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Regulation of synaptic plasticity in the mesocortical dopamine system Thompson, Jennifer Louise 2014

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Regulation of synaptic plasticity in the mesocortical dopamine system   by   Jennifer Louise Thompson   B.A., The University of Illinois at Chicago, 2006   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)      THE UNIVERSITY OF BRITISH COLUMBIA   (Vancouver)      October 2014       © Jennifer Louise Thompson, 2014  ii Abstract The mesocortical dopamine system is crucial for regulating goal-directed behavior.   Changes in synaptic transmission in brain regions included in this system underlie modifications in cognitive flexibility, and the learning of salient contextual cues to efficiently navigate one’s environment.  Chapter 1 of this thesis describes plasticity of dopamine neurons in the ventral tegmental area and how it contributes to reinforcement learning. Synaptic plasticity in several brain regions is modified by peptides associated with feeding and energy homeostasis. Leptin, an adipocyte derived cytokine, is able to modulate food and drug-seeking behaviors.   In addition, dopamine signaling in the orbitofrontal cortex and its role in decision-making is also reviewed. Chapter 2 examines how leptin modifies synaptic transmission at excitatory synapses in the ventral tegmental area (VTA).  Leptin caused a depression of evoked EPSCs mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and n-methyl-D-aspartate receptors (NMDARs).  This effect was presynaptic in nature and required signaling through phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K).  Dopamine signaling in the orbitofrontal cortex (OFC) is important for adaptive decision-making in the face of salient events.  In Chapter 3, we demonstrate how dopamine acts in the OFC to modify excitatory synaptic transmission mediated by NMDARs. In adult and juvenile rats, D1 receptor activation potentiated, while D2 receptor activation reduced evoked EPSCs in lateral OFC subregions.  Additionally, we showed that dopamine has differential effects in juvenile versus adult rats, because a PLC-coupled dopamine receptor pathway potentiates EPSCs in juvenile rats but is not present in adult animals.  Administration of SKF83959, a ligand for PLC-coupled dopamine receptors, potentiated NMDAR-mediated responses and facilitated performance on a task measuring cognitive flexibility in juvenile rats only.  Chapter 4 discusses the results from the current experiments,  iii and integrates possible ways that the actions of leptin in the VTA could influence dopamine signaling in the OFC. Future experiments to further investigate the precise mechanisms underlying the changes in synaptic transmission of the mesocortical dopamine system are also considered.      iv Preface Chapters 2 and 3 are based on research I performed in the Laboratory for the Study of Appetitive Motivation by Stephanie L. Borgland and Jennifer L. Thompson  • A version of Chapter 2 has been published:   Thompson JL & Borgland SL (2013). Presynaptic leptin action suppresses excitatory  synaptic transmission in the ventral tegmental area. Biol Psychiatry 73:860-8.   J.L.T worked in collaboration with S.L.B to design the experiments.  J.L.T performed  the experiments, and collected and analyzed the data.  J.L.T wrote the manuscript  with S.L.B  • A version of Chapter 3 is in preparation for publication   Thompson JL, Yang J, Kerr L & Borgland SL. Differential effects of dopamine on  excitatory synaptic transmission in the orbitofrontal cortex: the role of an age- dependent PLC-coupled pathway. In preparation.   J.L.T worked in collaboration with S.L.B to design the experiments.  J.L.T and J.Y.  performed the patch- clamp experiments and analyzed this data.  J.L.T and L.K.  performed the behavioral experiments, and J.L.T analyzed this data.  J.L.T wrote the  manuscript with S.L.B.  This research was approved by the University of British Columbia and University of Calgary animal care committees:    University of British Columbia • Certificate: A11-0089     Project title: Understanding impulse control disorders in Parkinson's Disease,   Parkinson's Society of Canada    Project title: Modulation of synaptic transmission in orbitofrontal cortex  neurons: a  possible relationship to cognitive flexibility, Mind Foundation of  BC  • Certificate: A08-0042    Project title: Role of feeding related peptides on brain reward pathways, NSERC  University of Calgary  • Certificate: AC13-0106   Project title: Role of feeding related peptides on brain reward pathways, NSERC   v Table of contents  Abstract ................................................................................................................................... ii	  Preface ..................................................................................................................................... iv	  Table of contents ...................................................................................................................... v	  List of figures ........................................................................................................................... x	  List of abbreviations ............................................................................................................. xii	  Acknowledgements ................................................................................................................ xv	  Dedication .............................................................................................................................. xvi	  Chapter 1: Introduction .......................................................................................................... 1	  1.1	  The	  role	  of	  dopamine	  in	  goal-­directed	  behavior	  ....................................................................	  1	  1.2	  Control	  of	  dopamine	  neuronal	  firing	  ..........................................................................................	  4	  1.3	  Excitatory	  synaptic	  transmission	  and	  changes	  in	  reward	  learning	  .................................	  5	  1.3.1 Long-term potentiation and long-term depression ................................................... 5	  1.3.2 Modulation of synaptic transmission by feeding peptides ....................................... 6	  1.4	  Leptin	  signaling	  in	  the	  brain	  .........................................................................................................	  7	  1.4.1 Leptin as an adiposity signal .................................................................................... 7	  1.4.2 Disruption of leptin signaling leads to hyperphagia ................................................. 8	  1.4.3 Leptin receptor signaling .......................................................................................... 9	  1.4.4 Leptin signaling in the hypothalamus ..................................................................... 10	  1.4.5 Effects of leptin on the mesolimbic system ............................................................ 11	       1.4.5.1. Leptin and reinforcing behaviors ................................................................... 11	       1.4.5.2. Effects of leptin on VTA neuron excitability ................................................ 13	   vi      1.4.5.3. Effect of leptin on dopamine release ............................................................. 13	       1.4.5.4. Effects of leptin on excitatory synaptic transmission .................................... 14	  1.5	  Role	  of	  the	  prefrontal	  cortex	  in	  executive	  function	  ............................................................	  16	  1.6	  The	  role	  of	  the	  orbitofrontal	  cortex	  in	  goal-­directed	  behavior	  ......................................	  17	  1.7	  The	  significance	  of	  dopamine	  in	  the	  mPFC/DLPFC	  .............................................................	  20	  1.8	  Dopamine	  receptor	  signaling	  pathways	  .................................................................................	  21	  1.9	  Dopamine	  receptor	  expression	  in	  the	  adult	  PFC	  .................................................................	  22	  1.9.1 Receptor expression in pyramidal neurons ............................................................. 23	  1.9.2 Receptor expression in GABA neurons .................................................................. 24	  1.9.3 Expression in presynaptic terminals ....................................................................... 25	  1.10	  Differential	  expression	  in	  prefrontal	  dopamine	  across	  development	  ......................	  26	  1.10.1 Dopamine terminal innervation in PFC ................................................................ 27	  1.11	  Dopamine	  signaling	  in	  the	  OFC	  ...............................................................................................	  28	  1.11.1 Dopamine release in the OFC during behavioral tasks ........................................ 28	  1.11.2 Behavioral effects of dopamine receptor signaling in the OFC ........................... 29	       1.11.2.1. Impulsive responding .................................................................................. 29	       1.11.2.2. Reversal-learning ......................................................................................... 30	       1.11.2.3. Progressive ratio responding ....................................................................... 31	  1.11.3 Selectivity of dopamine receptor ligands ............................................................. 31	  1.12	  Dopamine	  release	  in	  the	  prefrontal	  cortex	  .........................................................................	  32	  1.13	  The	  role	  of	  NMDA	  receptors	  in	  the	  PFC	  ................................................................................	  34	  1.13.1 Structure and function of NMDARs ..................................................................... 34	  1.13.2 Modulation of PFC NMDARs during behavior ................................................... 35	  1.13.3 Effect of dopamine on NMDARs in the PFC ....................................................... 36	   vii 1.14	  Overview	  and	  objectives	  ...........................................................................................................	  38	  Chapter 2: Presynaptic leptin action suppresses excitatory synaptic transmission onto ventral tegmental area dopamine neurons .......................................................................... 40	  2.1	  Introduction	  .....................................................................................................................................	  40	  2.2	  Methods	  .............................................................................................................................................	  42	  2.2.1 Animals ................................................................................................................... 42	  2.2.2 Slice preparation ..................................................................................................... 42	  2.2.3 Electrophysiology ................................................................................................... 43	  2.2.4 Drugs ...................................................................................................................... 45	  2.2.5 Statistical analysis ................................................................................................... 45	  2.2.6 Tyrosine hydroxylase immunocytochemistry ........................................................ 46	  2.3	  Results	  ................................................................................................................................................	  46	  2.3.1 Leptin suppresses excitatory synaptic transmission onto VTA dopamine neurons46	  2.3.2 Leptin-induced synaptic depression occurs by activation of the JAK2 - PI3K pathway ............................................................................................................................ 48	  2.4	  Discussion	  .........................................................................................................................................	  50	  2.4.1 Mechanisms behind reductions in food intake ....................................................... 50	  2.4.2 Role of leptin in reward-seeking ............................................................................ 51	  2.4.3 Modulation of synaptic transmission ...................................................................... 53	  2.4.4 LepRb expression in VTA ...................................................................................... 54	  2.4.5 Diversity of signaling pathways mediating changes in VTA neurons ................... 55	  2.4.6 Upregulation of excitatory amino acid transporters ............................................... 56	  2.4.7 Summary and conclusions ...................................................................................... 57	   viii Chapter 3: Activation of PLC-coupled dopamine receptors in the lateral orbitofrontal cortex alleviates cognitive impairment and potentiates excitatory synaptic transmission in juvenile rats ........................................................................................................................ 59	  3.1	  Introduction	  .....................................................................................................................................	  59	  3.2	  Methods	  .............................................................................................................................................	  61	  3.2.1 Subjects ................................................................................................................... 