composition of the VTA, corelease of excitatory orexin and inhibitory dynorphin in this region likely has important but complex modulatory actions on both neuronal function and behaviour. By demonstrating that orexin and dynorphin alter the activity of dopamine neurons in a projection target specific manner, we have started to break down some of this complexity. However, the picture is far from complete. Orexin and dynorphin may have important regulatory actions on dopamine neurons that are independent from changes in neuronal firing. For example, orexin alters excitatory synaptic transmission onto VTA dopamine neurons (Borgland et al., 2006, 2009), and given the results presented in chapter 2, is likely an important regulatory signal for  inhibitory transmission as well. Although, in Chapter 3 we demonstrated that orexin-mediated increase in firing rate was not due to altered excitatory synaptic transmission, synaptically driven changes in firing may not be detectable in the slice preparation used here, where many afferent inputs are severed. However, future studies should be aimed at assessing if orexin-mediated potentiation of excitatory synaptic transmission can alter the firing pattern of dopamine neurons in vivo.    Nevertheless, the biggest limitation of the work presented here is that we are exogenously applying orexin and dynorphin, rather than stimulating endogenous release of these peptides from orexin fibers in the VTA. This is now possible with optogenetic techniques and has been successful in other brain regions (Adamantidis et  96 al., 2007; Carter et al., 2009; Schöne et al., 2012, 2014; Apergis-Schoute et al., 2015). However, to date no one has exploited this technique to probe orexin or dynorphin release in the VTA. Adopting this technique will be critical to developing a proper understanding of orexin functions in this region. For example, although orexin and dynorphin are found in the same vesicles within the LH (Muschamp et al., 2014), it is unclear if these vesicles are transported to the VTA. Therefore, it is uncertain if orexin and dynorphin are in fact released together in the VTA, or perhaps released from the same neurons, but not necessarily at the same time or under the same conditions. This is particularly relevant given that orexin and dynorphin are encoded by different genes, which likely undergo different regulatory processes. Given the discrepancy in responding of different subpopulations of dopamine neurons to these peptides, differential production and or trafficking of orexin and dynorphin would likely induce distinct phenotypes. Moreover, although the low synaptic incidence of orexin fibers in the VTA (Balcita-Pedicino and Sesack, 2007) suggests that these neurons do not represent an important source of glutamate to the region, this has not yet been directly tested. Similarly, although it is clear that orexin is released in the VTA to drive drug-induced synaptic plasticity, the nature of this release is unknown. With optogenetic control, it will be possible to determine the pattern and level of activity required to induce synaptic plasticity within the VTA. This is particularly important given that it is currently unclear how drugs like cocaine and morphine activate orexin neurons.  Conclusions  The experiments described in this thesis examined mechanisms by which orexin neurons modulate the output of VTA dopamine neurons. First, we demonstrated that orexin signalling in the VTA gates morphine-induced synaptic plasticity at both  97 excitatory and inhibitory synapses onto VTA dopamine neurons. Furthermore, we found that orexin and dynorphin alter the firing activity of dopamine neurons in a projection target specific manner. The results in this thesis contribute to our growing understanding of the role of orexin neurons modulate the activity of dopamine neurons to promote goal directed motivated behaviour.     98 References  Abercrombie ED, Keefe KA, DiFrischia DS, Zigmond MJ (1989) Differential effect of stress on in vivo dopamine release in striatum, nucleus accumbens, and medial frontal cortex. J Neurochem 52:1655–1658. Abraham AD, Neve KA, Lattal KM (2016) Activation of D1/5 Dopamine Receptors: A Common Mechanism for Enhancing Extinction of Fear and Reward-Seeking Behaviors. Neuropsychopharmacology doi:10.1038/npp.2016.5 Adamantidis AR, Zhang F, Aravanis AM, Deisseroth K, de Lecea L (2007) Neural substrates of awakening probed with optogenetic control of hypocretin neurons. Nature 450:420–424. Adams WJ, Lorens SA, Mitchell CL (1972) Morphine enhances lateral hypothalamic self-stimulation in the rat. Proc Soc Exp Biol Med Soc Exp Biol Med N Y N 140:770–771. Agnati LF, Fuxe K, Zoli M, Ozini I, Toffano G, Ferraguti F (1986) A correlation analysis of the regional distribution of central enkephalin and beta-endorphin immunoreactive terminals and of opiate receptors in adult and old male rats. Evidence for the existence of two main types of communication in the central nervous system: the volume transmission and the wiring transmission. Acta Physiol Scand 128:201–207. Agnati LF, Guidolin D, Guescini M, Genedani S, Fuxe K (2010) Understanding wiring and volume transmission. Brain Res Rev 64:137–159. Agnati LF, Zoli M, Strömberg I, Fuxe K (1995) Intercellular communication in the brain: wiring versus volume transmission. Neuroscience 69:711–726. Akimoto H, Honda Y, Takahashi Y (1960) Pharmacotherapy in narcolepsy. Dis Nerv Syst 21:704–706. Aldrich JV, Patkar KA, McLaughlin JP (2009) Zyklophin, a systemically active selective kappa opioid receptor peptide antagonist with short duration of action. Proc Natl Acad Sci U S A 106:18396–18401. Al-Hasani R, McCall JG, Shin G, Gomez AM, Schmitz GP, Bernardi JM, Pyo C-O, Park SI, Marcinkiewcz CM, Crowley NA, Krashes MJ, Lowell BB, Kash TL, Rogers JA, Bruchas MR (2015) Distinct Subpopulations of Nucleus Accumbens Dynorphin Neurons Drive Aversion and Reward. Neuron 87:1063–1077. Anstrom KK, Woodward DJ (2005) Restraint increases dopaminergic burst firing in awake rats. Neuropsychopharmacology 30:1832–1840. Apergis-Schoute J, Iordanidou P, Faure C, Jego S, Schöne C, Aitta-Aho T, Adamantidis A, Burdakov D (2015) Optogenetic evidence for inhibitory signaling from orexin to MCH neurons via local microcircuits. J Neurosci 35:5435–5441.  99 Aragona BJ, Cleaveland NA, Stuber GD, Day JJ, Carelli RM, Wightman RM (2008) Preferential enhancement of dopamine transmission within the nucleus accumbens shell by cocaine is attributable to a direct increase in phasic dopamine release events. J Neurosci 28:8821–8831. Argilli E, Sibley DR, Malenka RC, England PM, Bonci A (2008) Mechanism and time course of cocaine-induced long-term potentiation in the ventral tegmental area. J Neurosci 28:9092–9100. Arvidsson U, Riedl M, Chakrabarti S, Vulchanova L, Lee JH, Nakano AH, Lin X, Loh HH, Law PY, Wessendorf MW (1995) The kappa-opioid receptor is primarily postsynaptic: combined immunohistochemical localization of the receptor and endogenous opioids. Proc Natl Acad Sci U S A 92:5062–5066. Aston-Jones G, Smith RJ, Sartor GC, Moorman DE, Massi L, Tahsili-Fahadan P, Richardson KA (2010) Lateral hypothalamic orexin/hypocretin neurons: A role in reward-seeking and addiction. Brain Res 1314:74–90. Baimel C, Bartlett SE, Chiou L-C, Lawrence AJ, Muschamp JW, Patkar O, Tung L-W, Borgland SL (2015) Orexin/hypocretin role in reward: implications for opioid and other addictions. Br J Pharmacol 172:334–348. Baimel C, Borgland SL (2015) Orexin Signaling in the VTA Gates Morphine-Induced Synaptic Plasticity. J Neurosci 35:7295–7303. Balcita-Pedicino JJ, Sesack SR (2007) Orexin axons in the rat ventral tegmental area synapse infrequently onto dopamine and gamma-aminobutyric acid neurons. J Comp Neurol 503:668–684. Baldo BA, Daniel RA, Berridge CW, Kelley AE (2003) Overlapping distributions of orexin/hypocretin- and dopamine-beta-hydroxylase immunoreactive fibers in rat brain regions mediating arousal, motivation, and stress. J Comp Neurol 464:220–237. Bals-Kubik R, Ableitner A, Herz A, Shippenberg TS (1993) Neuroanatomical sites mediating the motivational effects of opioids as mapped by the conditioned place preference paradigm in rats. J Pharmacol Exp Ther 264:489–495. Bassareo V, De Luca MA, Di Chiara G (2002) Differential Expression of Motivational Stimulus Properties by Dopamine in Nucleus Accumbens Shell versus Core and Prefrontal Cortex. J Neurosci 22:4709–4719. Beardsley PM, Howard JL, Shelton KL, Carroll FI (2005) Differential effects of the novel kappa opioid receptor antagonist, JDTic, on reinstatement of cocaine-seeking induced by footshock stressors vs cocaine primes and its antidepressant-like effects in rats. Psychopharmacology (Berl) 183:118–126. Beckstead MJ, Grandy DK, Wickman K, Williams JT (2004) Vesicular dopamine release elicits an inhibitory postsynaptic current in midbrain dopamine neurons. Neuron 42:939–946.  