61	  3.2.2 Slice preparation ..................................................................................................... 61	  3.2.3 Electrophysiology ................................................................................................... 62	  3.2.4 Immunohistochemistry ........................................................................................... 62	  3.2.5 Intracranial cannulations ......................................................................................... 63	  3.2.6 Behavioral training ................................................................................................. 63	  3.2.7 Drugs ...................................................................................................................... 66	  3.2.8 Statistical analysis ................................................................................................... 66	  3.3	  Results	  ................................................................................................................................................	  67	  3.3.1 Subregion-specific potentiation of NMDAR EPSCs .............................................. 67	  3.3.2 D2 receptor-mediated suppression of EPSCs ......................................................... 69	  3.3.3 Age-specific effects of D1:D2R co-activation ....................................................... 71	  3.3.4 Age-specific effects of a PLC-coupled dopamine receptor agonist ....................... 73	  3.3.5 Bidirectional effects of dopamine in young vs adult OFC ..................................... 74	  3.3.6 Amelioration of reversal learning deficits in juvenile rats ..................................... 76	  3.4	  Discussion	  .........................................................................................................................................	  77	  3.4.1 Regional differences in D1R-mediated effects on NMDARs ................................ 78	  3.4.2 Mechanisms behind D1:D2 cooperativity in juvenile OFC ................................... 79	  3.4.3 Age-related discrepancies in response to dopamine ............................................... 81	   ix 3.4.4 Summary and conclusions ...................................................................................... 81	  Chapter 4: General discussion .............................................................................................. 84	  4.1	  Net	  effects	  of	  leptin	  on	  firing	  activity	  of	  VTA	  dopamine	  neurons	  ..................................	  84	  4.2	  Leptin	  concentrations	  in	  vivo	  versus	  in	  vitro	  .......................................................................	  85	  4.3	  Potential	  orbitofrontal	  contributions	  to	  leptin	  signaling	  ................................................	  87	  4.4	  Activation	  of	  PLC-­coupled	  dopamine	  receptors	  and	  OFC	  dependent	  behavior	  ........	  88	  4.5	  Experimental	  limitations	  .............................................................................................................	  89	  4.5.1 Parsing behavioral changes induced by leptin in VTA .......................................... 90	  4.5.2 Actions of dopamine receptor agonists on non-pyramidal cortical neurons .......... 90	  4.6	  Future	  directions	  ............................................................................................................................	  91	  4.6.1 Targeted deletion of leptin receptors in the VTA ................................................... 91	  4.6.2 Region-specific effects of D1:D2 co-activation in the OFC .................................. 92	  4.6.3 Investigation of physical interactions between  DAR subtypes ............................. 93	  4.7	  Conclusions	  ......................................................................................................................................	  93	  Figures  ................................................................................................................................... 95	  References ............................................................................................................................. 114	     x List of figures Figure 1. Leptin depresses AMPAR- and NMDAR-mediated synaptic transmission onto   VTA dopamine neurons.......................................................................................................... 95  Figure 2. Leptin-induced synaptic inhibition is mediated presynaptically............................ 96 Figure 3. Leptin-induced synaptic inhibition requires activation of JAK2 signaling............ 98 Figure 4. Leptin-induced synaptic inhibition requires PI3K activation................................. 99 Figure 5. Putative mechanisms of leptin-induced synaptic depression onto VTA dopamine neurons.................................................................................................................................. 101 Figure 6. SKF38393, a D1R agonist, potentiates NMDAR EPSCs in pyramidal neurons of the lateral OFC of juvenile rats............................................................................................. 102 Figure 7.  SKF38393 potentiates NMDAR EPSCs in lateral OFC of juvenile rats via D1Rs coupled to PKA signaling..................................................................................................... 104 Figure 8. SKF38393 potentiates NMDAR EPSCs of pyramidal neurons selectively in lateral OFC of adult rats................................................................................................................... 105 Figure 9. Quinpirole, a D2R agonist, inhibits NMDAR EPSCs of lateral OFC pyramidal neurons from adult or juvenile rats....................................................................................... 107 Figure 10. SKF38393 and quinpirole have no effect on AMPAR-mediated EPSCs in juvenile lateral OFC pyramidal neurons............................................................................................. 108 Figure 11. Co-application of SKF38393 and quinpirole potentiates NMDAR EPSCs in juvenile but not adult lateral OFC pyramidal neurons.......................................................... 109 Figure 12. SKF83959, a D1:D2 heterodimer agonist, potentiated NMDAR EPSCs of juvenile but not adult lateral OFC pyramidal neurons.......................................................... 110 Figure 13. Dopamine has differential effects on NMDAR EPSCs of juvenile or adult lateral OFC pyramidal neurons........................................................................................................ 111  xi Figure 14. Intra-lateral OFC SKF83959 improves juvenile performance on a reversal learning task.......................................................................................................................... 112 Figure 15.  Cannula placements within juvenile and adult lateral OFC.............................. 113                       xii List of abbreviations  5HT    serotonin aCSF artificial cerebrospinal fluid ADHD    attention deficit/hyperactivity disorder AGRP    agouti-related peptide AKT protein kinase B AMPAR    α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid CaMKII    calcium and calmodulin-dependent protein kinase II CNS    central nervous system D1R    dopamine D1-like receptor D2R    dopamine D2-like receptor  DA    dopamine DAT dopamine transporter db/db  mice lacking gene encoding leptin receptor DLPFC   dorsolateral prefrontal cortex EAATs    excitatory amino acid transporters EPSC excitatory postsynaptic current ERK extracellular signal-regulated kinases fa/fa Zucker fatty rat fMRI    functional magnetic resonance imaging FRET    fluorescence resonance energy transfer GABA    γ-aminobutyric acid  GFP    green fluorescent protein  xiii GSK3β    glycogen synthase kinase 3 beta HCN    hyperpolarization-activated cyclic nucleotide–gated cation channels  JAK2    janus kinase 2 LEPR    leptin receptor gene LEPRb    leptin receptor LH    lateral hypothalamus LOFC    lateral orbitofrontal cortex LTD    long-term depression LTP    long-term potentiation MAPK    mitogen-activated protein kinase mEPSCs    miniature excitatory postsynaptic currents MOFC    medial orbitofrontal cortex mPFC    medial prefrontal cortex NAc    nucleus accumbens NMDAR    n-methyl-D-aspartate receptors  NPY neuropeptide Y Ob    gene encoding leptin ob/ob    mice lacking gene encoding leptin OFC    orbitofrontal cortex P (e.g. P60)    postnatal day PAG    periaqueductal grey PFC    prefrontal cortex PI3K   phosphatidylinositol-4,5-bisphosphate 3-kinase Pitx3    pituitary homeobox 3  xiv PKA protein kinase A PLC phospholipase C POA preoptic area pSTAT3    phosphorylated STAT3 SHP-2    SH2-containing tyrosine phosphatase shRNA    short-hairpin RNA SNpc    substantia nigra pars compacta STAT3    signal transducer and activator of transcription 3 STAT5    signal transducer and activator of transcription 5 TH    tyrosine hydroxylase VOFC    ventral orbitofrontal cortex VTA    ventral tegmental area   xv  Acknowledgements I would first like to thank my supervisor Dr. Stephanie Borgland, for her unconditional support and guidance throughout my PhD. I consider myself lucky for the valuable mentorship from such a respected female scientist.  In addition, I would like to thank the members and chairpersons of my present and past supervisory committees, Drs. Bernard McLeod, Yu Tian Wang, Harley Kurata, Jim Wright, Jeremy Seamans, and Catherine Winstanley for their support and invaluable critique of my research projects.   Thanks to the current and former staff members in the department of Anesthesiology, Pharmacology & Therapeutics at UBC for their generous favors, and moral and technical support:   Wynne Leung Andy Jeffries Christian Caritey Jessica Yu Marnie Bymak  Thanks to my former supervisors at the University of Illinois-Chicago, Drs. Michael Ragozzino and Mitchell Roitman, for inspiring me to pursue a career in neuroscience, and for putting UBC and Dr. Borgland's lab on the radar.  Thanks to all of my current and former lab mates for their help with experiments, scientific feedback, and moral support, especially Dr. Gwenael Labouebe, Dr. Joyce Yang, Corey Baimel, Kimberley Pitman, Dr. Lindsay Naef, Michael Drysdale, and Lauren Kerr  Thanks to my fellow students and friends in the pharmacology and neuroscience programs for their kindness and support, especially Ricardo Rivera, Anu Khurana, Steve Wainwright, and Jayant Shrava  Thanks to the Hill lab and the Pittman lab at the University of Calgary for their technical expertise and generous use of their equipment, without which I could not collect a lot of my data.  Last but not least, a special thanks to my family––Lori, Roy, Ashley and Mike for never questioning the long journey to complete my PhD.     xvi  Dedication    dedicated to my mom, Lori 1 Chapter 1: Introduction 1.1 The role of dopamine in goal-directed behavior    Dopamine, a monoamine derived from the amino acid tryptophan, is present in the brain of all vertebrate species (Camps et al., 1990).  After dopamine was initially determined to be a neurotransmitter (Carlsson et al. 1958; Carlsson, 1959), its role was relegated to the motor system (Fahn et al. 2008; Anden et al., 1964).  Subsequently, it was discovered that dopamine could encode the perception of rewarding stimuli such as food, and drugs of abuse (Yokel and Wise, 1975; Wise et al. 1978).  Over time, however, many elegant behavioral experiments, in combination with techniques that measure sub-second fluctuations in brain dopamine levels, have drastically changed the way we think about this monoamine.  Although remnants of dopamine as the ‘pleasure neurotransmitter’ still exist in popular culture, it is now established that dopamine acts as a learning signal to mediated goal-directed behavior for both rewarding and aversive events (Berridge and Robinson, 2003; Tobler et al., 2003; Bayer and Glimcher, 2005; Schultz, 2010).     Dopamine is released widely throughout the mammalian brain, yet the cell bodies that contain dopamine are located in very few regions––the midbrain, hypothalamus, and olfactory bulb (Chiodo and Bunney, 1983; van et al., 1984; Jahanshahi et al., 2013).  The role of dopamine in learning and decision-making behavior is predominantly controlled by neurons in the midbrain, which project to areas implicated in goal-directed behavior (Berger et al., 1976; Satoh et al., 2003; Morris et al., 2006; Balleine et al., 2007; Roesch et al., 2007; Salamone et al., 2007).   2    The ventral tegmental area (VTA) and substantia nigra pars compacta (SNpc) are two major cell populations in the midbrain that contain dopamine (Guyenet and Aghajanian, 1978; Wang, 1981).  The functional relevance for dopamine release from the VTA and SNpc has traditionally been segregated to reward and motor systems, respectively.  Although burgeoning evidence for dopamine in the SNpc is implicated in reinforcement learning (Ilango et al., 2014; Reynolds et al., 2001; Ramayya et al., 2014), much of the data implicate VTA dopamine as a predominant mediator of these phenomena (Bayer and Glimcher, 2005; Berridge, 2007; Tsai et al., 2009; Schultz, 2010; Kobayashi and Schultz, 2014; Hollerman and Schultz, 1998).  In addition, while projection targets of the VTA and SNpc are somewhat overlapping, the majority of dopaminergic innervation to target brain regions emphasized in this thesis, namely the prefrontal cortex (PFC) and nucleus accumbens (NAc), originates in the VTA (Fallon, 1988).   For these reasons, VTA dopamine neurons and the subsequent projection targets will be the current focus.        The VTA is comprised of diverse cell populations.  Most of neurons in the VTA contain dopamine.  However, cells that express γ-aminobutyric acid (GABA) and glutamate, the main inhibitory and excitatory neurotransmitters in the brain, are also located here and form synaptic connections with dopamine neurons (Bonci and Malenka, 1999; Nugent and Kauer, 2008; Yamaguchi et al., 2007; Hnasko et al., 2012).  Dopamine neurons also receive mixed excitatory and inhibitory inputs from distal brain regions including regions of PFC, ventral striatum, hypothalamus, and the amygdala (Watabe-Uchida et al., 2012). Dopaminergic efferents project back to many of the same regions that innervate the VTA, and are therefore reciprocal in nature (Swanson, 1982; Fallon, 1988).  The variety of inputs  3 that the VTA receives suggests a complex mechanism behind how the activity of dopamine neurons is regulated by outside sources to affect downstream release of dopamine.          Temporal patterns of terminal dopamine release are used to guide the responses of an animal in uncertain situations (Hollerman and Schultz, 1998; Fiorillo et al., 2003; Schultz et al., 1997).  Dopamine release and reuptake in the NAc is precisely time-locked to the receipt of reward, environmental cues that predict the reward, or the behavioral response required to obtain it (Ito et al., 2000; Roitman et al., 2004; Day et al., 2007; Mark et al., 1994). In contrast, pauses in dopamine release occur when expected rewards are omitted or during aversive stimuli (Oleson et al., 2012; Mark et al., 1991; Roitman et al., 2008). Thus, this temporal coding by dopamine occurs for both positive and negative events, although it has been most extensively studied under circumstances that predict positive, rewarding outcomes.      The amount of dopamine released is directly related to the magnitude of the reward received and how well an animal can predict a rewarding outcome using external cues from the environment (Kiyatkin and Gratton, 1994; Roitman et al., 2004; Day et al., 2007; Phillips et al., 2003; Oleson et al., 2012).  Furthermore, modulating the relative concentrations of dopamine release by electrically stimulating or inhibiting VTA dopamine neurons, promotes behaviors related to obtaining rewarding or avoiding aversive outcomes, respectively (Phillips et al., 2003; Ilango et al., 2014).  This illustrates that dopamine is not only directly involved in signaling the valence of an outcome, but in orchestrating goal-directed behavior to obtain those rewards. Thus, dopamine neurons are responsible for encoding  ‘prediction  4 errors’—or the perceived difference between an expected versus actual outcome––when responding to a stimulus with an ambiguous end result (Hollerman and Schultz, 1998).  1.2 Control of dopamine neuronal firing    The amount of dopamine released into target brain regions is controlled by firing patterns of VTA dopamine neurons (Garris et al., 1993; Garris and Wightman, 1994; Zhang and Sulzer, 2004; Bass et al., 2010). The majority of dopamine neurons exhibit spontaneous firing at low frequencies (Grace and Bunney, 1983), and contribute to background (tonic) dopamine concentrations. However, upon prolonged depolarization, phasic (burst-like) firing of VTA dopamine neurons occurs (Bayer and Glimcher, 2005), in which several action potentials cluster together, thus resulting in high frequency firing for several hundred milliseconds (Grace and Bunney, 1984). This bursting activity can greatly increase the concentrations of dopamine, because the rate of release is greater than that of degradation or reuptake mechanisms (Chergui et al., 1994; Floresco et al., 2003; Venton et al., 2003).     The firing patterns of dopamine neurons are driven by intrinsic conductances in addition to excitatory (glutamatergic and cholinergic) and inhibitory (GABAergic) inputs (Komendantov et al., 2004). Conductances mediated by inwardly rectifying and calcium-regulated potassium channels, and hyperpolarization-activated cyclic nucleotide–gated cation channels (HCN) regulate basal levels of excitability by controlling tonic firing (Grace, 1991; Hopf et al., 2007; Tateno and Robinson, 2011). Bursting activity of dopamine neurons typically requires activation of glutamatergic n-methyl-D-aspartate receptors (NMDARs) (Overton and Clark, 1992; Chergui et al., 1993; Komendantov et al., 2004; Zweifel et al.,  5 2009). Although some glutamate-containing neurons exist in the VTA (Yamaguchi et al., 2007), activation of NMDARs expressed on VTA dopamine neurons is predominantly controlled by excitatory inputs from distal brain regions, including the PFC and pedunculopontine tegmental nucleus (Grace, 1991; Carr and Sesack, 2000; Floresco et al., 2003).   1.3 Excitatory synaptic transmission and changes in reward learning 1.3.1 Long-term potentiation and long-term depression     Long-term changes in the strength of glutamatergic synapses occur widely throughout the brain and are believed to underlie several learning and memory processes (Kirkwood et al., 1995; Abel et al., 1997; Tsvetkov et al., 2002; Collingridge et al., 2004; Migues et al., 2010). Long-term potentiation (LTP) or long-term depression (LTD) is reflected by experience-dependent changes in the number or function of postsynaptic glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and/or the magnitude of presynaptic glutamate release (Kauer and Malenka, 2007; Migues et al., 2010). These effects can last for several weeks (Sigurdsson et al., 2007; Pu et al., 2006).            LTP and LTD occurs in dopamine neurons of the VTA (Thomas et al., 2000; Bonci and Malenka, 1999; Malenka and Bear, 2004; Overton et al., 1999). Excitatory inputs play a large role in regulating the excitability of dopamine neurons (Kalivas, 1993; White, 1996). Experience dependent plasticity of glutamatergic synapses on VTA dopamine neurons is a key element in the development of learned, salient environmental cues that predict rewarding events (Kauer and Malenka, 2007; Stuber et al., 2008c).  6  Long-term potentiation of glutamatergic synapses onto VTA neurons occurs through activation of NMDA receptors when animals learn to approach a cue light that predicts delivery of sucrose (Stuber et al., 2008b). Although this plasticity is transient for natural rewards such as sucrose, long-lasting changes occur through administration of drugs of abuse (Ungless et al., 2001; Saal et al., 2003; Borgland et al., 2004; Chen et al., 2008; Stuber et al., 2008a; Stuber et al., 2008b).   1.3.2 Modulation of synaptic transmission by feeding peptides    Interestingly, endogenous feeding-related peptides such as insulin, orexin, and ghrelin (Abizaid et al., 2006; Borgland et al., 2006; Borgland et al., 2009; Labouebe et al., 2013) can also modulate synaptic efficacy at glutamatergic synapses onto VTA dopamine neurons. Insulin, a peripheral hormone released from the pancreas, crosses the blood brain barrier (Frank and Pardridge, 1981) to act in the ventral medial hypothalamus to suppress feeding (Bruning et al., 2000). Insulin also induces an endocannabinoid-mediated LTD selectively at excitatory synapses onto dopamine neurons (Labouebe et al., 2013). Whereas insulin suppresses feeding, orexigenic (appetite stimulating) peptides such as ghrelin or orexin act in the VTA to potentiate synaptic strength, albeit by different mechanisms. Ghrelin, when administered peripherally, increases synapse number and glutamate release onto VTA dopamine neurons (Abizaid et al., 2006) whereas orexin potentiates postsynaptic NMDA receptors on dopamine neurons (Borgland et al., 2006).       7 1.4 Leptin signaling in the brain     In general, peptides that stimulate or inhibit aspects of anticipatory behavior that precede foraging also potentiate or reduce glutamatergic signaling in the VTA (Overton et al., 1999; Borgland et al., 2009; Labouebe et al., 2013). Leptin is a circulating peptide released from adipocytes that has been shown to decrease reward-seeking behaviors.  However, is unknown if concomitant changes in excitatory synaptic transmission in the VTA also occur in response to leptin. Leptin receptors are expressed in the VTA (Figlewicz et al., 2003; Korotkova et al., 2006; Leshan et al., 2010).  We hypothesized that leptin decreases synaptic efficacy at excitatory synapses onto VTA dopamine neurons. Chapter 2 explores the mechanism by which leptin regulates synaptic efficacy in the VTA.    1.4.1 Leptin as an adiposity signal    Tonic leptin activity in the brain can control the quantity of food consumed (Halaas et al., 1995; Mistry et al., 1997; Niswender et al., 2001). Although often considered a ‘satiety’ hormone, it is more accurate to describe the actions of leptin as an adiposity signal that negatively regulates food consumption—the most drastic change in feeding behavior occurs when leptin concentrations drop through fasting or fat loss, which promotes feeding (Ahima et al., 1996). In contrast, increases in circulating leptin only moderately reduce food consumption, quite possibly because it is relatively easy to become leptin resistant. Rats maintained on a high sugar diet become leptin resistant in as little as two weeks, which occurs before significant changes in body weight are apparent (Vasselli et al., 2013; Apolzan and Harris, 2013). The development of leptin resistance may be one reason why peripheral  8 leptin administration in obese individuals does not readily attenuate food consumption (Unger and Scherer, 2010). The ability of leptin to act in the CNS can also be regulated by other peptides implicated in feeding behavior, such as cholecystokinin (Cano et al., 2008).  Additionally, leptin is able to reduce both the gastric secretion of ghrelin and its ability to promote food consumpion (Kalra et al., 2005).  Thus, the role of leptin in regulating energy  consumption is multi-faceted, as it can signal directly in the CNS in addition to regulating other peripherally circulating peptides.   1.4.2 Disruption of leptin signaling leads to hyperphagia    Because tonic leptin signaling decreases food consumption, removing leptin signaling, either by knocking out leptin receptors or in animals that do not express preproleptin, promotes increased food intake and subsequent obesity. Rare cases of congenital leptin deficiency in humans induced by either mutations in the Ob gene responsible for leptin synthesis (Fatima et al., 2011), or the gene that encodes the leptin receptor, LEPR (Mazen et al., 2011), causes severe early-onset obesity.  Similar phenotypes have been demonstrated in animal models.  In Zucker (fa/fa) rats, a mutation in LEPR renders an obese phenotype that is also hyperlipidemic and hyperglycemic (Zucker and Zucker, 1961). The mutated leptin receptors allow leptin to bind but prevent downstream signaling (Phillips et al., 1996).  Additionally, two well-utilized mouse models of obesity, ob/ob and db/db mice, both involve disruptions in leptin signaling.  Whereas db/db mice (Chen et al., 1996; Lee et al., 1996) have a splice variant in the leptin receptor transcript that prevents intracellular signaling, the ob/ob mouse produces an abnormal leptin protein, but signaling through the receptor is intact (Weigle et al., 1995; Vaisse et al., 1996). Taken  9 together, the absence of leptin signaling removes the tonic ‘brake’ on food consumption, and thus obesity results from hyperphagia (Becker and Grinker, 1977; Coleman, 1978; Pelleymounter et al., 1995; Fatima et al., 2011; Mazen et al., 2011). In contrast, exogenous leptin administration in lean rodents and humans inhibits feeding (Seeley et al., 1996; Halaas et al., 1997; Heymsfield et al., 1999; Krugel et al., 2003; Patel and Ebenezer, 2008).     Hyperphagia can occur through disruptions in either homeostatic or hedonic regulation of food intake. While homeostatic feeding exists in order to fulfill an animal’s basal energy requirements, hedonic feeding can occur irrespective of energy needs and solely for the rewarding effects (Scheggi et al., 2013).  Evidence exists for the ability of leptin to regulate both types of feeding behavior, and may be determined by the particular brain region where leptin is acting (Ahima et al., 1996; Sahu, 2003; Figlewicz et al., 2004; Hommel et al., 2006; Domingos et al., 2011; Davis et al., 2011).   1.4.3 Leptin receptor signaling     Leptin crosses the blood-brain barrier through a saturable transport mechanism (Banks et al., 1996). Several short form splice variants of LEPR encode the receptors responsible for leptin transport across the blood brain barrier (Kastin et al., 1999; Maresh et al., 2001). Short-form leptin receptors are highly expressed in microvessels throughout the brain (Bjorbaek et al., 1998b; Burguera et al., 2000). In contrast, Koltesky rats are devoid of the short-form leptin receptor splice variant, yet radiolabeled leptin can still enter the brain, suggesting a separate transport mechanism may be responsible for leptin entering the central nervous system (CNS) (Kastin et al., 1999; Banks et al., 2002). However, compensatory  10 mechanisms behind persistent transport of leptin in Koltesky rats, such as changes in diffusion rates at the median eminence (Zlokovic, 1995), were not investigated.     Once leptin crosses the blood-brain barrier, it binds to the long-form splice variant responsible for intracellular signaling, LepRb. Expression of leptin receptors (LepRb) is widespread throughout the brain (Huang et al., 1996), and belongs to the class I family of janus kinase 2 (JAK2)- associated cytokine receptors (Tartaglia et al., 1995). Once leptin is bound, LepRb dimerizes and JAK2 undergoes autophosphorylation (Phillips et al., 1996). This activates various signal transduction pathways such as extracellular-signaling related kinase/mitogen activated protein kinase (ERK/MAPK), phosphatidylinositol 3-kinase/protein kinase B PI3K/Akt, and signal transducer and activator of transcription 3 and 5 (STAT3 and STAT5), both transcription factors (Munzberg and Myers, 2005; Mutze et al., 2007).  