100 Beier KT, Steinberg EE, DeLoach KE, Xie S, Miyamichi K, Schwarz L, Gao XJ, Kremer EJ, Malenka RC, Luo L (2015) Circuit Architecture of VTA Dopamine Neurons Revealed by Systematic Input-Output Mapping. Cell 162:622–634. Bellone C, Lüscher C (2006) Cocaine triggered AMPA receptor redistribution is reversed in vivo by mGluR-dependent long-term depression. Nat Neurosci 9:636–641. Bentzley BS, Aston-Jones G (2015) Orexin-1 receptor signaling increases motivation for cocaine-associated cues. Eur J Neurosci 41:1149–1156. Berglind WJ, Case JM, Parker MP, Fuchs RA, See RE (2006) Dopamine D1 or D2 receptor antagonism within the basolateral amygdala differentially alters the acquisition of cocaine-cue associations necessary for cue-induced reinstatement of cocaine-seeking. Neuroscience 137:699–706. Berridge KC, Venier IL, Robinson TE (1989) Taste reactivity analysis of 6-hydroxydopamine-induced aphagia: implications for arousal and anhedonia hypotheses of dopamine function. Behav Neurosci 103:36–45. Bielajew C, Shizgal P (1986) Evidence implicating descending fibers in self-stimulation of the medial forebrain bundle. J Neurosci 6:919–929. Bissière S, Humeau Y, Lüthi A (2003) Dopamine gates LTP induction in lateral amygdala by suppressing feedforward inhibition. Nat Neurosci 6:587–592. Bissonette GB, Roesch MR (2016) Development and function of the midbrain dopamine system: what we know and what we need to. Genes Brain Behav 15:62–73. Bocklisch C, Pascoli V, Wong JCY, House DRC, Yvon C, de Roo M, Tan KR, Lüscher C (2013) Cocaine disinhibits dopamine neurons by potentiation of GABA transmission in the ventral tegmental area. Science 341:1521–1525. Borgland SL, Chang S-J, Bowers MS, Thompson JL, Vittoz N, Floresco SB, Chou J, Chen BT, Bonci A (2009) Orexin A/hypocretin-1 selectively promotes motivation for positive reinforcers. J Neurosci 29:11215–11225. Borgland SL, Malenka RC, Bonci A (2004) Acute and chronic cocaine-induced potentiation of synaptic strength in the ventral tegmental area: electrophysiological and behavioral correlates in individual rats. J Neurosci 24:7482–7490. Borgland SL, Storm E, Bonci A (2008) Orexin B/hypocretin 2 increases glutamatergic transmission to ventral tegmental area neurons. Eur J Neurosci 28:1545–1556. Borgland SL, Taha SA, Sarti F, Fields HL, Bonci A (2006) Orexin A in the VTA is critical for the induction of synaptic plasticity and behavioral sensitization to cocaine. Neuron 49:589–601.  101 Boutrel B, Kenny PJ, Specio SE, Martin-Fardon R, Markou A, Koob GF, de Lecea L (2005) Role for hypocretin in mediating stress-induced reinstatement of cocaine-seeking behavior. Proc Natl Acad Sci U S A 102:19168–19173. Brady KT, Sinha R (2005) Co-occurring mental and substance use disorders: the neurobiological effects of chronic stress. Am J Psychiatry 162:1483–1493. Brauer LH, De Wit H (1997) High dose pimozide does not block amphetamine-induced euphoria in normal volunteers. Pharmacol Biochem Behav 56:265–272. Brischoux F, Chakraborty S, Brierley DI, Ungless MA (2009) Phasic excitation of dopamine neurons in ventral VTA by noxious stimuli. Proc Natl Acad Sci U S A 106:4894–4899. Britt JP, Benaliouad F, McDevitt RA, Stuber GD, Wise RA, Bonci A (2012) Synaptic and behavioral profile of multiple glutamatergic inputs to the nucleus accumbens. Neuron 76:790–803. Brown MTC, Bellone C, Mameli M, Labouèbe G, Bocklisch C, Balland B, Dahan L, Luján R, Deisseroth K, Lüscher C (2010) Drug-driven AMPA receptor redistribution mimicked by selective dopamine neuron stimulation. PloS One 5:e15870. Burgess CR, Scammell TE (2012) Narcolepsy: neural mechanisms of sleepiness and cataplexy. J Neurosci 32:12305–12311. Burlet S, Tyler CJ, Leonard CS (2002) Direct and indirect excitation of laterodorsal tegmental neurons by Hypocretin/Orexin peptides: implications for wakefulness and narcolepsy. J Neurosci 22:2862–2872. Cameron DL, Wessendorf MW, Williams JT (1997) A subset of ventral tegmental area neurons is inhibited by dopamine, 5-hydroxytryptamine and opioids. Neuroscience 77:155–166. Carey RJ, Goodal E (1975) Differential effects of amphetamine and food deprivation of self-stimulation of the lateral hypothalamus and medial frontal cortex. J Comp Physiol Psychol 88:224–230. Carlezon WA, Boundy VA, Haile CN, Lane SB, Kalb RG, Neve RL, Nestler EJ (1997) Sensitization to morphine induced by viral-mediated gene transfer. Science 277:812–814. Carlezon WA, Rasmussen K, Nestler EJ (1999) AMPA antagonist LY293558 blocks the development, without blocking the expression, of behavioral sensitization to morphine. Synap N Y N 31:256–262. Carter ME, Adamantidis A, Ohtsu H, Deisseroth K, de Lecea L (2009) Sleep homeostasis modulates hypocretin-mediated sleep-to-wake transitions. J Neurosci 29:10939–10949.  102 Cazala P, Darracq C, Saint-Marc M (1987) Self-administration of morphine into the lateral hypothalamus in the mouse. Brain Res 416:283–288. Chavkin C, James IF, Goldstein A (1982) Dynorphin is a specific endogenous ligand of the kappa opioid receptor. Science 215:413–415. Chefer VI, Bäckman CM, Gigante ED, Shippenberg TS (2013) Kappa opioid receptors on dopaminergic neurons are necessary for kappa-mediated place aversion. Neuropsychopharmacology 38:2623–2631. Chemelli RM, Willie JT, Sinton CM, Elmquist JK, Scammell T, Lee C, Richardson JA, Williams SC, Xiong Y, Kisanuki Y, Fitch TE, Nakazato M, Hammer RE, Saper CB, Yanagisawa M (1999) Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation. Cell 98:437–451. Chen BT, Bowers MS, Martin M, Hopf FW, Guillory AM, Carelli RM, Chou JK, Bonci A (2008) Cocaine but not natural reward self-administration nor passive cocaine infusion produces persistent LTP in the VTA. Neuron 59:288–297. Chergui K, Suaud-Chagny MF, Gonon F (1994) Nonlinear relationship between impulse flow, dopamine release and dopamine elimination in the rat brain in vivo. Neuroscience 62:641–645. Chieng B, Azriel Y, Mohammadi S, Christie MJ (2011) Distinct cellular properties of identified dopaminergic and GABAergic neurons in the mouse ventral tegmental area. J Physiol 589:3775–3787. Chou TC, Lee CE, Lu J, Elmquist JK, Hara J, Willie JT, Beuckmann CT, Chemelli RM, Sakurai T, Yanagisawa M, Saper CB, Scammell TE (2001) Orexin (hypocretin) neurons contain dynorphin. J Neurosci 21:RC168. Chu H-Y, Ito W, Li J, Morozov A (2012) Target-specific suppression of GABA release from parvalbumin interneurons in the basolateral amygdala by dopamine. J Neurosci 32:14815–14820. Collins AGE, Frank MJ (2015) Surprise! Dopamine signals mix action, value and error. Nat Neurosci 19:3–5. Corbett AD, Paterson SJ, McKnight AT, Magnan J, Kosterlitz HW (1982) Dynorphin and dynorphin are ligands for the kappa-subtype of opiate receptor. Nature 299:79–81. Cornish JL, Kalivas PW (2001) Repeated cocaine administration into the rat ventral tegmental area produces behavioral sensitization to a systemic cocaine challenge. Behav Brain Res 126:205–209. Correa M, Carlson BB, Wisniecki A, Salamone JD (2002) Nucleus accumbens dopamine and work requirements on interval schedules. Behav Brain Res 137:179–187.  103 Couteaux R, Pécot-Dechavassine M (1970) [Synaptic vesicles and pouches at the level of “active zones” of the neuromuscular junction]. Comptes Rendus Hebd Séances Académie Sci Sér Sci Nat 271:2346–2349. Creed MC, Lüscher C (2013) Drug-evoked synaptic plasticity: beyond metaplasticity. Curr Opin Neurobiol 23:553–558. Crocker A, España RA, Papadopoulou M, Saper CB, Faraco J, Sakurai T, Honda M, Mignot E, Scammell TE (2005) Concomitant loss of dynorphin, NARP, and orexin in narcolepsy. Neurology 65:1184–1188. Dacher M, Nugent FS (2011) Morphine-induced modulation of LTD at GABAergic synapses in the ventral tegmental area. Neuropharmacology 61:1166–1171. Day JJ, Roitman MF, Wightman RM, Carelli RM (2007) Associative learning mediates dynamic shifts in dopamine signaling in the nucleus accumbens. Nat Neurosci 10:1020–1028. de Lecea L, Kilduff TS, Peyron C, Gao X, Foye PE, Danielson PE, Fukuhara C, Battenberg EL, Gautvik VT, Bartlett FS, Frankel WN, van den Pol AN, Bloom FE, Gautvik KM, Sutcliffe JG (1998) The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity. Proc Natl Acad Sci U S A 95:322–327. de Oliveira AR, Reimer AE, de Macedo CEA, de Carvalho MC, Silva MA de S, Brandão ML (2011) Conditioned fear is modulated by D2 receptor pathway connecting the ventral tegmental area and basolateral amygdala. Neurobiol Learn Mem 95:37–45. DePaoli AM, Hurley KM, Yasada K, Reisine T, Bell G (1994) Distribution of kappa opioid receptor mRNA in adult mouse brain: an in situ hybridization histochemistry study. Mol Cell Neurosci 5:327–335. Deroche-Gamonet V, Piazza PV (2014) Psychobiology of cocaine addiction: Contribution of a multi-symptomatic animal model of loss of control. Neuropharmacology 76 Pt B:437–449. de Souza Caetano KA, de Oliveira AR, Brandão ML (2013) Dopamine D2 receptors modulate the expression of contextual conditioned fear: role of the ventral tegmental area and the basolateral amygdala. Behav Pharmacol 24:264–274. Devine DP, Leone P, Pocock D, Wise RA (1993) Differential involvement of ventral tegmental mu, delta and kappa opioid receptors in modulation of basal mesolimbic dopamine release: in vivo microdialysis studies. J Pharmacol Exp Ther 266:1236–1246. Di Chiara G, Imperato A (1988) Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proc Natl Acad Sci U S A 85:5274–5278.  104 Dimitrova A, Fronczek R, Van der Ploeg J, Scammell T, Gautam S, Pascual-Leone A, Lammers GJ (2011) Reward-seeking behavior in human narcolepsy. J Clin Sleep Med JCSM Off Publ Am Acad Sleep Med 7:293–300. Dodt H-U, Eder M, Schierloh A, Zieglgänsberger W (2002) Infrared-guided laser stimulation of neurons in brain slices. Sci STKE Signal Transduct Knowl Environ 2002:pl2. Dong H-W, Swanson LW (2004) Projections from bed nuclei of the stria terminalis, posterior division: implications for cerebral hemisphere regulation of defensive and reproductive behaviors. J Comp Neurol 471:396–433. Douglas RJ, Martin KAC (2004) Neuronal circuits of the neocortex. Annu Rev Neurosci 27:419–451. Dube MG, Kalra SP, Kalra PS (1999) Food intake elicited by central administration of orexins/hypocretins: identification of hypothalamic sites of action. Brain Res 842:473–477. Durstewitz D, Seamans JK (2002) The computational role of dopamine D1 receptors in working memory. Neural Netw Off J Int Neural Netw Soc 15:561–572. Ehrich JM, Messinger DI, Knakal CR, Kuhar JR, Schattauer SS, Bruchas MR, Zweifel LS, Kieffer BL, Phillips PEM, Chavkin C (2015) Kappa Opioid Receptor-Induced Aversion Requires p38 MAPK Activation in VTA Dopamine Neurons. J Neurosci 35:12917–12931. Engblom D, Bilbao A, Sanchis-Segura C, Dahan L, Perreau-Lenz S, Balland B, Parkitna JR, Luján R, Halbout B, Mameli M, Parlato R, Sprengel R, Lüscher C, Schütz G, Spanagel R (2008) Glutamate receptors on dopamine neurons control the persistence of cocaine seeking. Neuron 59:497–508. Eriksson KS, Sergeeva OA, Selbach O, Haas HL (2004) Orexin (hypocretin)/dynorphin neurons control GABAergic inputs to tuberomammillary neurons. Eur J Neurosci 19:1278–1284. España RA, Melchior JR, Roberts DCS, Jones SR (2011a) Hypocretin 1/orexin A in the ventral tegmental area enhances dopamine responses to cocaine and promotes cocaine self-administration. Psychopharmacology (Berl) 214:415–426. España RA, Melchior JR, Roberts DCS, Jones SR (2011b) Hypocretin 1/orexin A in the ventral tegmental area enhances dopamine responses to cocaine and promotes cocaine self-administration. Psychopharmacology (Berl) 214:415–426. España RA, Oleson EB, Locke JL, Brookshire BR, Roberts DCS, Jones SR (2010a) The hypocretin-orexin system regulates cocaine self-administration via actions on the mesolimbic dopamine system. Eur J Neurosci 31:336–348.  105 España RA, Oleson EB, Locke JL, Brookshire BR, Roberts DCS, Jones SR (2010b) The hypocretin-orexin system regulates cocaine self-administration via actions on the mesolimbic dopamine system. Eur J Neurosci 31:336–348. Fadel J, Deutch AY (2002) Anatomical substrates of orexin-dopamine interactions: lateral hypothalamic projections to the ventral tegmental area. Neuroscience 111:379–387. Fadok JP, Darvas M, Dickerson TMK, Palmiter RD (2010) Long-term memory for pavlovian fear conditioning requires dopamine in the nucleus accumbens and basolateral amygdala. PloS One 5:e12751. Faleiro LJ, Jones S, Kauer JA (2003) Rapid AMPAR/NMDAR response to amphetamine: a detectable increase in AMPAR/NMDAR ratios in the ventral tegmental area is detectable after amphetamine injection. Ann N Y Acad Sci 1003:391–394. Fallon JH, Leslie FM, Cone RI (1985) Dynorphin-containing pathways in the substantia nigra and ventral tegmentum: a double labeling study using combined immunofluorescence and retrograde tracing. Neuropeptides 5:457–460. Flores Á, Valls-Comamala V, Costa G, Saravia R, Maldonado R, Berrendero F (2014) The hypocretin/orexin system mediates the extinction of fear memories. Neuropsychopharmacology 39:2732–2741. Floresco SB, West AR, Ash B, Moore H, Grace AA (2003) Afferent modulation of dopamine neuron firing differentially regulates tonic and phasic dopamine transmission. Nat Neurosci 6:968–973. Ford CP, Mark GP, Williams JT (2006) Properties and opioid inhibition of mesolimbic dopamine neurons vary according to target location. J Neurosci 26:2788–2797. Frank ST, Krumm B, Spanagel R (2008) Cocaine-induced dopamine overflow within the nucleus accumbens measured by in vivo microdialysis: a meta-analysis. Synap N Y N 62:243–252. Fuchs RA, See RE (2002) Basolateral amygdala inactivation abolishes conditioned stimulus- and heroin-induced reinstatement of extinguished heroin-seeking behavior in rats. Psychopharmacology (Berl) 160:425–433. Fujiki N, Yoshida Y, Ripley B, Honda K, Mignot E, Nishino S (2001) Changes in CSF hypocretin-1 (orexin A) levels in rats across 24 hours and in response to food deprivation. Neuroreport 12:993–997. Galeote L, Berrendero F, Bura SA, Zimmer A, Maldonado R (2009) Prodynorphin gene disruption increases the sensitivity to nicotine self-administration in mice. Int J Neuropsychopharmacol Off Sci J Coll Int Neuropsychopharmacol CINP 12:615–625. Gantz SC, Bunzow JR, Williams JT (2013) Spontaneous inhibitory synaptic currents mediated by a G protein-coupled receptor. Neuron 78:807–812.  106 Gao X-B, Hermes G (2015) Neural plasticity in hypocretin neurons: the basis of hypocretinergic regulation of physiological and behavioral functions in animals. Front Syst Neurosci 9:142. Gawin FH (1986) Neuroleptic reduction of cocaine-induced paranoia but not euphoria? Psychopharmacology (Berl) 90:142–143. Georgescu D, Zachariou V, Barrot M, Mieda M, Willie JT, Eisch AJ, Yanagisawa M, Nestler EJ, DiLeone RJ (2003) Involvement of the lateral hypothalamic peptide orexin in morphine dependence and withdrawal. J Neurosci 23:3106–3111. Glick SD, Maisonneuve IM, Raucci J, Archer S (1995) Kappa opioid inhibition of morphine and cocaine self-administration in rats. Brain Res 681:147–152. Gonon FG (1988) Nonlinear relationship between impulse flow and dopamine released by rat midbrain dopaminergic neurons as studied by in vivo electrochemistry. Neuroscience 24:19–28. González JA, Jensen LT, Fugger L, Burdakov D (2012) Convergent inputs from electrically and topographically distinct orexin cells to locus coeruleus and ventral tegmental area. Eur J Neurosci 35:1426–1432. Good CH, Lupica CR (2010) Afferent-specific AMPA receptor subunit composition and regulation of synaptic plasticity in midbrain dopamine neurons by abused drugs. J Neurosci 30:7900–7909. Gore BB, Soden ME, Zweifel LS (2014) Visualization of plasticity in fear-evoked calcium signals in midbrain dopamine neurons. Learn Mem Cold Spring Harb N 21:575–579. Grace AA (1991) Regulation of spontaneous activity and oscillatory spike firing in rat midbrain dopamine neurons recorded in vitro. Synap N Y N 7:221–234. Grace AA, Bunney BS (1980) Nigral dopamine neurons: intracellular recording and identification with L-dopa injection and histofluorescence. Science 210:654–656. Grace AA, Bunney BS (1984a) The control of firing pattern in nigral dopamine neurons: single spike firing. J Neurosci 4:2866–2876. Grace AA, Bunney BS (1984b) The control of firing pattern in nigral dopamine neurons: burst firing. J Neurosci 4:2877–2890. Grace AA, Floresco SB, Goto Y, Lodge DJ (2007) Regulation of firing of dopaminergic neurons and control of goal-directed behaviors. Trends Neurosci 30:220–227. Grace AA, Onn SP (1989) Morphology and electrophysiological properties of immunocytochemically identified rat dopamine neurons recorded in vitro. J Neurosci 9:3463–3481.  