Because of the diversity of expression in different regions, LepRb can regulate several different neurological functions in addition to homeostatic feeding, including memory formation, physical mobility, and circadian rhythm (Wayner et al., 2004; Oomura et al., 2006; Ahima et al., 1998; Sahu, 2003; Irving et al., 2006; Kanoski et al., 2011). Consistent with these additional functions, high levels of LepRb expression are found in the hippocampus, cerebellum, and hypothalamus (Elmquist et al., 1998; Hakansson et al., 1998; Irving et al., 2006; Leshan et al., 2010).  1.4.4 Leptin signaling in the hypothalamus          While studies investigating global disruption of leptin signaling show robust elevations in feeding behavior, they unfortunately preclude analysis of which brain areas are  11 affected, or whether changes in hedonic or homeostatic mechanisms underlie the hyperphagia-induced obesity. Mechanisms of action of leptin in the hypothalamus have been well characterized (Gautron and Elmquist, 2011). In the arcuate nucleus of the hypothalamus, leptin inhibits firing of agouti-related peptide (AGRP) neurons that produce the orexigenic peptide neuropeptide Y (NPY), and increases firing of adjacent proopiomelanocortin neurons that produce anorectic peptides, including α-melanocyte-stimulating-hormone (Stephens et al., 1995; Schwartz et al., 1997; Kristensen et al., 1998; Oswal and Yeo, 2010; Gautron and Elmquist, 2011). Hence, leptin signaling can regulate feeding through bidirectional control of two separate populations of arcuate neurons in the hypothalamus.    In addition to the hypothalamus, leptin modulates the mesolimbic dopamine circuit, suggesting that its role in feeding is not solely linked to energy homeostasis. Indeed, selective deletions in hypothalamic neurons do not produce the drastically obese, hyperphagic phenotype typically seen in global LepRb knockouts (Pelleymounter et al., 1995; Xu et al., 2007). The actions of leptin on the mesolimbic system may be important for the motivation and/or preference for highly palatable foods, and thus, disrupted leptin signaling in the VTA might underlie non-homeostatic feeding behavior in animals and humans.    1.4.5 Effects of leptin on the mesolimbic system  1.4.5.1. Leptin and reinforcing behaviors    Growing evidence for leptin action in mesolimbic circuitry points to discrete signaling within the VTA. Specific knockdown of LepRb in the VTA intensifies preference for palatable foods (Hommel et al., 2006) and increases motivation to consume them (Davis  12 et al., 2011). In contrast, exogenous leptin reduces the rewarding value of sucrose through actions in the VTA (Domingos et al., 2011). Indirectly modulating leptin signaling can also change the preference for highly palatable foods. For instance, voluntary exercise has been shown to increase LepRb signaling in the VTA and paradoxically, is correlated with attenuation of a preference for high fat food over regular chow (Scarpace et al., 2010; Shapiro et al., 2011). It seems counterintuitive that increased energy expenditure would decrease rather than increase preference for energy-dense food in order to balance an energy deficit through exercise. However, wheel running, which modifies dopamine signaling in the NAc (Roberts et al., 2012), mitigates the preference for high fat food over regular chow (Scarpace et al., 2010). Importantly, these experiments suggest that leptin can regulate the hedonic and motivational properties of food, separately from consumption of food for energy requirement.     Leptin receptor knockout in the VTA is an important tool to isolate the effects of leptin signaling on mesolimbic circuitry. However, in the studies described above, leptin receptor knockdown in the VTA, and potentiations in LepRb signaling after wheel running were not specific to dopamine neurons (Hommel et al., 2006; Scarpace et al., 2010; Davis et al., 2011). Because leptin receptors are expressed on both GABAergic and dopaminergic VTA neurons as well as presynaptic inputs to the VTA, the cellular locus behind these effects on reward-seeking behavior remains unclear (Figlewicz et al., 2003; Fulton et al., 2006; Hommel et al., 2006; Leinninger et al., 2009; Leshan et al., 2010; Davis et al., 2011; Liu et al., 2011).  Interestingly, knockout of leptin receptors specifically on dopamine neurons produces an anxiogenic phenotype but does not increase hedonic feeding or total food  13 consumption (Liu et al., 2011). This suggests that leptin’s ability to reduce the motivation to eat might occur indirectly, through actions at non-dopaminergic cells or afferents to the VTA.  1.4.5.2. Effects of leptin on VTA neuron excitability      Systemic leptin reduces basal firing rates during in vivo recordings of VTA dopamine cells, which were identified as having a characteristic triphasic wave form with low firing frequency (Hommel et al., 2006). In contrast, selective knockdown of leptin receptors in dopaminergic neurons increases the percentage of neurons that spontaneously burst fire, but does not change basal firing rate (Liu et al., 2011), possibly owing to the specificity of leptin receptor knockdown as compared to non-specific actions of systemic leptin on multiple cell types (Hommel et al., 2006). Results from VTA brain slice recordings are less clear. Hommel et al. demonstrated reduced spontaneous firing in putative dopamine neurons after leptin, but did not observe a significant change in resting membrane potential (Hommel et al., 2006). Another study failed to observe changes in firing rate using slice recordings (Korotkova et al., 2006). In this study, leptin concentrations were several-fold higher than in Hommel et al. (Hommel et al., 2006).  1.4.5.3. Effect of leptin on dopamine release      The effect of leptin on dopamine release is not well defined. Acute ventricular leptin infusions reduce basal and feeding-evoked increases in NAc dopamine concentrations in adult rats (Krugel et al., 2003). In contrast, chronic systemic leptin replacement reverses impairments in evoked dopamine release in NAc brain slices of ob/ob mice by increasing  14 tyrosine hydroxylase (TH) expression, the rate-limiting enzyme in the production of dopamine synthesis (Fulton et al., 2006). More recent work establishes that leptin signaling in a subpopulation of GABAergic neurons in the lateral hypothalamus (LH), and not specifically in the VTA, is responsible for rescuing the reduced TH content in VTA neurons and dopamine levels in the NAc of ob/ob mice (Leinninger et al., 2009). It is plausible that these contradictory findings may be due to compensatory changes occurring in leptin-deficient ob/ob mice. Notably, animals that have selective deletions of LepRb in pro-opiomelanocortin neurons of the hypothalamus have reduced baseline inhibitory synaptic transmission in these cells (Chun and Jo, 2010). Although changes in synaptic transmission in ob/ob mice with global LepRb deletions have not been characterized, it is possible that other brain regions would also display reduced synaptic inputs.  Changes in neuronal activation of midbrain dopamine neurons affects the amount of TH expression (Aumann et al., 2011), which could be one reason for reduced TH levels observed in ob/ob mice.   1.4.5.4. Effects of leptin on excitatory synaptic transmission    LepRb activation can modulate glutamatergic synaptic transmission in various brain regions.  In general, leptin can increase or decrease synaptic strength, depending on the signal cascade activated via LepRb.  In the hippocampus, leptin potentiates NMDAR currents via PI3K and MAPK signaling (Shanley et al., 2001). However, leptin effects on AMPA receptor-mediated excitatory postsynaptic currents (EPSCs) are mixed. In juvenile rat hippocampal slices, leptin suppresses evoked AMPAR currents (Durakoglugil et al., 2005; Moult and Harvey, 2011).  In another study by the same group, leptin reverses LTP in the same hippocampal region, but has no effect on basal synaptic transmission (Moult et al.,  15 2009).  Leptin concentration, rat age, and patch-clamp methods were similar between these studies, and therefore, are unlikely to account for the discrepancies in findings. Finally, in older rats and hippocampal cultures, leptin increases evoked AMPAR EPSCs through PI3K (Moult et al., 2010), ERK pathways (Moult and Harvey, 2011) or MAPK in cerebellar granular neurons (Irving et al., 2006). Other groups have reported opposing effects of leptin on excitatory synaptic transmission.  In slices taken from rat hippocampus, reductions in evoked AMPAR-mediated EPSCs by leptin occurs (Xu et al., 2008). Similarly, NPY-containing neurons in the arcuate nucleus of the hypothalamus exhibit transient reductions in evoked EPSCs (Glaum et al., 1996). These two studies use a mix of juvenile and adolescent animals (Glaum et al., 1996; Xu et al., 2008).  Leptin concentrations were also in the low nanomolar range, at least one order of magnitude lower than studies illustrating potentiated EPSCs by leptin. Therefore, effects of age and concentration of leptin could have differential effects on synaptic transmission. To date however, the mechanisms that underlie these effects have not been investigated.     Leptin signaling in the VTA mediates several reward-related behaviors and the excitability of dopamine neurons (Fulton et al., 2000; Figlewicz et al., 2001; Figlewicz et al., 2004; Hommel et al., 2006; Davis et al., 2011; Trinko et al., 2011). Surprisingly, it is unknown if leptin can alter synaptic transmission within this region. Because excitatory drive onto dopamine neurons influences the firing modality and output of dopamine at projection targets of the VTA (Komendantov et al., 2004; Floresco et al., 2003), an understanding of how leptin can influence excitatory synaptic transmission is warranted. Therefore, in Chapter 2, we demonstrate how leptin suppresses excitatory synaptic transmission onto dopamine neurons.   16 1.5 Role of the prefrontal cortex in executive function   The PFC makes up the anterior portion of the mammalian cerebral cortex, and is an amalgamation of several distinct subregions responsible for planning, introspection, imagination, and many other aspects of executive function (Burgess, 2010). Although the primate PFC has a higher degree of anatomical segregation, the rodent PFC is similar in terms of both structure and function (Uylings et al., 2003).  The PFC receives substantial inputs from midbrain dopamine neurons (Berger et al., 1976; Van Eden et al., 1987; Kalsbeek et al., 1988; Lewis et al., 1988; Goldman-Rakic et al., 1992; Rosenberg and Lewis, 1994), and stimulation of dopamine receptors expressed here permit regulation of decision-making, attention, and working memory (Zahrt et al., 1997; Granon et al., 2000; Seamans and Yang, 2004; Tunbridge et al., 2004).     The most well-characterized prefrontal region in primates is the dorsolateral prefrontal cortex (DLPFC) and the medial prefrontal cortex (mPFC), which is the analogous region in the rodent cortex.  Behaviors classically assigned to this area are working memory, attention, and performance monitoring (Carter et al., 1998; Levy and Goldman-Rakic, 2000).  The firing frequency of cortical neurons required for the mPFC to properly encode attention is controlled by balanced inhibitory and excitatory tone.  Disrupting the relative contribution in signaling between these two inputs, such as modulating GABAergic signaling, causes deficits in prefrontal function (Pezze et al., 2014; Yizhar et al., 2011).      The other main region in the PFC is the orbitofrontal cortex (OFC). Unlike the mPFC, the OFC is less involved in working memory and attention processes, and is instead required  17 for updating information about expected outcomes (Schoenbaum et al., 2009b; Takahashi et al., 2011). Unlike the wealth of data on modulation of synaptic transmission in the mPFC, little is known about alterations in synaptic function in OFC neurons and how it might differ from other PFC regions.        The OFC can be further broken down into lateral, ventral, and medial (LOFC, VOFC, and MOFC) subregions in both primates and rodents (Ongür and Price, 2000; Uylings et al., 2003).  The function of the LOFC is best understood. However, growing evidence suggests that these subregions differentially contribute to different features of OFC-dependent behavior (Rudebeck et al., 2008; Mar et al., 2011; Stopper et al., 2014).  Less is known about VOFC, but evidence suggests opposing roles for MOFC and LOFC in the rat. Interestingly, lesions of rat MOFC reduce cocaine-seeking, while lesions in LOFC cause a perseveration of this response (Fuchs et al., 2004). Others have shown that similar lesions in MOFC increase preference for larger delayed rewards, where LOFC lesions decrease it (Mar et al., 2011).  This indicates that these subregions might have differential roles in decision- making tasks.    1.6 The role of the orbitofrontal cortex in goal-directed behavior    Both the mPFC in rodents (or DLPFC in primates) and the OFC are implicated in goal-directed behavior. Whereas the mPFC is important for attending to a given task, the OFC is crucial for predicting the benefit of an outcome resulting from a chosen strategy (Tait et al., 2007; Schoenbaum et al., 2009a). Given that attention is required for efficient performance during decision-making tasks, it explains why disruptions in mPFC activity can  18 impair performance on tasks that are classically assigned to the OFC (Hitchcott et al., 2007; Salazar et al., 2004).      Several debilitating mental illnesses including, but not limited to drug addiction, attention deficit hyperactivity disorder (ADHD), eating disorders, and obsessive-compulsive disorder OCD are linked to OFC dysfunction (Volkow et al., 1991; Hesslinger et al., 2002; Chamberlain et al., 2008; Suda et al., 2010). Underlying these disorders is rigid cognitive inflexibility that stems from an inability to restrain unwanted thoughts or actions (Suda et al., 2010).      