107 Graziane NM, Polter AM, Briand LA, Pierce RC, Kauer JA (2013) Kappa opioid receptors regulate stress-induced cocaine seeking and synaptic plasticity. Neuron 77:942–954. Gremel CM, Cunningham CL (2009) Involvement of amygdala dopamine and nucleus accumbens NMDA receptors in ethanol-seeking behavior in mice. Neuropsychopharmacology 34:1443–1453. Guilleminault C, Carskadon M, Dement WC (1974) On the treatment of rapid eye movement narcolepsy. Arch Neurol 30:90–93. Gulia KK, Mallick HN, Kumar VM (2003) Orexin A (hypocretin-1) application at the medial preoptic area potentiates male sexual behavior in rats. Neuroscience 116:921–923. Guyenet PG, Aghajanian GK (1978) Antidromic identification of dopaminergic and other output neurons of the rat substantia nigra. Brain Res 150:69–84. Hamid AA, Pettibone JR, Mabrouk OS, Hetrick VL, Schmidt R, Vander Weele CM, Kennedy RT, Aragona BJ, Berke JD (2016) Mesolimbic dopamine signals the value of work. Nat Neurosci 19:117–126. Haney M, Ward AS, Foltin RW, Fischman MW (2001) Effects of ecopipam, a selective dopamine D1 antagonist, on smoked cocaine self-administration by humans. Psychopharmacology (Berl) 155:330–337. Hara J, Beuckmann CT, Nambu T, Willie JT, Chemelli RM, Sinton CM, Sugiyama F, Yagami K, Goto K, Yanagisawa M, Sakurai T (2001) Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity. Neuron 30:345–354. Harris GC, Aston-Jones G (2006) Arousal and reward: a dichotomy in orexin function. Trends Neurosci 29:571–577. Harris GC, Wimmer M, Aston-Jones G (2005) A role for lateral hypothalamic orexin neurons in reward seeking. Nature 437:556–559. Harris GC, Wimmer M, Randall-Thompson JF, Aston-Jones G (2007) Lateral hypothalamic orexin neurons are critically involved in learning to associate an environment with morphine reward. Behav Brain Res 183:43–51. Haynes AC, Jackson B, Overend P, Buckingham RE, Wilson S, Tadayyon M, Arch JR (1999) Effects of single and chronic intracerebroventricular administration of the orexins on feeding in the rat. Peptides 20:1099–1105. Heath FC, Jurkus R, Bast T, Pezze MA, Lee JLC, Voigt JP, Stevenson CW (2015) Dopamine D1-like receptor signalling in the hippocampus and amygdala modulates the acquisition of contextual fear conditioning. Psychopharmacology (Berl) 232:2619–2629.  108 Heath RG (1963) ELECTRICAL SELF-STIMULATION OF THE BRAIN IN MAN. Am J Psychiatry 120:571–577. Hedlund E, Pruszak J, Lardaro T, Ludwig W, Viñuela A, Kim K-S, Isacson O (2008) Embryonic stem cell-derived Pitx3-enhanced green fluorescent protein midbrain dopamine neurons survive enrichment by fluorescence-activated cell sorting and function in an animal model of Parkinson’s disease. Stem Cells Dayt Ohio 26:1526–1536. Heikkinen AE, Möykkynen TP, Korpi ER (2009) Long-lasting modulation of glutamatergic transmission in VTA dopamine neurons after a single dose of benzodiazepine agonists. Neuropsychopharmacology 34:290–298. Hemby SE, Co C, Koves TR, Smith JE, Dworkin SI (1997) Differences in extracellular dopamine concentrations in the nucleus accumbens during response-dependent and response-independent cocaine administration in the rat. Psychopharmacology (Berl) 133:7–16. Hernandez G, Breton Y-A, Conover K, Shizgal P (2010) At what stage of neural processing does cocaine act to boost pursuit of rewards? PloS One 5:e15081. Hnasko TS, Hjelmstad GO, Fields HL, Edwards RH (2012) Ventral tegmental area glutamate neurons: electrophysiological properties and projections. J Neurosci 32:15076–15085. Hollander JA, Lu Q, Cameron MD, Kamenecka TM, Kenny PJ (2008) Insular hypocretin transmission regulates nicotine reward. Proc Natl Acad Sci U S A 105:19480–19485. Hopf FW, Martin M, Chen BT, Bowers MS, Mohamedi MM, Bonci A (2007) Withdrawal from intermittent ethanol exposure increases probability of burst firing in VTA neurons in vitro. J Neurophysiol 98:2297–2310. Horvath TL, Gao X-B (2005) Input organization and plasticity of hypocretin neurons: possible clues to obesity’s association with insomnia. Cell Metab 1:279–286. Hyman SE, Malenka RC, Nestler EJ (2006) Neural mechanisms of addiction: the role of reward-related learning and memory. Annu Rev Neurosci 29:565–598. Jalabert M, Bourdy R, Courtin J, Veinante P, Manzoni OJ, Barrot M, Georges F (2011) Neuronal circuits underlying acute morphine action on dopamine neurons. Proc Natl Acad Sci U S A 108:16446–16450. Johnson SW, North RA (1992a) Two types of neurone in the rat ventral tegmental area and their synaptic inputs. J Physiol 450:455–468. Johnson SW, North RA (1992b) Opioids excite dopamine neurons by hyperpolarization of local interneurons. J Neurosci 12:483–488.  109 Johnson SW, Seutin V, North RA (1992) Burst firing in dopamine neurons induced by N-methyl-D-aspartate: role of electrogenic sodium pump. Science 258:665–667. Jones S, Kauer JA (1999) Amphetamine depresses excitatory synaptic transmission via serotonin receptors in the ventral tegmental area. J Neurosci 19:9780–9787. Kalivas PW, Alesdatter JE (1993) Involvement of N-methyl-D-aspartate receptor stimulation in the ventral tegmental area and amygdala in behavioral sensitization to cocaine. J Pharmacol Exp Ther 267:486–495. Kalivas PW, Duffy P (1995) Selective activation of dopamine transmission in the shell of the nucleus accumbens by stress. Brain Res 675:325–328. Kalivas PW, Volkow ND (2005) The neural basis of addiction: a pathology of motivation and choice. Am J Psychiatry 162:1403–1413. Kauer JA, Malenka RC (2007) Synaptic plasticity and addiction. Nat Rev Neurosci 8:844–858. Kelley AE (2004) Memory and addiction: shared neural circuitry and molecular mechanisms. Neuron 44:161–179. Kmiotek EK, Baimel C, Gill KJ (2012) Methods for intravenous self administration in a mouse model. J Vis Exp JoVE:e3739. Komendantov AO, Komendantova OG, Johnson SW, Canavier CC (2004) A modeling study suggests complementary roles for GABAA and NMDA receptors and the SK channel in regulating the firing pattern in midbrain dopamine neurons. J Neurophysiol 91:346–357. Koob GF, Volkow ND (2010) Neurocircuitry of addiction. Neuropsychopharmacology 35:217–238. Korotkova TM, Sergeeva OA, Eriksson KS, Haas HL, Brown RE (2003) Excitation of ventral tegmental area dopaminergic and nondopaminergic neurons by orexins/hypocretins. J Neurosci 23:7–11. Kotz CM, Teske JA, Levine JA, Wang C (2002) Feeding and activity induced by orexin A in the lateral hypothalamus in rats. Regul Pept 104:27–32. Kourrich S, Rothwell PE, Klug JR, Thomas MJ (2007) Cocaine experience controls bidirectional synaptic plasticity in the nucleus accumbens. J Neurosci 27:7921–7928. Kukkonen JP, Leonard CS (2014) Orexin/hypocretin receptor signalling cascades. Br J Pharmacol 171:314–331. Kunii K, Yamanaka A, Nambu T, Matsuzaki I, Goto K, Sakurai T (1999) Orexins/hypocretins regulate drinking behaviour. Brain Res 842:256–261.  110 Kuzmin AV, Semenova S, Gerrits MA, Zvartau EE, Van Ree JM (1997) Kappa-opioid receptor agonist U50,488H modulates cocaine and morphine self-administration in drug-naive rats and mice. Eur J Pharmacol 321:265–271. Kwon O-B, Lee JH, Kim HJ, Lee S, Lee S, Jeong M-J, Kim S-J, Jo H-J, Ko B, Chang S, Park SK, Choi Y-B, Bailey CH, Kandel ER, Kim J-H (2015) Dopamine Regulation of Amygdala Inhibitory Circuits for Expression of Learned Fear. Neuron 88:378–389. Lacey MG, Mercuri NB, North RA (1989) Two cell types in rat substantia nigra zona compacta distinguished by membrane properties and the actions of dopamine and opioids. J Neurosci 9:1233–1241. Lambe EK, Aghajanian GK (2003) Hypocretin (orexin) induces calcium transients in single spines postsynaptic to identified thalamocortical boutons in prefrontal slice. Neuron 40:139–150. Lammel S, Hetzel A, Häckel O, Jones I, Liss B, Roeper J (2008) Unique properties of mesoprefrontal neurons within a dual mesocorticolimbic dopamine system. Neuron 57:760–773. Lammel S, Ion DI, Roeper J, Malenka RC (2011) Projection-specific modulation of dopamine neuron synapses by aversive and rewarding stimuli. Neuron 70:855–862. Lammel S, Lim BK, Ran C, Huang KW, Betley MJ, Tye KM, Deisseroth K, Malenka RC (2012) Input-specific control of reward and aversion in the ventral tegmental area. Nature 491:212–217. Lane DA, Jaferi A, Kreek MJ, Pickel VM (2010) Acute and chronic cocaine differentially alter the subcellular distribution of AMPA GluR1 subunits in region-specific neurons within the mouse ventral tegmental area. Neuroscience 169:559–573. Laorden ML, Ferenczi S, Pintér-Kübler B, González-Martín LL, Lasheras MC, Kovács KJ, Milanés MV, Núñez C (2012) Hypothalamic orexin--a neurons are involved in the response of the brain stress system to morphine withdrawal. PloS One 7:e36871. Latimer LG, Duffy P, Kalivas PW (1987) Mu opioid receptor involvement in enkephalin activation of dopamine neurons in the ventral tegmental area. J Pharmacol Exp Ther 241:328–337. Lecca S, Melis M, Luchicchi A, Muntoni AL, Pistis M (2012) Inhibitory inputs from rostromedial tegmental neurons regulate spontaneous activity of midbrain dopamine cells and their responses to drugs of abuse. Neuropsychopharmacology 37:1164–1176. Lee JH, Kim J-H (2016) Dopamine-dependent synaptic plasticity in an amygdala inhibitory circuit controls fear memory expression. BMB Rep 49:1–2.  111 Lee MG, Hassani OK, Jones BE (2005) Discharge of identified orexin/hypocretin neurons across the sleep-waking cycle. J Neurosci 25:6716–6720. Leonard CS, Kukkonen JP (2014) Orexin/hypocretin receptor signalling: a functional  perspective. Br J Pharmacol 171:294-313.    Leone P, Pocock D, Wise RA (1991) Morphine-dopamine interaction: ventral tegmental morphine increases nucleus accumbens dopamine release. Pharmacol Biochem Behav 39:469–472. Leyton M, Casey KF, Delaney JS, Kolivakis T, Benkelfat C (2005) Cocaine craving, euphoria, and self-administration: a preliminary study of the effect of catecholamine precursor depletion. Behav Neurosci 119:1619–1627. Leyton M, Rajabi H, Stewart J (1992) U-50,488H into A10 reduces haloperidol-induced elevations of accumbens dopamine. Neuroreport 3:1127–1130. Lindholm S, Werme M, Brené S, Franck J (2001) The selective kappa-opioid receptor agonist U50,488H attenuates voluntary ethanol intake in the rat. Behav Brain Res 120:137–146. Lin L, Faraco J, Li R, Kadotani H, Rogers W, Lin X, Qiu X, de Jong PJ, Nishino S, Mignot E (1999) The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene. Cell 98:365–376. Lintas A, Chi N, Lauzon NM, Bishop SF, Gholizadeh S, Sun N, Tan H, Laviolette SR (2011) Identification of a dopamine receptor-mediated opiate reward memory switch in the basolateral amygdala-nucleus accumbens circuit. J Neurosci 31:11172–11183. Liu Q, Pu L, Poo M (2005) Repeated cocaine exposure in vivo facilitates LTP induction in midbrain dopamine neurons. Nature 437:1027–1031. Liu SJ, Zukin RS (2007) Ca2+-permeable AMPA receptors in synaptic plasticity and neuronal death. Trends Neurosci 30:126–134. Li X, Marchant NJ, Shaham Y (2014) Opposing roles of cotransmission of dynorphin and hypocretin on reward and motivation. Proc Natl Acad Sci U S A 111:5765–5766. Li Y, Gao XB, Sakurai T, van den Pol AN (2002) Hypocretin/Orexin excites hypocretin neurons via a local glutamate neuron-A potential mechanism for orchestrating the hypothalamic arousal system. Neuron 36:1169–1181. Li Y, van den Pol AN (2006) Differential target-dependent actions of coexpressed inhibitory dynorphin and excitatory hypocretin/orexin neuropeptides. J Neurosci 26:13037–13047. Li Y, van den Pol AN (2008) Mu-opioid receptor-mediated depression of the hypothalamic hypocretin/orexin arousal system. J Neurosci 28:2814–2819.  112 Lobb CJ, Wilson CJ, Paladini CA (2011) High-frequency, short-latency disinhibition bursting of midbrain dopaminergic neurons. J Neurophysiol 105:2501–2511. Lodge DJ, Grace AA (2006) The laterodorsal tegmentum is essential for burst firing of ventral tegmental area dopamine neurons. Proc Natl Acad Sci U S A 103:5167–5172. Logrip ML, Janak PH, Ron D (2009) Blockade of ethanol reward by the kappa opioid receptor agonist U50,488H. Alcohol Fayettev N 43:359–365. Loweth JA, Scheyer AF, Milovanovic M, LaCrosse AL, Flores-Barrera E, Werner CT, Li X, Ford KA, Le T, Olive MF, Szumlinski KK, Tseng KY, Wolf ME (2014) Synaptic depression via mGluR1 positive allosteric modulation suppresses cue-induced cocaine craving. Nat Neurosci 17:73–80. Lujan R, Nusser Z, Roberts JD, Shigemoto R, Somogyi P (1996) Perisynaptic location of metabotropic glutamate receptors mGluR1 and mGluR5 on dendrites and dendritic spines in the rat hippocampus. Eur J Neurosci 8:1488–1500. Lüscher C, Pascoli V, Creed M (2015) Optogenetic dissection of neural circuitry: from synaptic causalities to blue prints for novel treatments of behavioral diseases. Curr Opin Neurobiol 35:95–100. Lüscher C, Ungless MA (2006) The mechanistic classification of addictive drugs. PLoS Med 3:e437. Mahler SV, Moorman DE, Smith RJ, James MH, Aston-Jones G (2014) Motivational activation: a unifying hypothesis of orexin/hypocretin function. Nat Neurosci 17:1298–1303. Mahler SV, Smith RJ, Aston-Jones G (2013) Interactions between VTA orexin and glutamate in cue-induced reinstatement of cocaine seeking in rats. Psychopharmacology (Berl) 226:687–698. Mameli M, Balland B, Luján R, Lüscher C (2007) Rapid synthesis and synaptic insertion of GluR2 for mGluR-LTD in the ventral tegmental area. Science 317:530–533. Mameli M, Bellone C, Brown MTC, Lüscher C (2011) Cocaine inverts rules for synaptic plasticity of glutamate transmission in the ventral tegmental area. Nat Neurosci 14:414–416. Mameli M, Halbout B, Creton C, Engblom D, Parkitna JR, Spanagel R, Lüscher C (2009) Cocaine-evoked synaptic plasticity: persistence in the VTA triggers adaptations in the NAc. Nat Neurosci 12:1036–1041. Mansour A, Burke S, Pavlic RJ, Akil H, Watson SJ (1996) Immunohistochemical localization of the cloned kappa 1 receptor in the rat CNS and pituitary. Neuroscience 71:671–690.  113 Mantsch JR, Baker DA, Funk D, Lê AD, Shaham Y (2016) Stress-Induced Reinstatement of Drug Seeking: 20 Years of Progress. Neuropsychopharmacology 41:335–356. Marcus JN, Aschkenasi CJ, Lee CE, Chemelli RM, Saper CB, Yanagisawa M, Elmquist JK (2001) Differential expression of orexin receptors 1 and 2 in the rat brain. J Comp Neurol 435:6–25. Margolis EB, Coker AR, Driscoll JR, Lemaître A-I, Fields HL (2010) Reliability in the identification of midbrain dopamine neurons. PloS One 5:e15222. Margolis EB, Hjelmstad GO, Bonci A, Fields HL (2003) Kappa-opioid agonists directly inhibit midbrain dopaminergic neurons. J Neurosci 23:9981–9986. Margolis EB, Hjelmstad GO, Bonci A, Fields HL (2005) Both kappa and mu opioid agonists inhibit glutamatergic input to ventral tegmental area neurons. J Neurophysiol 93:3086–3093. Margolis EB, Hjelmstad GO, Fujita W, Fields HL (2014) Direct bidirectional μ-opioid control of midbrain dopamine neurons. J Neurosci 34:14707–14716. Margolis EB, Lock H, Chefer VI, Shippenberg TS, Hjelmstad GO, Fields HL (2006a) Kappa opioids selectively control dopaminergic neurons projecting to the prefrontal cortex. Proc Natl Acad Sci U S A 103:2938–2942. Margolis EB, Lock H, Hjelmstad GO, Fields HL (2006b) The ventral tegmental area revisited: is there an electrophysiological marker for dopaminergic neurons? J Physiol 577:907–924. Margolis EB, Mitchell JM, Ishikawa J, Hjelmstad GO, Fields HL (2008) Midbrain dopamine neurons: projection target determines action potential duration and dopamine D(2) receptor inhibition. J Neurosci 28:8908–8913. Margolis EB, Toy B, Himmels P, Morales M, Fields HL (2012) Identification of rat ventral tegmental area GABAergic neurons. PloS One 7:e42365. Marowsky A, Yanagawa Y, Obata K, Vogt KE (2005) A specialized subclass of interneurons mediates dopaminergic facilitation of amygdala function. Neuron 48:1025–1037. Matsui A, Jarvie BC, Robinson BG, Hentges ST, Williams JT (2014) Separate GABA afferents to dopamine neurons mediate acute action of opioids, development of tolerance, and expression of withdrawal. Neuron 82:1346–1356. Matsui A, Williams JT (2011) Opioid-sensitive GABA inputs from rostromedial tegmental nucleus synapse onto midbrain dopamine neurons. J Neurosci 31:17729–17735. McGregor A, Baker G, Roberts DC (1994) Effect of 6-hydroxydopamine lesions of the amygdala on intravenous cocaine self-administration under a progressive ratio schedule of reinforcement. Brain Res 646:273–278.  