An early theory of orbitofrontal function was its role in encoding reward value, or the subjective magnitude of a rewarding stimulus or event in the near or distant future (Tremblay and Schultz, 1999; Schultz et al., 2000; Izquierdo et al., 2004; Schoenbaum et al., 2009a). However, neural activity in the OFC encodes many contextual aspects of the environment that are not necessarily based on assessment of value, such as spatial location (Feierstein et al., 2006), uncertainty (Kepecs et al., 2008), length of time to reward receipt (Roesch et al., 2006), and prediction of risk (O'Neill and Schultz, 2013). Thus, the OFC does not calculate the absolute value of a reward per se, but instead forms abstract representations of motivationally salient events (O'Doherty et al., 2001; Wilson et al., 2014) that are used to encode changes in expected outcomes during decision-making (Takahashi et al., 2009; Wilson et al., 2014).     Several behavioral paradigms including, but not limited to reversal learning, delay discounting, reinforcer devaluation and others have implicated the OFC. All of these tasks  19 involve updating mental representations upon changes to the environment or interoceptive cues, and are required for a behavioral change to occur (Wilson et al., 2014).  Thus, the OFC must integrate information about current context while calling on previous experience and predicting future outcomes (Bowman et al., 2012; Takahashi et al., 2013).     The ability of OFC neurons to encode changes in expected outcomes is often measured by a sensory-specific satiety paradigm. This is easily investigated by changing the motivation level of a hungry animal by feeding it a meal beyond satiety (Kringelbach et al., 2003). For example, when human subjects are slowly fed chocolate to excess during a positron emission tomography (PET) scan, reduced activation of the OFC reflects the changes in subjective evaluations as the food, which was once pleasurable, eventually becomes aversive (Small et al., 2001). Similar findings have been demonstrated in rodents and non-human primates. As animals become sated on a particular meal, the firing of OFC neurons is drastically attenuated, suggesting that they may encode the changes in the expected outcome of the food presented to them (Rolls et al., 1989). In addition, the general ability to inhibit responding to a food that is no longer reinforcing is dependent upon intact connections to the OFC (Zeeb and Winstanley, 2013).  Thus, researchers are able to artificially manipulate the subjective magnitude of a rewarding outcome, and these manipulations are reflected by reductions in activity in the OFC.      As the above findings demonstrate, OFC activation is associated with updating the predicted outcome of salient stimuli. Participants need not consume food in order to change subjective ratings about sensory stimuli, however.  Predictive-reward value can be manipulated by merely giving opposing information about a stimulus. Thus, previous  20 knowledge can change whether the stimulus is perceived as pleasant or aversive.  For example, OFC activation can be tightly correlated with subjective ratings of an odor. During an fMRI scan, OFC activation increases when participants are told the odor is cheddar cheese, which is rated as pleasant, but is reduced when told it is body odor, which is rated as aversive (de Araujo et al., 2005).   1.7 The significance of dopamine in the mPFC/DLPFC    Dopamine release in the PFC is able to regulate goal-directed behavior. This process involves both mPFC and OFC regions that encode attention and outcome expectancies, respectively (Schoenbaum and Roesch, 2005; Paine et al., 2009). Although dopaminergic signaling in the OFC has been characterized at the behavioral level, extensive research on the effects of dopamine on neurons in the cortex has only been carried out in mPFC/DLPFC of rodent/primate (Seamans and Yang, 2004; Hitchcott et al., 2007; Gao and Wolf, 2008; Durstewitz and Seamans, 2008; Kroener et al., 2009; Gonzalez-Islas and Hablitz, 2001; Bandyopadhyay et al., 2005).      In the rat mPFC, dopamine provides long-lasting modulation of pyramidal cell firing, the glutamatergic neurons that make up the majority of neurons in the cortex (Santana et al., 2009). This modulates cognitive flexibility (Lim and Goldman, 2014). Unlike the NAc, dopamine release and reuptake in the PFC is less time-locked to rewards or environmental cues due to lower levels of the dopamine transporter (DAT) here (Sesack et al., 1998c). In a decision-making context or during anticipation of highly palatable food, dopamine is released and remains elevated for longer time periods (Ahn and Phillips, 1999; Watanabe et al., 1997).  21 One hypothesis is that dopamine signaling in the cortex is less about encoding temporally-dependent information about environmental context, as it does in the NAc, and more about tonic modulation of networks of cortical neurons in order to modify attention to relevant stimuli (Tunbridge et al., 2004; Lavin et al., 2005). In this way, dopamine acts as a neuromodulator to fine-tune the efficacy of inhibitory and excitatory synaptic connections in neurons of the PFC (Lim and Goldman, 2014; Seamans and Yang, 2004), and is associated with proficiency at working memory tasks (Vijayraghavan et al., 2007; Zahrt et al., 1997).  1.8 Dopamine receptor signaling pathways   The ability of dopamine to guide behavior ultimately depends upon changes in neuronal excitability through activation of dopamine receptors. The five different receptor subtypes that exist are all encoded by different genes but are separated into two distinct families, corresponding to the G protein with which they associate (Jackson and Westlind-Danielsson, 1994).  The D1-like receptors (D1Rs) include D1 and D5 isoforms and couple to Gs/Golf  proteins which activate adenylyl cyclase and protein kinase A (PKA)-dependent signaling cascades (Monsma et al., 1990; Zhuang et al., 2000).  The vast majority of D1Rs signal in this way. However, evidence for atypical Gq coupled D1Rs that activate phospholipase C (PLC) also exists (Wang et al., 1995; Jin et al., 2001; So et al., 2009). Recently, it was confirmed that D5 is the receptor subtype in the D1-like family that is responsible for signaling via PLC (Paspalas and Goldman-Rakic, 2004; So et al., 2009). In contrast, D2-like receptors (D2Rs) are inhibitory in nature and include D2, D3, and D4 receptors.  These receptors couple to Gi/o proteins that inhibit adenylyl cyclase-dependent  22 signaling cascades (Senogles, 1994; Obadiah et al., 1999). Therefore, the main net effects of D1R and D2R activation are excitatory and inhibitory, respectively.    1.9  Dopamine receptor expression in the adult PFC     There are no published papers that specifically focus on dopamine receptor expression in regions of the OFC.  However, many investigate overall receptor expression in the cortex without differentiating between prefrontal subregions. In the rat mPFC and more lateral regions near the OFC, D1 receptor mRNA and radiolabeled D1Rs are localized to all cortical layers (Al-Tikriti et al., 1992; Gaspar et al., 1995). While D2 receptors are distributed uniformly throughout cortical layers in putative orbitofrontal regions, D2 receptor mRNA in mPFC is found primarily in deeper layers V and VI (Bouthenet et al., 1991; Santana et al., 2009).     Relatively similar expression patterns in the mPFC and DLPFC have been demonstrated, including subcellular localization of receptor expression. In primates, immunolabeling for D1Rs and D2Rs is observed in glutmatergic pyramidal neurons and GABAergic interneurons (Bergson et al., 1995; Glausier et al., 2009). Parallel levels of expression in rat are also found using radiolabels for both receptor subtypes (Vincent et al., 1993; Vincent et al., 1995).       23 1.9.1 Receptor expression in pyramidal neurons    The highest expression for all dopamine receptor subtypes in the cortex is on glutamatergic pyramidal neurons.  Postsynaptic D2R expression in pyramidal neurons parallels that of D1Rs—receptors are found on dendritic spines that receive excitatory inputs, and are peri- and extrasynaptic (Negyessy and Goldman-Rakic, 2005).   In macaques, D1 and D5 receptors are predominantly peri- and extrasynaptic and highly expressed in pyramidal neurons, although rarely on the same neuron (Paspalas and Goldman-Rakic, 2004). The extrasynaptic nature of D1 receptor expression and reduced DAT expression in the PFC suggests that dopamine can activate D1 receptors even when it has diffused relatively far from the release site (Smiley et al., 1994; Seamans and Yang, 2004; Paspalas and Goldman-Rakic, 2005). D1 and D5Rs are differentially localized to the spines and shafts of pyramidal cell dendrites, respectively. In this way, D1Rs appear to regulate both excitatory and inhibitory inputs, under the assertion that excitatory terminals target dendritic spines versus inhibitory terminals that target dendritic shafts. However, this may be an oversimplification due to the diversity of inhibitory synaptic contacts that are made onto the various dendritic locations of pyramidal neurons (Somogyi and Cowey, 1981; Jones, 1993; DeFelipe and Fariñas, 1992).     D1 and D2 receptor expression on pyramidal neurons in mPFC is predominantly restricted to discrete cell populations.  However, portions of excitatory neurons co-express both receptors (Vincent et al., 1993; Vincent et al., 1995; Lee et al., 2004). In mPFC neurons that co-express D1Rs and D2Rs, assembly of a functional D1:D2 heterodimer is possible (Pei et al., 2010), although this has not been demonstrated in other areas of the PFC. These  24 receptor complexes are preferentially coupled to Gq proteins, and can be activated by dopamine or ligands mainly used to study D1 or D2 homomers (Rashid et al., 2007; Perreault et al., 2011; O'Dowd et al., 2012). Both D1:D2 and D5:D2 complexes have been identified by various techniques including fluorescence resonance energy transfer (FRET) and co-immunoprecipitation (Hasbi et al., 2009; Pei et al., 2010).  Like Gq-coupled D5 receptors, activation of D1:D2 or D5:D2 heterodimers leads to PLC-mediated increased internal calcium concentrations. These receptor heterodimers signal in a similar manner to PLC-coupled D5 receptors, but intracellular signaling is more rapid (So et al., 2009).  Unfortunately, subcellular localization of dopamine receptors in the OFC has not yet been characterized.   1.9.2 Receptor expression in GABA neurons    In general, D1R expression on GABAergic interneurons is lower than in pyramidal neurons in the PFC (Bergson et al., 1995; Gaspar et al., 1995). Vincent et al. reported higher D1R expression for interneurons, but cellular identification was based only on soma size, a poor indicator of cell type (Vincent et al., 1995).  Others have also reported that a greater proportion of total interneurons in the cortex express D1Rs, but the greater number of pyramidal neurons in the cortex means absolute expression of D1Rs is still higher in these excitatory cells (Santana et al., 2009).     D2R expression is also lower in GABAergic neurons in the cortex (Khan et al., 1998; Santana et al., 2009). While some have reported that D4 receptors are preferentially expressed on pyramidal neurons in the primate PFC (Khan et al., 1998), others have  25 suggested that interneurons exhibit higher expression of this receptor subtype (Mrzljak et al., 1996). D2R expression is also found on presynaptic terminals of GABAergic interneurons in the PFC of rats and non-human primates, which make symmetric synaptic connections with pyramidal cells (Seamans et al., 2001b; Negyessy and Goldman-Rakic, 2005). Thus, it is possible that dopamine can modulate the excitability of pyramidal neurons indirectly, by altering GABA release from interneurons that express either D1Rs or D2Rs (Le Moine and Gaspar, 1998).    1.9.3 Expression in presynaptic terminals   Dopamine receptors are also located presynaptically, on axon terminals. In the primate DLPFC, D1R expression is present on putative glutamatergic terminals, and D5R expression has been found on both putative glutamatergic and GABAergic terminals (Bergson et al., 1995; Paspalas and Goldman-Rakic, 2005). Presynaptic D1 receptor expression has also been demonstrated in rat mPFC (Gao et al., 2001; Seamans et al., 2001a; Wang et al., 2002; Feng et al., 2004).  The pre- and postsynaptic localization of D1Rs receptors on both GABAergic and glutamatergic neurons in the PFC indicates that dopamine can cause a stimulatory response that ultimately regulates how neurons respond to synaptic inputs, in addition to possibly regulating the release of endogenous neurotransmitters (Gao et al., 2001).      Dopamine is also able to regulate presynaptic release of glutamate through D2Rs, which are located on excitatory presynaptic terminals (Seamans et al., 2001a; Negyessy and Goldman-Rakic, 2005), and often act as auto-receptors on dopamine terminals in the PFC, as  26 a negative feedback mechanism to regulate subsequent dopamine release (Cass and Gerhardt, 1994; Gobert et al., 1996).  1.10 Differential expression in prefrontal dopamine across development     Most studies investigating the role of dopamine on cognitive performance use adult animals. Yet, the electrophysiological data elucidating dopamine’s role in modulating neuronal physiology in the PFC is often obtained from juvenile animals.  However, dopamine innervation and receptor expression in PFC regions changes across development. In the mPFC and DLPFC in rats and primates, respectively, both terminal innervation and receptor expression are relatively conserved across mammals (Kalsbeek et al., 1988).    D1 and D2 receptors both appear to peak during the perinatal period, but are relatively stable after that (Lidow and Rakic, 1992).  The pattern of D1Rs receptor expression in juvenile/adult rat, the predominant dopamine receptor in the medial PFC (Tarazi and Baldessarini, 2000; Vincent et al., 1993), is similar to the findings in homologous regions in developing rhesus monkeys such that D1 receptors peak around 2-3 weeks of age and then decline and stabilize in adulthood (Leslie et al., 1991).      