114 McGregor A, Roberts DC (1993) Dopaminergic antagonism within the nucleus accumbens or the amygdala produces differential effects on intravenous cocaine self-administration under fixed and progressive ratio schedules of reinforcement. Brain Res 624:245–252. McPherson CS, Featherby T, Krstew E, Lawrence AJ (2007) Quantification of phosphorylated cAMP-response element-binding protein expression throughout the brain of amphetamine-sensitized rats: activation of hypothalamic orexin A-containing neurons. J Pharmacol Exp Ther 323:805–812. Mead AN, Brown G, Le Merrer J, Stephens DN (2005) Effects of deletion of gria1 or gria2 genes encoding glutamatergic AMPA-receptor subunits on place preference conditioning in mice. Psychopharmacology (Berl) 179:164–171. Mello NK, Negus SS (1998) Effects of kappa opioid agonists on cocaine- and food-maintained responding by rhesus monkeys. J Pharmacol Exp Ther 286:812–824. Mendizábal V, Zimmer A, Maldonado R (2006) Involvement of kappa/dynorphin system in WIN 55,212-2 self-administration in mice. Neuropsychopharmacology 31:1957–1966. Meredith GE (1999) The synaptic framework for chemical signaling in nucleus accumbens. Ann N Y Acad Sci 877:140–156. Mileykovskiy BY, Kiyashchenko LI, Siegel JM (2005) Behavioral correlates of activity in identified hypocretin/orexin neurons. Neuron 46:787–798. Mirenowicz J, Schultz W (1994) Importance of unpredictability for reward responses in primate dopamine neurons. J Neurophysiol 72:1024–1027. Moorman DE, Aston-Jones G (2010) Orexin/hypocretin modulates response of ventral tegmental dopamine neurons to prefrontal activation: diurnal influences. J Neurosci 30:15585–15599. Mucha RF, Herz A (1985) Motivational properties of kappa and mu opioid receptor agonists studied with place and taste preference conditioning. Psychopharmacology (Berl) 86:274–280. Muschamp JW, Dominguez JM, Sato SM, Shen R-Y, Hull EM (2007) A role for hypocretin (orexin) in male sexual behavior. J Neurosci 27:2837–2845. Muschamp JW, Hollander JA, Thompson JL, Voren G, Hassinger LC, Onvani S, Kamenecka TM, Borgland SL, Kenny PJ, Carlezon WA (2014) Hypocretin (orexin) facilitates reward by attenuating the antireward effects of its cotransmitter dynorphin in ventral tegmental area. Proc Natl Acad Sci U S A 111:E1648–E1655. Nann-Vernotica E, Donny EC, Bigelow GE, Walsh SL (2001) Repeated administration of the D1/5 antagonist ecopipam fails to attenuate the subjective effects of cocaine. Psychopharmacology (Berl) 155:338–347.  115 Narita M, Nagumo Y, Hashimoto S, Narita M, Khotib J, Miyatake M, Sakurai T, Yanagisawa M, Nakamachi T, Shioda S, Suzuki T (2006) Direct involvement of orexinergic systems in the activation of the mesolimbic dopamine pathway and related behaviors induced by morphine. J Neurosci 26:398–405. Narita M, Nagumo Y, Miyatake M, Ikegami D, Kurahashi K, Suzuki T (2007) Implication of protein kinase C in the orexin-induced elevation of extracellular dopamine levels and its rewarding effect. Eur J Neurosci 25:1537–1545. Navratilova E, Xie JY, Okun A, Qu C, Eyde N, Ci S, Ossipov MH, King T, Fields HL, Porreca F (2012) Pain relief produces negative reinforcement through activation of mesolimbic reward-valuation circuitry. Proc Natl Acad Sci U S A 109:20709–20713. Negus SS, Mello NK, Portoghese PS, Lin CE (1997) Effects of kappa opioids on cocaine self-administration by rhesus monkeys. J Pharmacol Exp Ther 282:44–55. Nestby P, Schoffelmeer AN, Homberg JR, Wardeh G, De Vries TJ, Mulder AH, Vanderschuren LJ (1999) Bremazocine reduces unrestricted free-choice ethanol self-administration in rats without affecting sucrose preference. Psychopharmacology (Berl) 142:309–317. Nicola SM, Surmeier J, Malenka RC (2000) Dopaminergic modulation of neuronal excitability in the striatum and nucleus accumbens. Annu Rev Neurosci 23:185–215. Niehaus JL, Murali M, Kauer JA (2010) Drugs of abuse and stress impair LTP at inhibitory synapses in the ventral tegmental area. Eur J Neurosci 32:108–117. Nishino S, Mignot E (1997) Pharmacological aspects of human and canine narcolepsy. Prog Neurobiol 52:27–78. Nugent FS, Penick EC, Kauer JA (2007) Opioids block long-term potentiation of inhibitory synapses. Nature 446:1086–1090. O’Connor EC, Chapman K, Butler P, Mead AN (2011) The predictive validity of the rat self-administration model for abuse liability. Neurosci Biobehav Rev 35:912–938. Olds J, Milner P (1954) Positive reinforcement produced by electrical stimulation of septal area and other regions of rat brain. J Comp Physiol Psychol 47:419–427. Overton P, Clark D (1992) Iontophoretically administered drugs acting at the N-methyl-D-aspartate receptor modulate burst firing in A9 dopamine neurons in the rat. Synap N Y N 10:131–140. Paladini CA, Roeper J (2014) Generating bursts (and pauses) in the dopamine midbrain neurons. Neuroscience 282C:109–121. Paladini CA, Williams JT (2004) Noradrenergic inhibition of midbrain dopamine neurons. J Neurosci 24:4568–4575.  116 Palmiter RD (2008) Dopamine signaling in the dorsal striatum is essential for motivated behaviors: lessons from dopamine-deficient mice. Ann N Y Acad Sci 1129:35–46. Pascoli V, Terrier J, Hiver A, Lüscher C (2015) Sufficiency of Mesolimbic Dopamine Neuron Stimulation for the Progression to Addiction. Neuron 88:1054–1066. Perugini M, Vezina P (1994) Amphetamine administered to the ventral tegmental area sensitizes rats to the locomotor effects of nucleus accumbens amphetamine. J Pharmacol Exp Ther 270:690–696. Peyron C et al. (2000) A mutation in a case of early onset narcolepsy and a generalized absence of hypocretin peptides in human narcoleptic brains. Nat Med 6:991–997. Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Sutcliffe JG, Kilduff TS (1998) Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci 18:9996–10015. Pfeiffer A, Brantl V, Herz A, Emrich HM (1986) Psychotomimesis mediated by kappa opiate receptors. Science 233:774–776. Pierce RC, Born B, Adams M, Kalivas PW (1996) Repeated intra-ventral tegmental area administration of SKF-38393 induces behavioral and neurochemical sensitization to a subsequent cocaine challenge. J Pharmacol Exp Ther 278:384–392. Poulin J-F, Arbour D, Laforest S, Drolet G (2009) Neuroanatomical characterization of endogenous opioids in the bed nucleus of the stria terminalis. Prog Neuropsychopharmacol Biol Psychiatry 33:1356–1365. Poulin J-F, Zou J, Drouin-Ouellet J, Kim K-YA, Cicchetti F, Awatramani RB (2014) Defining midbrain dopaminergic neuron diversity by single-cell gene expression profiling. Cell Rep 9:930–943. Prince CD, Rau AR, Yorgason JT, España RA (2015) Hypocretin/Orexin regulation of dopamine signaling and cocaine self-administration is mediated predominantly by hypocretin receptor 1. ACS Chem Neurosci 6:138–146. Przewłocki R, Lasón W, Konecka AM, Gramsch C, Herz A, Reid LD (1983) The opioid peptide dynorphin, circadian rhythms, and starvation. Science 219:71–73. Quarta D, Valerio E, Hutcheson DM, Hedou G, Heidbreder C (2010) The orexin-1 receptor antagonist SB-334867 reduces amphetamine-evoked dopamine outflow in the shell of the nucleus accumbens and decreases the expression of amphetamine sensitization. Neurochem Int 56:11–15. Rao Y, Liu Z-W, Borok E, Rabenstein RL, Shanabrough M, Lu M, Picciotto MR, Horvath TL, Gao X-B (2007) Prolonged wakefulness induces experience-dependent  117 synaptic plasticity in mouse hypocretin/orexin neurons. J Clin Invest 117:4022–4033. Rao Y, Lu M, Ge F, Marsh DJ, Qian S, Wang AH, Picciotto MR, Gao X-B (2008) Regulation of synaptic efficacy in hypocretin/orexin-containing neurons by melanin concentrating hormone in the lateral hypothalamus. J Neurosci 28:9101–9110. Rao Y, Mineur YS, Gan G, Wang AH, Liu Z-W, Wu X, Suyama S, de Lecea L, Horvath TL, Picciotto MR, Gao X-B (2013) Repeated in vivo exposure of cocaine induces long-lasting synaptic plasticity in hypocretin/orexin-producing neurons in the lateral hypothalamus in mice. J Physiol 591:1951–1966. Redila VA, Chavkin C (2008) Stress-induced reinstatement of cocaine seeking is mediated by the kappa opioid system. Psychopharmacology (Berl) 200:59–70. Richardson KA, Aston-Jones G (2012) Lateral hypothalamic orexin/hypocretin neurons that project to ventral tegmental area are differentially activated with morphine preference. J Neurosci 32:3809–3817. Robinson JD, McDonald PH (2015) The orexin 1 receptor modulates kappa opioid receptor function via a JNK-dependent mechanism. Cell Signal 27:1449–1456. Robinson TE, Berridge KC (1993) The neural basis of drug craving: an incentive-sensitization theory of addiction. Brain Res Brain Res Rev 18:247–291. Rosin DL, Weston MC, Sevigny CP, Stornetta RL, Guyenet PG (2003) Hypothalamic orexin (hypocretin) neurons express vesicular glutamate transporters VGLUT1 or VGLUT2. J Comp Neurol 465:593–603. Saal D, Dong Y, Bonci A, Malenka RC (2003) Drugs of abuse and stress trigger a common synaptic adaptation in dopamine neurons. Neuron 37:577–582. Sakurai T et al. (1998) Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92:573–585. Salamone JD, Correa M (2002) Motivational views of reinforcement: implications for understanding the behavioral functions of nucleus accumbens dopamine. Behav Brain Res 137:3–25. Salamone JD, Wisniecki A, Carlson BB, Correa M (2001) Nucleus accumbens dopamine depletions make animals highly sensitive to high fixed ratio requirements but do not impair primary food reinforcement. Neuroscience 105:863–870. Sarti F, Zhang Z, Schroeder J, Chen L (2013) Rapid suppression of inhibitory synaptic transmission by retinoic acid. J Neurosci 33:11440–11450.  118 Sartor GC, Aston-Jones GS (2012) A septal-hypothalamic pathway drives orexin neurons, which is necessary for conditioned cocaine preference. J Neurosci 32:4623–4631. Schenk S, Partridge B, Shippenberg TS (1999) U69593, a kappa-opioid agonist, decreases cocaine self-administration and decreases cocaine-produced drug-seeking. Psychopharmacology (Berl) 144:339–346. Schenk S, Partridge B, Shippenberg TS (2001) Effects of the kappa-opioid receptor agonist, U69593, on the development of sensitization and on the maintenance of cocaine self-administration. Neuropsychopharmacology 24:441–450. Schenk S, Snow S (1994) Sensitization to cocaine’s motor activating properties produced by electrical kindling of the medial prefrontal cortex but not of the hippocampus. Brain Res 659:17–22. Schilström B, Yaka R, Argilli E, Suvarna N, Schumann J, Chen BT, Carman M, Singh V, Mailliard WS, Ron D, Bonci A (2006) Cocaine enhances NMDA receptor-mediated currents in ventral tegmental area cells via dopamine D5 receptor-dependent redistribution of NMDA receptors. J Neurosci 26:8549–8558. Schöne C, Apergis-Schoute J, Sakurai T, Adamantidis A, Burdakov D (2014) Coreleased orexin and glutamate evoke nonredundant spike outputs and computations in histamine neurons. Cell Rep 7:697–704. Schöne C, Cao ZFH, Apergis-Schoute J, Adamantidis A, Sakurai T, Burdakov D (2012) Optogenetic probing of fast glutamatergic transmission from hypocretin/orexin to histamine neurons in situ. J Neurosci 32:12437–12443. Schultz W, Dayan P, Montague PR (1997) A neural substrate of prediction and reward. Science 275:1593–1599. Seamans JK, Floresco SB, Phillips AG (1998) D1 receptor modulation of hippocampal-prefrontal cortical circuits integrating spatial memory with executive functions in the rat. J Neurosci 18:1613–1621. Sears RM, Fink AE, Wigestrand MB, Farb CR, de Lecea L, LeDoux JE (2013) Orexin/hypocretin system modulates amygdala-dependent threat learning through the locus coeruleus. Proc Natl Acad Sci U S A 110:20260–20265. Sesack SR, Grace AA (2010) Cortico-Basal Ganglia reward network: microcircuitry. Neuropsychopharmacology 35:27–47. Shippenberg TS, Herz A (1986) Differential effects of mu and kappa opioid systems on motivational processes. NIDA Res Monogr 75:563–566. Shippenberg TS, Zapata A, Chefer VI (2007) Dynorphin and the pathophysiology of drug addiction. Pharmacol Ther 116:306–321.  119 Shoji S, Simms D, McDaniel WC, Gallagher JP (1997) Chronic cocaine enhances gamma-aminobutyric acid and glutamate release by altering presynaptic and not postsynaptic gamma-aminobutyric acidB receptors within the rat dorsolateral septal nucleus. J Pharmacol Exp Ther 280:129–137. Shoji S, Simms D, Yamada K, Gallagher JP (1998) Cocaine administered in vitro to brain slices from rats treated with cocaine chronically in vivo results in a gamma-aminobutyric acid receptor-mediated hyperpolarization recorded from the dorsolateral septum. J Pharmacol Exp Ther 286:509–518. Smidt MP, van Schaick HS, Lanctôt C, Tremblay JJ, Cox JJ, van der Kleij AA, Wolterink G, Drouin J, Burbach JP (1997) A homeodomain gene Ptx3 has highly restricted brain expression in mesencephalic dopaminergic neurons. Proc Natl Acad Sci U S A 94:13305–13310. Smith BN, Davis SF, Van Den Pol AN, Xu W (2002) Selective enhancement of excitatory synaptic activity in the rat nucleus tractus solitarius by hypocretin 2. Neuroscience 115:707–714. Smith RJ, Aston-Jones G (2012) Orexin  /  hypocretin 1 receptor antagonist reduces heroin self-administration and cue-induced heroin seeking. Eur J Neurosci 35:798–804. Smith RJ, See RE, Aston-Jones G (2009) Orexin/hypocretin signaling at the orexin 1 receptor regulates cue-elicited cocaine-seeking. Eur J Neurosci 30:493–503. Soya S, Shoji H, Hasegawa E, Hondo M, Miyakawa T, Yanagisawa M, Mieda M, Sakurai T (2013) Orexin receptor-1 in the locus coeruleus plays an important role in cue-dependent fear memory consolidation. J Neurosci 33:14549–14557. Spanagel R, Herz A, Shippenberg TS (1990) The effects of opioid peptides on dopamine release in the nucleus accumbens: an in vivo microdialysis study. J Neurochem 55:1734–1740. Spanagel R, Herz A, Shippenberg TS (1992) Opposing tonically active endogenous opioid systems modulate the mesolimbic dopaminergic pathway. Proc Natl Acad Sci U S A 89:2046–2050. Speciale SG, Manaye KF, Sadeq M, German DC (1993) Opioid receptors in midbrain dopaminergic regions of the rat. II. Kappa and delta receptor autoradiography. J Neural Transm Gen Sect 91:53–66. Steffensen SC, Svingos AL, Pickel VM, Henriksen SJ (1998) Electrophysiological characterization of GABAergic neurons in the ventral tegmental area. J Neurosci 18:8003–8015. Steinberg EE, Keiflin R, Boivin JR, Witten IB, Deisseroth K, Janak PH (2013) A causal link between prediction errors, dopamine neurons and learning. Nat Neurosci 16:966–973.  120 Stuber GD, Klanker M, de Ridder B, Bowers MS, Joosten RN, Feenstra MG, Bonci A (2008) Reward-predictive cues enhance excitatory synaptic strength onto midbrain dopamine neurons. Science 321:1690–1692. Stuber GD, Roitman MF, Phillips PEM, Carelli RM, Wightman RM (2005) Rapid dopamine signaling in the nucleus accumbens during contingent and noncontingent cocaine administration. Neuropsychopharmacology 30:853–863. Suaud-Chagny MF, Chergui K, Chouvet G, Gonon F (1992) Relationship between dopamine release in the rat nucleus accumbens and the discharge activity of dopaminergic neurons during local in vivo application of amino acids in the ventral tegmental area. Neuroscience 49:63–72. Südhof TC (2012a) The presynaptic active zone. Neuron 75:11–25. Südhof TC (2012b) Calcium control of neurotransmitter release. Cold Spring Harb Perspect Biol 4:a011353. Svingos AL, Chavkin C, Colago EE, Pickel VM (2001) Major coexpression of kappa-opioid receptors and the dopamine transporter in nucleus accumbens axonal profiles. Synap N Y N 42:185–192. Swanson LW (1982) The projections of the ventral tegmental area and adjacent regions: a combined fluorescent retrograde tracer and immunofluorescence study in the rat. Brain Res Bull 9:321–353. Syed ECJ, Grima LL, Magill PJ, Bogacz R, Brown P, Walton ME (2016) Action initiation shapes mesolimbic dopamine encoding of future rewards. Nat Neurosci 19:34–36. Tallent M (2008) Presynaptic inhibition of glutamate release by neuropeptides: use- dependent synaptic modification. Results and Problems in Cell Differentiation  44:177-200.  Tan KR, Yvon C, Turiault M, Mirzabekov JJ, Doehner J, Labouèbe G, Deisseroth K, Tye KM, Lüscher C (2012) GABA neurons of the VTA drive conditioned place aversion. Neuron 73:1173–1183. Tateno T, Robinson HPC (2011) The mechanism of ethanol action on midbrain dopaminergic neuron firing: a dynamic-clamp study of the role of I(h) and GABAergic synaptic integration. J Neurophysiol 106:1901–1922. Thannickal TC, Moore RY, Nienhuis R, Ramanathan L, Gulyani S, Aldrich M, Cornford M, Siegel JM (2000) Reduced number of hypocretin neurons in human narcolepsy. Neuron 27:469–474. Thomas MJ, Beurrier C, Bonci A, Malenka RC (2001) Long-term depression in the nucleus accumbens: a neural correlate of behavioral sensitization to cocaine. Nat Neurosci 4:1217–1223.  