Investigations into dopamine receptor expression in the PFC have been largely ignored in regions outside the mPFC. In the frontal pole of the rat, which is predominantly comprised of orbitofrontal cortex (Paxinos and Watson, 2007), one study demonstrated relative stability of D1 receptor expression from P21 onwards (Leslie et al., 1991). Other studies have indicated lower D1 receptor expression in the lateral OFC juveniles compared to  27 adults (Garske et al., 2013). Increasing levels of expression of both D1 and D2 receptor subtypes compared to neonatal rats are also observed in juvenile frontal cortex (including putative mPFC and OFC) and hippocampus, and appear to increase until adulthood. However, it is unclear whether levels of dopamine receptor expression between juveniles and adults are statistically significant in this paper (Tarazi and Baldessarini, 2000).  1.10.1 Dopamine terminal innervation in PFC     In the primate PFC, dopamine terminal innervation of the most superficial and deepest cortical layers is fairly complete perinatally (Berger et al., 1991; Rosenberg and Lewis, 1995). Dopamine fibers, as measured by TH immunolabeling, innervate superficial layers II/III and peak during adolescence. Subsequently, the density of these fibers declines until adulthood where they become stable, mirroring patterns of dopamine receptor expression.  Because of this decline post-adolescence, it appears that the density of dopaminergic fibers is similar in juveniles and adults (Rosenberg and Lewis, 1994). While norepinephrine terminals, abundant in the PFC, could also be labeled by TH antibodies, only a small percentage of TH positive terminals in the PFC contain norepinephrine (Noack and Lewis, 1989; Akil and Lewis, 1993; Miner et al., 2003).     In rat medial prefrontal and orbitofrontal regions, the density of dopamine innervation by adulthood is similar to that of primates (Berger et al., 1991). While the distribution of dopamine terminal is similar in juveniles and adults, total dopamine content increases during the adolescent period (Leslie et al., 1991). In addition the density of dopamine-containing  28 fibers in rat PFC show a gradual increase throughout the perinatal period until adulthood (Verney et al., 1982; Kalsbeek et al., 1988; Berger et al., 1991).   1.11  Dopamine signaling in the OFC 1.11.1 Dopamine release in the OFC during behavioral tasks    Like the mPFC, dopaminergic tone in the orbitofrontal cortex is also mediates decision-making tasks. Here, dopamine is responsible for integrating subjective information about rewards. For example, in rats and humans, OFC dopamine concentration increases when performing tasks that measure decision-making and impulse control (Winstanley et al., 2006; Albrecht et al., 2014). In rats, this is accomplished by using a delay-discounting task, where rats choose between a small, immediately available reward or a larger one after a long delay. As the delay period to the large reward is increased, animals begin to ‘discount’ the long delay choice, and select the small immediate rewards more often. This is associated with increased orbitofrontal dopamine release as the task progresses. Interestingly, this only occurs if animals are faced with the option to choose between multiple rewards, and not because of general operant responding for food rewards (Winstanley et al., 2006). Additionally, lesions to the OFC or depletion of dopamine post-training make animals more tolerant of longer delays (Kheramin et al., 2004; Winstanley et al., 2004). Taken together, these results indicate that decision-making about rewarding events is associated with increases in dopamine release in the OFC.      29 1.11.2 Behavioral effects of dopamine receptor signaling in the OFC    Although it is clear that dopamine plays a role in OFC function, the contribution of dopamine receptor subtypes for modulating behavior is unclear. Dopamine signaling in the OFC is restricted to behavioral data and the use of intracranial infusions of dopamine receptor agonists and antagonists, which could modulate both interneurons and pyramidal neurons. Baseline levels of OFC dopamine, innate differences in levels of impulsive responding, behavioral paradigms used, and specific sites of OFC infusion may all contribute to the ambiguous findings from in vivo experiments described below.   1.11.2.1. Impulsive responding    Impulsive responding for food reward is associated with dopamine signaling in the OFC. Blockade of dopamine receptor signaling can affect different types of impulsivity. Impulsive choice, or choosing small immediate rewards over larger delayed ones, is typically measured through bevhavioral paradigms such as the delayed discounting task (Zeeb et al., 2010).  Impulsive action, on the other hand, is the inability to inhibit performing a specific motor behavior, and is often assessed by the amount of premature respponses made during the 5-choice serial reaction time task (Winstanley et al. 2010). Intra-OFC administration of D1 receptor antagonists can affect performance on both tasks, illustratng a role for dopamine signaling in both impulsive choice and impulsive action.   In rats that exhibit high baseline levels of impulsivity, blockade of D1 receptors in the OFC reduced premature responding, and hence reduced impulsive action . However, decreased impulsivity was not observed in rats that were less impulsive with intra-OFC  30 administration of a D1 antagonist. Inhibiting D2 receptors on this task did not affect impulsivity per se, but a D2R agonist caused deficits in response accuracy and the number of trials completed (Winstanley et al., 2010).  Thus, reducing dopamine signaling through D1 receptors appears to reduce high baseline levels of impulsive action, and activation of D2 receptors causes more generalized impairments in performance.  The same D1 antagonist in the OFC can increase choice of small immediate rewards during delay discounting.  Interestingly, this only occurs when the delay to the large reward is signaled by a cue light, where animals are given immediate feedback about their choice (Zeeb et al., 2010). In contrast, D2 antagonists in the OFC did not significantly affect impulsive choice behavior.  Thus, the ability of dopamine receptor signaling to modulate impulsive responding not only depends on the type of impulsivity being measured, but also baseline levels of impulsive action, in addition to specific features of the task itself.    1.11.2.2. Reversal-learning     Other studies have demonstrated that both dopamine receptor subtypes might be involved in reversal learning, which measures the ability to exhibit adaptive responding under changing reward contingencies. Blockade of either D1 or D2 receptors can impair performance on a variation of a reversal-learning task, whereby stimuli previously associated with small or large rewards are switched (Calaminus and Hauber, 2008). Interestingly, in another study examining reversal learning, significantly higher concentrations of intra-OFC D1 but not D2 receptor antagonists also impaired performance, but this did occur when blocking D2 receptors (Winter et al., 2009).     31 1.11.2.3. Progressive ratio responding    A role for both receptor subtypes is also observed when rats are tested on a progressive ratio schedule of reinforcement, used to measure levels of motivation (Cetin et al., 2004). The number of lever presses required to obtain food reward increases on an exponential scale. Thus, this task requires the rats to engage in progressively more effort to receive the same reward (Richardson and Roberts, 1996).  OFC-administration of D1 or D2 receptor antagonists reduces the break-point, or the maximal effort the animals will expend to receive a reinforcer. Because the exponential nature of required lever presses, one hypothesis is that blockade of dopamine receptors further enhances the subjective disparity between the predicted and actual outcomes, thus reducing the effort the rat is motivated to put forth (Cetin et al., 2004).   1.11.3 Selectivity of dopamine receptor ligands    One important caveat with many of the above behavioral studies is the use of very high concentrations of dopamine receptor agonists and antagonists, which likely exhibit off-target effects when infused into the brain. The D1 agonist SKF81297 and antagonist SCH23390 are both widely used in behavioral studies that examine the role of D1 receptors in PFC function.  For example, Mizoguchi et al. (Mizoguchi et al., 2010) reported that SKF81297 ameliorates reversal-learning deficits in aged rats. Subsequently, SCH23390 was able to block the effect. However, SKF81297 blocks the pore of the NMDA receptor at depolarized potentials at concentrations used in these studies (Cui et al., 2006). This is an important consideration when exploring effects of this agonist on cognition, as NMDARs  32 play an important role in OFC functioning (described below). Furthermore, SCH23390, a D1 antagonist, is also a potent agonist of serotonin (5HT) 2C receptors, which are expressed in the OFC (Bischoff et al., 1986; Millan et al., 2001; Pompeiano et al., 1994).  Several studies infuse these drugs into the brain at millimolar concentrations (Zahrt et al., 1997; Granon et al., 2000; Mizoguchi et al., 2010; Zeeb et al., 2010), which will undoubtedly modulate 5HT signaling. Although it is likely that the required concentrations of antagonists used in vivo is higher than those used in slice preparations or expression systems, it is unlikely that 1000-fold higher concentrations are needed (Friden et al., 2007). Therefore, care should be taken when interpreting findings using these pharmacological agents.     Despite numerous in vivo studies investigating the role of dopamine in the OFC, it is unknown how dopamine modulates synaptic transmission in this region. The OFC is interconnected with the amygdala, ventral striatum, VTA, and other brain regions responsible for regulating goal-directed behavior (Watabe-Uchida et al., 2012; Krettek and Price, 1977; Cranford et al., 1976; Mailly et al., 2013; Schilman et al., 2008). In addition, synaptic transmission in pyramidal neurons of the mPFC is heavily regulated by dopamine. Therefore, we hypothesize that dopamine also modifies synaptic transmission onto pyramidal neurons of the OFC. Chapter 3 will describe how dopamine receptor signaling modulates excitatory synaptic transmission onto pyramidal neurons in 3 subregions of the OFC.   1.12 Dopamine release in the prefrontal cortex    To date, studies examining the role of dopamine release in the PFC are restricted to the mPFC/DLPFC in the rodent/primate.  Few have explored dopamine release in other parts  33 of the PFC, including the orbitofrontal region.   Both primate and rat PFC receives innervation from dopamine neurons of the VTA and the SNpc (Lindvall et al., 1974; Berger et al., 1976; Van Eden et al., 1987). The density of innervation is significantly less in the PFC compared to other regions such as the striatum (Andén et al., 1966; Abercrombie et al., 1989). Yet, significantly lower levels of dopamine terminal innervation do not necessarily indicate lower concentrations of dopamine. DAT, the main reuptake mechanism that clears dopamine from the synapse, has significantly lower expression in the PFC (Ciliax et al., 1995).  Therefore, instead of reuptake through DAT, approximately 50% of dopamine clearance is controlled by catechol-o-methyltransferase, while monoamine oxidase and the norephinephrine transporter contribute to the remainder (Karoum et al., 1994; Käenmäki et al. 2010; Móron et al., 2002). Compensatory changes in each mechanism’s contribution to dopamine clearance is observed if one is not available (e.g. through the use of knockout animals), or if dopamine concentrations are pharmacologically elevated (Tunbridge et al., 2004) illustrating that that clearance mechanisms work to maintain a balanced level of dopaminergic tone.  This cortex-specific combination of metabolism and reuptake allows dopamine to diffuse farther from its release site relative to the striatum and nucleus accumbens (Garris et al., 1993; Sesack et al., 1998a; Sesack et al., 1998b). This is one reason why dopamine's role in the cortex is not heavily dependent upon the frequency of VTA/SN firing (Garris and Wightman, 1994). Thus, unlike mesolimbic circuits where concentrations are tightly controlled by frequency-dependent release and highly efficient reuptake, dopamine in the PFC exhibits a modulatory ‘tone’ due to its unique diffusion properties (Seamans and Yang, 2004; Grace, 1991).      34 1.13 The role of NMDA receptors in the PFC    One way dopamine can influence decision-making and other cognitive tasks is by modulating glutamatergic synaptic transmission in the PFC. Electrophysiological experiments examining network activity are restricted to mPFC/DLPFC regions and have not yet been investigated in the OFC. Populations of neurons in the PFC form networks that respond to environmental or internal sensory perceptions can underlie several cognitive processes, including decision-making and working memory (Fuster et al., 2000; Wang, 2002). These networks can become more depolarized to form 'up’ states or cortical oscillations resulting from synchronous firing of pyramidal neurons (Sanchez-Vives and McCormick, 2000; Sohal, 2012). This persistent, self-regulating firing activity is thought to underlie working memory and cognitive control. While AMPA receptors can be important for sustaining cell firing induced by asynchronous activity or extra-network inputs, maintaining consistent depolarization and maintenance of up-states is highly dependent upon NMDARs (Wang, 1999).    1.13.1 Structure and function of NMDARs    NMDARs are non-specific cation channels that are activated by glutamate, with glycine as a co-agonist (Jahr and Stevens, 1987; Akaike et al., 1988; Cull-Candy and Usowicz, 1987; Johnson and Ascher, 1987).  