121 Thompson AC, Zapata A, Justice JB, Vaughan RA, Sharpe LG, Shippenberg TS (2000) Kappa-opioid receptor activation modifies dopamine uptake in the nucleus accumbens and opposes the effects of cocaine. J Neurosci 20:9333–9340. Tidey JW, Miczek KA (1996) Social defeat stress selectively alters mesocorticolimbic dopamine release: an in vivo microdialysis study. Brain Res 721:140–149. Tong ZY, Overton PG, Clark D (1996) Antagonism of NMDA receptors but not AMPA/kainate receptors blocks bursting in dopaminergic neurons induced by electrical stimulation of the prefrontal cortex. J Neural Transm Vienna Austria 1996 103:889–904. Trivedi P, Yu H, MacNeil DJ, Van der Ploeg LH, Guan XM (1998) Distribution of orexin receptor mRNA in the rat brain. FEBS Lett 438:71–75. Ungless MA, Grace AA (2012) Are you or aren’t you? Challenges associated with physiologically identifying dopamine neurons. Trends Neurosci 35:422–430. Ungless MA, Whistler JL, Malenka RC, Bonci A (2001) Single cocaine exposure in vivo induces long-term potentiation in dopamine neurons. Nature 411:583–587. Uramura K, Funahashi H, Muroya S, Shioda S, Takigawa M, Yada T (2001) Orexin-a activates phospholipase C- and protein kinase C-mediated Ca2+ signaling in dopamine neurons of the ventral tegmental area. Neuroreport 12:1885–1889. Valenstein ES, Cox VC, Kakolewski JW (1970) Reexamination of the role of the hypothalamus in motivation. Psychol Rev 77:16–31. van den Pol AN (2012) Neuropeptide transmission in brain circuits. Neuron 76:98–115. van den Pol AN, Gao XB, Obrietan K, Kilduff TS, Belousov AB (1998) Presynaptic and postsynaptic actions and modulation of neuroendocrine neurons by a new hypothalamic peptide, hypocretin/orexin. J Neurosci 18:7962–7971. Vanderschuren LJ, Kalivas PW (2000) Alterations in dopaminergic and glutamatergic transmission in the induction and expression of behavioral sensitization: a critical review of preclinical studies. Psychopharmacology (Berl) 151:99–120. van Zessen R, Phillips JL, Budygin EA, Stuber GD (2012) Activation of VTA GABA neurons disrupts reward consumption. Neuron 73:1184–1194. Venton BJ, Zhang H, Garris PA, Phillips PEM, Sulzer D, Wightman RM (2003) Real-time decoding of dopamine concentration changes in the caudate-putamen during tonic and phasic firing. J Neurochem 87:1284–1295. Venugopalan VV, Casey KF, O’Hara C, O’Loughlin J, Benkelfat C, Fellows LK, Leyton M (2011) Acute phenylalanine/tyrosine depletion reduces motivation to smoke cigarettes across stages of addiction. Neuropsychopharmacology 36:2469–2476.  122 Vezina P (1993) Amphetamine injected into the ventral tegmental area sensitizes the nucleus accumbens dopaminergic response to systemic amphetamine: an in vivo microdialysis study in the rat. Brain Res 605:332–337. Vezina P, Queen AL (2000) Induction of locomotor sensitization by amphetamine requires the activation of NMDA receptors in the rat ventral tegmental area. Psychopharmacology (Berl) 151:184–191. Vezina P, Stewart J (1989) The effect of dopamine receptor blockade on the development of sensitization to the locomotor activating effects of amphetamine and morphine. Brain Res 499:108–120. Vittoz NM, Berridge CW (2006) Hypocretin/orexin selectively increases dopamine efflux within the prefrontal cortex: involvement of the ventral tegmental area. Neuropsychopharmacology 31:384–395. Vittoz NM, Schmeichel B, Berridge CW (2008) Hypocretin /orexin preferentially activates caudomedial ventral tegmental area dopamine neurons. Eur J Neurosci 28:1629–1640. Wachtel SR, Ortengren A, de Wit H (2002) The effects of acute haloperidol or risperidone on subjective responses to methamphetamine in healthy volunteers. Drug Alcohol Depend 68:23–33. Wanat MJ, Hopf FW, Stuber GD, Phillips PE, Bonci A (2008) Corticotropin-releasing  factor increases mouse ventral tegmental area dopamine neuron firing through a  protein kinase C-dependent enhancement of Ih. J Physiol 586:2157-2170.  Wang B, You Z-B, Wise RA (2009) Reinstatement of cocaine seeking by hypocretin (orexin) in the ventral tegmental area: independence from the local corticotropin-releasing factor network. Biol Psychiatry 65:857–862. Wang H, Treadway T, Covey DP, Cheer JF, Lupica CR (2015) Cocaine-Induced Endocannabinoid Mobilization in the Ventral Tegmental Area. Cell Rep 12:1997–2008. Wassum KM, Izquierdo A (2015) The basolateral amygdala in reward learning and addiction. Neurosci Biobehav Rev 57:271–283. White FJ, Hu XT, Zhang XF, Wolf ME (1995) Repeated administration of cocaine or amphetamine alters neuronal responses to glutamate in the mesoaccumbens dopamine system. J Pharmacol Exp Ther 273:445–454. Willie JT, Chemelli RM, Sinton CM, Tokita S, Williams SC, Kisanuki YY, Marcus JN, Lee C, Elmquist JK, Kohlmeier KA, Leonard CS, Richardson JA, Hammer RE, Yanagisawa M (2003) Distinct narcolepsy syndromes in Orexin receptor-2 and Orexin null mice: molecular genetic dissection of Non-REM and REM sleep regulatory processes. Neuron 38:715–730.  123 Willie JT, Chemelli RM, Sinton CM, Yanagisawa M (2001) To eat or to sleep? Orexin in the regulation of feeding and wakefulness. Annu Rev Neurosci 24:429–458. Wise RA (1978) Catecholamine theories of reward: a critical review. Brain Res 152:215–247. Wolf ME (1998) The role of excitatory amino acids in behavioral sensitization to psychomotor stimulants. Prog Neurobiol 54:679–720. Wolf ME, Jeziorski M (1993) Coadministration of MK-801 with amphetamine, cocaine or morphine prevents rather than transiently masks the development of behavioral sensitization. Brain Res 613:291–294. Wolf ME, Sun X, Mangiavacchi S, Chao SZ (2004) Psychomotor stimulants and neuronal plasticity. Neuropharmacology 47 Suppl 1:61–79. Xie JY, Qu C, Patwardhan A, Ossipov MH, Navratilova E, Becerra L, Borsook D, Porreca F (2014) Activation of mesocorticolimbic reward circuits for assessment of relief of ongoing pain: a potential biomarker of efficacy. Pain 155:1659–1666. Xie X, Crowder TL, Yamanaka A, Morairty SR, Lewinter RD, Sakurai T, Kilduff TS (2006) GABA(B) receptor-mediated modulation of hypocretin/orexin neurones in mouse hypothalamus. J Physiol 574:399–414. Yamaguchi T, Sheen W, Morales M (2007) Glutamatergic neurons are present in the rat ventral tegmental area. Eur J Neurosci 25:106–118. Yang B, Samson WK, Ferguson AV (2003) Excitatory effects of orexin-A on nucleus tractus solitarius neurons are mediated by phospholipase C and protein kinase C. J Neurosci 23:6215–6222. Yeoh JW, James MH, Jobling P, Bains JS, Graham BA, Dayas CV (2012) Cocaine potentiates excitatory drive in the perifornical/lateral hypothalamus. J Physiol 590:3677–3689. Yoshida K, McCormack S, España RA, Crocker A, Scammell TE (2006) Afferents to the orexin neurons of the rat brain. J Comp Neurol 494:845–861. Yuan T, Mameli M, O’ Connor EC, Dey PN, Verpelli C, Sala C, Perez-Otano I, Lüscher C, Bellone C (2013) Expression of cocaine-evoked synaptic plasticity by GluN3A-containing NMDA receptors. Neuron 80:1025–1038. Zhang S, Tong Y, Tian M, Dehaven RN, Cortesburgos L, Mansson E, Simonin F, Kieffer B, Yu L (1998) Dynorphin A as a potential endogenous ligand for four members of the opioid receptor gene family. J Pharmacol Exp Ther 286:136–141. Zhang TA, Placzek AN, Dani JA (2010) In vitro identification and electrophysiological characterization of dopamine neurons in the ventral tegmental area. Neuropharmacology 59:431–436.  124 Zhang XF, Hu XT, White FJ, Wolf ME (1997) Increased responsiveness of ventral tegmental area dopamine neurons to glutamate after repeated administration of cocaine or amphetamine is transient and selectively involves AMPA receptors. J Pharmacol Exp Ther 281:699–706. Zhang Y, Butelman ER, Schlussman SD, Ho A, Kreek MJ (2004) Effect of the endogenous kappa opioid agonist dynorphin A(1-17) on cocaine-evoked increases in striatal dopamine levels and cocaine-induced place preference in C57BL/6J mice. Psychopharmacology (Berl) 172:422–429. Zhao S, Maxwell S, Jimenez-Beristain A, Vives J, Kuehner E, Zhao J, O’Brien C, de Felipe C, Semina E, Li M (2004) Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons. Eur J Neurosci 19:1133–1140. Zheng F, Johnson SW (2001) Glycine receptor-mediated inhibition of dopamine and non-dopamine neurons of the rat ventral tegmental area in vitro. Brain Res 919:313–317. Zweifel LS, Fadok JP, Argilli E, Garelick MG, Jones GL, Dickerson TMK, Allen JM, Mizumori SJY, Bonci A, Palmiter RD (2011) Activation of dopamine neurons is critical for aversive conditioning and prevention of generalized anxiety. Nat Neurosci 14:620–626. Zweifel LS, Parker JG, Lobb CJ, Rainwater A, Wall VZ, Fadok JP, Darvas M, Kim MJ, Mizumori SJY, Paladini CA, Phillips PEM, Palmiter RD (2009) Disruption of NMDAR-dependent burst firing by dopamine neurons provides selective assessment of phasic dopamine-dependent behavior. Proc Natl Acad Sci U S A 106:7281–7288.      

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.24.1-0303473/manifest

Comment

Related Items