They are composed of 4 individual subunits: 2 requisite GluN1 subunits (Behe et al., 1995) in conjunction with 2 other subunits, GluN2A-D and GluN3 that can exist in many different combinations (Monyer et al., 1994). NMDARs expressed in the PFC primarily contain GluN2A and GluN2B (Scherzer et al., 1998).  Unlike  35 other ionotropic glutamate receptors, NMDARs are voltage dependent, because magnesium ions block the channel pore at hyperpolarized potentials. This allows the receptors to act as 'coincidence detectors' and only open when there is concomitant presynaptic glutamate release and postsynaptic depolarization of the cell expressing the NMDARs (Nowak et al., 1984).  Thus, the unique biophysical properties and high levels of expression in pyramidal neurons make NMDARs ideal candidates for controlling the activity of specific populations of neurons in the PFC.    1.13.2 Modulation of PFC NMDARs during behavior     ‘Up’-states of pyramidal cell networks occur in many areas of the cerebral cortex. Yet, the PFC appears particularly well suited to this type of activity due to high levels of GluN2B containing NMDARs. The slow decay kinetics of GluN2B-containing NMDAR currents, which can be up to two-folds longer than other areas of the cortex, allows prefrontal cell networks to maintain stable firing activity (Wang, 1999; Wang et al., 2008). Therefore, blocking NMDARs in the PFC should suppress the firing of pyramidal neurons encoding information used to guide decision-making, leading to cognitive impairments. Intra-PFC administration of NMDAR, or GluN2B subunit antagonists produces deficits in working memory function and cognitive flexibility (Aura and Riekkinen, 1999; Stefani et al., 2003; Murphy et al., 2005). However, these manipulations are not specific to cell type, and likely cause cognitive impairment by increasing the firing of neurons, through potential inhibition of GABAergic interneurons (Jackson et al., 2004; Homayoun and Moghaddam, 2007).    Yet, more specific manipulations of GluN2B-containing NMDARs on putative pyramidal neurons (Wang and Gao, 2009) can enhance or inhibit cognitive performance by  36 overexpressing (Cui et al., 2011) or blocking (Dalton et al., 2011) these subunits, respectively. In another study, specific deletion of GluN2B-containing NMDA receptors in pyramidal neurons of the OFC impaired cognitive flexibility, as measured by reversal learning (Brigman et al., 2013).  Taken together, NMDA receptors expressed on pyramidal neurons from different regions of the PFC play an important role in executive functioning.   1.13.3  Effect of dopamine on NMDARs in the PFC      Most evidence describing dopaminergic effects on synaptic transmission are restricted to the mPFC in rodents.  Modulation of NMDAR-mediated excitatory synaptic transmission by dopamine in the mPFC is thought to underlie changes in cognition (Verma and Moghaddam, 1996; Nakako et al., 2013). Bidirectional modulation of NMDAR-mediated synaptic transmission are a function of dopamine concentration and the subsequent extent of D1 and D2 receptor activation (Zheng et al., 1999; Seamans et al., 2001a; Seamans et al., 2001b; Wang and O'Donnell, 2001; Gonzalez-Islas and Hablitz, 2003; Wang et al., 2003).   In the rodent mPFC, postsynaptic D1R stimulation in pyramidal neurons typically potentiates currents through NMDARs (Seamans et al., 2001a; Wang and O'Donnell, 2001). While the effects of D1R stimulation on glutamatergic synaptic transmission are typically regarded as postsynaptic, presynaptic changes have also been demonstrated.  Reductions in presynaptic glutamate release can also occur through stimulation of presynaptic D1 receptors located on pyramidal neurons (Law-Tho et al., 1994; Gao et al., 2001; Seamans et al., 2001a; Wang et al. 2002). D1 receptors inhibit high-voltage activated calcium channels, which prevents presynaptic calcium entry and ultimately neurotransmitter release . (Gao et al., 2001; Yang and Seamans, 1996; Wang et al. 2002; Seamans et al. 2001a). In addition, PKA  37 activation by D1 agonists also reduces sodium channel conductances in cultured neurons, without affecting voltage dependence or activation kinetics (Cantrell et al., 1997).  Although further investigation is required,  reductions in glutamate release through D1 receptor activation in the PFC might occur through one of these mechanisms.     Direct modulation of NMDARs typically occurs through activation of kinases, which can alter membrane trafficking of NMDAR subunits, or phosphorylate existing membrane-bound subunits (Swope et al., 1992; Wang et al., 2004). Activation of PKA, protein kinase C, calcium and calmodulin-dependent protein kinase II (CaMKII), and ERK1/2 (Gonzalez-Islas and Hablitz, 2003; Chen et al., 2004b; Tseng and O'Donnell, 2004; Kruse et al., 2009; Sarantis et al., 2009; Li et al., 2010) can all increase NMDA receptor function. Modulation of NMDARs can also occur through PKA-independent mechanisms.  For example, phosphorylation of tyrosine residues on GluN2B subunits is induced by D1R stimulation, and causes increased membrane expression of NMDARs on pyramidal neurons in the PFC (Gao and Wolf, 2008). In contrast, D2Rs decrease NMDAR currents in the mPFC, but this effect is not as well characterized as that for D1 receptors. One study demonstrated a PKA-independent role in the D2 receptor-induced suppression of NMDARs whereby internalization of GluN2B subunits occurs through activation of protein phosphatase 2A and glycogen synthase kinase 3β (Liu et al., 2009). Activation of D4 receptors decreases NMDARs by diminished PKA signaling that results in increased protein phosphatase 1, which inhibits CaMKII. This leads to endocytosis of NMDARs (Wang et al., 2003; Gao and Wolf, 2008). Thus, it is possible that dopamine receptor modulation of NMDARs can underlie the effect of dopamine on behaviors dependent on prefrontal function. While dopamine modulation of synaptic transmission of the rodent mPFC is well characterized,  38 there is a lack of knowledge of how dopamine can modulate NMDARs in the OFC, despite discrepancies in function, anatomical connections, and dopamine receptor expression exist between it and other prefrontal regions (Gaspar et al., 1995; McAlonan and Brown, 2003; Crombag et al., 2005; Homayoun and Moghaddam, 2006; Ragozzino, 2007; Lodge, 2011; Simon et al., 2011). Therefore, in Chapter 3 we demonstrate the mechanism by which selective activation of dopamine receptors can modulate NMDAR currents in the OFC.   1.14 Overview and objectives    Dopamine signaling in the brain is critical for guiding behavior related to rewarding or aversive outcomes. How dopamine is released may depend on synaptic transmission onto VTA dopamine neurons. Excitatory inputs from distal brain regions release glutamate onto these cells, increasing their excitability and subsequent amount of dopamine release into target regions.     Recent data illustrates that leptin signaling in the VTA can modify the motivation and preference for palatable foods, as well as other rewarding stimuli such as drugs of abuse (Hommel et al., 2006; Davis et al., 2011; Matheny et al., 2011).  Leptin signaling can also modulate the amount of dopamine release into brain regions critical for the expression of goal-directed behaviors (Roseberry et al., 2007; Brunetti et al., 1999; Fulton et al., 2006; Domingos et al., 2011). In addition, exogenous leptin reduces firing rate of VTA dopamine neurons (Hommel et al., 2006; Trinko et al., 2011).  In Chapter 2, we aim to determine the mechanism by which leptin can alter excitatory synaptic transmission onto VTA dopamine neurons.   39    Dopamine from the VTA is released into brain regions such as the orbitofrontal cortex. Through the use of behavioral neuropharmacology and microdialysis, dopamine signaling in this region has been shown to regulate decision-making and impulsivity (Winstanley et al., 2006; Winstanley et al., 2010; Zeeb et al., 2010; Calaminus and Hauber, 2008; Winter et al., 2009; Mizoguchi et al., 2010). Whereas the effects of dopamine in other prefrontal subregions have been well-characterized at both the behavioral and synaptic level, we do not yet know how dopamine modulates neurons in the OFC.  In Chapter 3, we characterize the role of dopamine receptor agonists on NMDA receptor currents in pyramidal neurons in the OFC.  Because the juvenile and adult OFC have differential expression of dopamine receptors and innervation of terminals, we investigate if dopamine modulates NMDA receptor currents differentially in juvenile versus adult pyramidal neurons.             40 Chapter 2: Presynaptic leptin action suppresses excitatory synaptic transmission onto ventral tegmental area dopamine neurons  2.1 Introduction    Leptin is a cytokine released from adipocytes that circulates throughout the CNS to convey status of body energy stores and to control feeding and energy expenditure.  Leptin readily crosses the blood brain barrier through a saturable transport system (Banks et al., 1996; Elmquist et al., 1998; Oswal and Yeo, 2010) and binds to LepRbs in many brain regions (Elmquist et al., 1998; Munzberg and Myers, 2005) including the VTA (Figlewicz et al., 2003; Fulton et al., 2006; Hommel et al., 2006; Leshan et al., 2010).  VTA dopamine neurons and their widespread neural targets in the striatum, amygdala, PFC, and elsewhere mediate the incentive salience of food and other rewards (Berridge, 2012).  Leptin signaling in the VTA may be one mechanism used to alter the motivation to consume food or drugs of abuse.  Indeed, intra-VTA leptin decreases consumption of palatable food (Hommel et al., 2006) and LepRb knockdown in the VTA increases effort to obtain palatable food reinforcers (Davis et al., 2011). Systemic leptin reduces the rewarding value of sucrose that is dependent on dopamine neuron activation (Domingos et al., 2011).  Taken together, a wealth of evidence suggests that leptin signaling in the mesolimbic dopamine circuit can modulate natural and drug reward-seeking behaviors (Narayanan et al., 2010).     So far, there is little understanding of the mechanism behind leptin modulation of these effects. Supraphysiological doses of systemic leptin decreases firing rate of putative VTA dopamine neurons in anesthetized rats (Hommel et al., 2006). Furthermore, bath  41 application of leptin to midbrain slices decreases firing rate of putative dopamine neurons by approximately 20% (Hommel et al., 2006) in an ERK-dependent manner (Trinko et al., 2011).  Leptin-induced decrease in neuronal firing may be due to a change in intrinsic properties of dopamine neurons or a result of altered pre- or post-synaptic effects of glutamate or GABA onto dopamine neurons. However, it is unknown how leptin modulates excitatory or inhibitory synaptic transmission onto VTA dopamine neurons. Here, we test the hypothesis that leptin modulates excitatory synaptic transmission onto dopamine neurons.    Glutamatergic synapses onto VTA dopamine neurons can undergo both LTP and LTD to regulate their synaptic strength and consequent dopaminergic output (Bonci and Malenka, 1999; Overton et al., 1999; Chergui et al., 1994; Overton and Clark, 1997; Komendantov et al., 2004 ).  Enhanced synaptic efficacy onto dopamine neurons has not only been linked to exposure to drugs of abuse, but to feeding-related peptides, such as hypocretin/orexin (Borgland et al., 2006; Borgland et al., 2009) and ghrelin (Abizaid et al., 2006).  Potentiation of synaptic strength onto dopamine neurons by feeding-promoting peptides may underlie motivation to obtain food (Borgland et al., 2009) (Thompson and Borgland, 2011; Blum et al., 2009).  Therefore, modulation of excitatory inputs by anorectic feeding peptides such as leptin could be particularly important in decreasing dopamine neuron firing activity and ultimately dopamine release.    Leptin modulation of glutamatergic synaptic transmission in other brain regions has been demonstrated in the hippocampus (Moult et al., 2009; Durakoglugil et al., 2005; Shanley et al., 2001), hypothalamus (Glaum et al., 1996), and cerebellum (Irving  42 et al., 2006).  Thus, we hypothesized that leptin action in the VTA can modulate glutamatergic synaptic transmission onto VTA dopamine neurons.  Here, we used whole-cell patch clamp electrophysiology in VTA brain slices to determine the effect of leptin on excitatory synaptic transmission.  2.2 Methods  2.2.1 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 British Columbia Animal Care Committee. Male C57Bl/6 mice (p21-25; University of BritishColumbia) were housed in groups or 3-5.  For some experiments, pituitary homeobox 3-green fluorescent protein (Pitx3-GFP) knock-in mice (p21-25; bred in-house) were used to easily identify VTA dopaminergic neurons, as pitx3 is a transcription factor necessary for the development of midbrain dopamine neurons (Zhao et al., 2004).  Data from C57Bl/6 mice and Pitx3-GFP mice were not significantly different and therefore grouped together. Mice were maintained on a 12 h light:dark schedule (lights on at 7:00 am), and given food and water ad libitum. All experiments were performed during the animals light cycle.  2.2.2 Slice preparation  Briefly, animals were deeply anaesthetized with isoflurane, decapitated, and brains rapidly extracted into ice-cold sucrose solution containing (in mM):  75 sucrose,  43 87 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 7 MgCl2, 0.95 CaCl2, 1-1.5 ascorbic acid. Horizontal brain sections containing the VTA were cut at 250 µm on a vibratome (Leica, Nussloch, Germany).  Slices were transferred to 250 mL bicarbonate-buffered artificial cerebrospinal fluid (aCSF) containing (in mM) 126 NaCl, 1.6 KCl, 1.1 NaH2PO4, 1.4 MgCl2, 26 NaHCO3, 11 glucose, 2.4 CaCl2, and incubated for a minimum of 45 minutes at 31.4-33˚C prior to recording.  All solutions were continuously saturated with 95% O2/5% CO2.  2.2.3 Electrophysiology   Slices were placed in the recording chamber and perfused with aCSF with the addition of picrotoxin (100 µM) to block GABAA receptor-mediated inhibitory postsynaptic currents.  Cells were visualized on an upright microscope using ‘Dodt-type’ gradient contrast infrared optics (Dodt et al., 2002).  Whole-cell voltage clamp recordings were made using a Multiclamp 700B amplifier (Molecular devices, Union City, California). Recording electrodes (3-5 MΩ) were filled with (in mM): 120 cesium methanesulfonate, 20 HEPES, 0.4 EGTA, 5 TEA-Cl, 2 MgCl2, 2.5 MgATP, 0.3 NaGTP, with a pH of 7.2-7.3 and 273-285 mOsm. Putative dopaminergic VTA neurons were identified by the presence of a large hyperpolarization-activated, cyclic nucleotide-regulated cation (Ih) current (Grace and Onn, 1989; Lacey et al., 1990; Johnson and North, 1992; Cameron et al., 1997; Ford et al., 2006).  A subset of C57Bl/6 neurons was identified by biocytin (0.2%) filling and post hoc staining for TH; see below.  Of the 11 biocytin-filled neurons expressing Ih, only 2 did not express TH and were excluded from the analysis. Dopamine neurons expressing Ih from Pitx3-GFP mice were identified by  44 GFP fluorescence.  Of 15 VTA dopamine neurons recorded from Pitx3-GFP mice, 3 cells were used for the experiments with leptin on AMPAR EPSCs, 2 cells with wortmannin in the internal solution, 2 cells were used with wortmannin in the external solution, 1 cell for the paired pulse ratio, 3 cells for the mEPSCs with leptin, and 3 cells for the mEPSCs with vehicle. To stimulate local presynaptic terminals, a bipolar stimulating electrode was placed 100-300 µm rostral to the recording electrode and was used to stimulate excitatory afferents at 0.1 Hz. Neurons were voltage-clamped at –70 mV and +40 mV to record AMPAR- and NMDAR-mediated EPSCs, respectively.  EPSCs were filtered at 2 kHz, digitized at 5-10 kHz and collected on-line using pClamp10 software (Molecular Devices, CA). Series resistance (6-30 MΩ) and input resistance were monitored on-line with a 5-mV depolarizing step (50 ms) given just after every afferent stimulus.   Paired-pulse ratio was elicited by evoking 2 EPSCs with an interval of 50 ms. Miniature EPSCs (mEPSCs) were recorded in the presence tetrodotoxin (500 nM) to block action potentials driven by spontaneous events as well as picrotoxin (100 µM) and AP-5 (50 µM).  For these experiments, mEPSCs were recorded before and 15 min after a 15-min application of leptin (100 nM).  mEPSCs were selected based on their amplitude (>10 pA), decay time (<3 ms) and rise time (<3 ms) using the MiniAnalysis60 program (Synaptosoft). Average noise levels were 4 ± 0.2 pA. Example traces for evoked EPSCs were constructed from the averaged trace of 12 sweeps (2 minutes).     45 2.2.4 Drugs    Recombinant mouse leptin (R&D systems, Minneapolis), was reconstituted in PBS (pH 8.0-8.2) and stored at -80° C.  Leptin was bath applied at 100 nM concentrations for 15 minutes in all experiments.  Picrotoxin (Sigma, St. Louis) was dissolved in H20 and was present in the external solution throughout all recordings. U0126 (Promega, Madison), wortmannin (Tocris, Ellisville), or AG490 (Calbiochem, Billerica) were dissolved in DMSO and used at 1/1000 working concentrations. AG490 was protected from light throughout experiments. Signal transduction inhibitors were pre- incubated with slices for a minimum of 20 minutes prior to recording, and were also present throughout the experiment.  In some experiments wortmannin (100 nM) or AG490 (50 µM) was included in the patch pipette.  2.2.5 Statistical analysis    All data expressed as mean percent change in baseline levels ± s.e.m.  To determine statistical significance for time-course data, a section of the baseline (average of 5 minutes [30 sweeps] at minutes 4 through 8) was compared to the effects at of leptin (average of 5 minutes [30 sweeps] at minutes 19 through 23) using a paired t-test. Paired-pulse ratios were calculated by averaging the peak amplitude of 2 evoked currents throughout the 10 minute baseline period. The mean value of the second peak was then divided by the first to obtain the paired-pulse ratio. This was also calculated during leptin application (approximately minutes 12 through 22). Differences between paired-pulse ratios during baseline and leptin application were then analyzed by a paired t-test.  A two-way, repeated measures ANOVA with planned comparisons (using Bonferroni posthoc tests) was used  46 to analyze mini EPSCs.  “N” refers to the number of cells recorded from at least three rats expressed as (n = cells/rats).  2.2.6 Tyrosine hydroxylase immunocytochemistry    Brain slices from patch-clamp recording were fixed overnight in cold 4% paraformaldehyde, rinsed in PBS, blocked in 10% normal donkey serum, incubated with monoclonal mouse anti-TH antibody (Sigma, 1:1000) for 48 hours at 4oC.   DyLight 594 streptavidin (Jackson Labs; 1:200) was applied overnight at 4oC.  Secondary donkey anti- mouse FITC antibody (1:50) was applied for two hours at 4oC.  Slices were mounted using Fluoromount (Sigma, St. Louis).  2.3 Results 2.3.1 Leptin suppresses excitatory synaptic transmission onto VTA dopamine neurons  AMPAR EPSCs were recorded from VTA dopamine neurons of mouse midbrain slices voltage-clamped at -70 mV. Bath application of leptin for 15 min caused a long- lasting depression of AMPAR EPSCs (16 ± 5% reduction from baseline; n=9/8,             p < 0.05, paired t-test, t=3.4, df=8; Figure 1A, B).  Leptin depressed AMPAR EPSCs in all 5 neurons that were confirmed to be positive for TH in post hoc staining. To measure NMDAR EPSCs, neurons were voltage clamped at +40 mV and measurements were taken 20 ms after the stimulus artefact, a time point at which the glutamatergic EPSC is mediated primarily by NMDARs (Bonci and Malenka, 1999). Leptin caused a depression of NMDAR EPSCs (17 ± 6 % reduction from baseline; n=7/5, p < 0.05,  47 paired t-test, t=2.7, df=6; Figure 1C, D). The effects of leptin on AMPAR or NMDAR-mediated EPSCs were not significantly different (p > 0.05, paired t-test t=0.12 df=14). Furthermore, of the 9 biocytin-labeled neurons included in the analysis, leptin depressed AMPAR EPSCs in 5 neurons and NMDAR in 4 neurons expressing TH, suggesting that leptin inhibition of glutamatergic synaptic transmission can occur onto VTA dopamine neurons.  Because leptin suppressed AMPAR or NMDARs with equal magnitude, leptin may be decreasing glutamate release. Therefore, to investigate whether leptin altered the number and/or function of postsynaptic AMPARs or caused a presynaptic inhibition of glutamate release, we measured AMPAR-mediated mEPSCs, a standard method for determining the locus of synaptic change (Ford et al., 2006). Analysis of mEPSC frequency at baseline and 15-18 minutes after bath application of leptin (100 nM, 15 min) or vehicle revealed a significant time x treatment interaction (F(1,22)=6.2, p<0.05, 2-way ANOVA; Fig 2A, C, D). Post hoc tests revealed that mEPSC frequency was significantly reduced (1.2 ± 0.3 Hz) compared to baseline mEPSCs (1.9 ± 0.4 Hz; n=12/8, p < 0.05, t=3.5, df=1; Fig 2A,C,D). This effect was not due to rundown over time as there was no significant difference between the baseline and application of vehicle (baseline:1.7 ± 0.3 Hz vs. vehicle: 1.7 ± 0.3 Hz, n=12/7, p > 0.05, t=0.05, df=1; Fig 2A, C, D). Leptin did not alter mEPSC amplitude (F(1,22)=0.03, p >0.05, 2-way  ANOVA; Fig 2B), with either leptin (baseline: 16.6 ± 0.9 pA vs. leptin: 16.0 ± 0.9 pA; n=12/8, p>0.05, t=1.9 df=1) or vehicle application (baseline: 18.6 ± 1.1 pA vs. vehicle: 17.9 ± 0.9 pA; n=12/7, p > 0.05, t=2.1, df=1; Fig 2B,C,E).   48  A reduction of mEPSCs frequency concomitant with unchanged amplitude likely reflects a decrease in the probability of presynaptic neurotransmitter release (Katz, 1971).  To further verify if leptin-mediated depression of AMPARs was mediated pre- or postsynaptically, we examined the effects of leptin on the probability of transmitter release, comparing the response to paired pulses, a measure that changes in a highly predictable fashion with release probability.  We recorded AMPAR EPSCs at –70 mV using a paired-pulse stimulation protocol with a 50-ms interval and observed significant paired pulse facilitation after leptin bath application (baseline: 0.85 ± 0.06 vs leptin 0.92 ± 0.05; n =7/6, p < 0.05, paired t-test, t=3.2, df=6; Fig 2F,G). Taken together, these data suggest that leptin-induced synaptic depression is consistent with a reduction in the probability of presynaptic glutamate release.  2.3.2 Leptin-induced synaptic depression occurs by activation of the JAK2 - PI3K pathway  LepRb receptor activation triggers 3 main signal transduction cascades upon association with JAK2 tyrosine kinase. First, Tyr(985) of LepRb recruits SH2-containing tyrosine phosphatase (SHP-2) resulting in ERK activation. Secondly, Tyr(1138) of LepRb recruits STAT3; and thirdly, tyrosine phosphorylation sites on the receptor-associated JAK2 leads to activation of PI3K (Oswal and Yeo, 2010). To determine if JAK2 signaling was required for leptin-induced synaptic depression, we bath applied the JAK2 inhibitor, AG-490 (50 µM (Jo et al., 2005)) to midbrain slices. Bath application of AG-490 significantly inhibited leptin-induced depression of AMPAR EPSCs (1.6 ± 6% reduction from baseline; n=7/6, p>0.05, paired t-test, t=0.26, df=6; Fig 3A, B).   49   Reductions in synaptic transmission can occur through retrograde signaling via endocannabinoids or nitric oxide, which are produced in the postsynaptic cell, but result in a depression of presynaptic neurotransmitter release (Melis et al., 2004; Gerdeman et al. 2002; Nugent and Kauer, 2008). Therefore, leptin-induced synaptic depression may be due to activation of postsynaptic leptin receptors on dopamine neurons leading to presynaptic inhibition of glutamate release via retrograde mediators. Another possibility is that activation of presynaptic LepRbs induces a suppression of glutamate release. To investigate if postsynaptic receptor activation was required for leptin-induced synaptic depression, we applied  AG490 via the patch pipette. Leptin-induced a significantsynaptic depression of AMPAR- mediated synaptic transmission with intracellular inhibition of JAK2 in dopamine neurons (23 ± 8 % reduction from baseline; n=5/3, paired t-test, t=2.8, df=4; p < 0.05 Fig 3C, D).  To test the involvement of the ERK pathway, we preincubated slices with U0126 (10 µM; (Trinko et al., 2011)) a MEK2 inhibitor upstream of ERK activation. In the presence of U0126, leptin significantly depressed AMPAR-mediated EPSCs (21 ± 6% reduction from baseline,  n = 5/4, p < 0.05, t=3.4, df=4; Fig 4A, B). These data indicate that unlike leptin suppression of firing rate (Trinko et al., 2011), leptin-induced synaptic depression occurs through a different signaling pathway.  We next tested the involvement of the JAK2 /PI3K pathway using the PI3K inhibitor, wortmannin.  Extracellular application of wortmannin (100 nM, (Hommel et al., 2006; Trinko et al., 2011)) abolished the leptin-induced synaptic depression 1 ± 6 % reduction from baseline; n=7/4, p > 0.05, paired t-test, t=0.16, df=6; Fig 4C, D).  Furthermore, with only postsynaptic inhibition of PI3K, leptin significantly depressed AMPAR EPSCs (14± 5 % reduction from baseline; n  50 = 7/5, p < 0.05, paired t-test, 3.0, df=6; Fig 4E, F). These data suggest that leptin likely activates presynaptic LepRbs to inhibit glutamate release and that leptin activation of JAK2-PI3K is necessary to induce a reduction in synaptic efficacy of VTA dopamine neuron inputs.  2.4 Discussion   The data presented here establish a novel mechanism for leptin action in the mesolimbic incentive motivation/reward system. Leptin reduced both NMDAR and AMPAR-mediated synaptic transmission onto dopamine neurons by reducing glutamate release from presynaptic terminals.  This effect was mediated by presynaptic Jak2 and PI3K signaling, suggesting that leptin is not directly acting on dopamine neurons to induce synaptic inhibition. A model of leptin inhibition of excitatory synaptic transmission onto VTA dopamine neurons is illustrated in Figure 5.   2.4.1 Mechanisms behind reductions in food intake    Previous studies have implicated leptin action in the VTA in reducing food intake (Hommel et al., 2006; Morton et al., 2009; Bruijnzeel et al., 2011). Interestingly, Morton et al. (Morton et al., 2009) demonstrated that leptin’s effect on food intake was not sensitive to PI3K inhibition, suggesting that our results may dissociate from the effects of leptin on food intake. To inhibit PI3K, this study used a concentration of LY-294002 (2 mM) intra-VTA which may have several off-target effects including action at several ion channels (Welling  51 et al., 2005; Sun et al., 2004; Wu et al., 2009), phosphodiesterases (Abbott and Thompson, 2004) and the estrogen receptor (Pasapera Limón et al., 2003), potentially confounding the feeding results. Furthermore, it is important to consider that leptin acting on GABAergic inputs to VTA neurons (excluded in our study) may play a role in feeding. Indeed, leptin potentiates inhibitory synaptic transmission in the hippocampus (Solovyova et al., 2009).  Future studies will be aimed to directly test leptin modulation of GABAergic inputs to VTA dopamine neurons.  Interestingly, knocking down LepRb expression within the VTA (using shRNA) or knocking out LepRb on VTA dopamine neurons yielded differential effects with regards to feeding. While neither method alters body weight, shRNA to LepRb in VTA increased consumption of high fat food and increased the work required to obtain sucrose (Hommel et al., 2006; Davis et al., 2011).  In contrast, there was no difference in regular or high fat food intake in mice with a conditional LepRb knockout in dopamine transporter expressing neurons compared to wildtype mice (Liu et al., 2011). Possible discrepancies between these studies may be because LepRb is removed only from dopamine neurons of the substantia nigra and VTA in the conditional knockout mouse, whereas the LepRb shRNA would target dopamine as well as GABAergic neurons only in the VTA.  Furthermore, the conditional mouse is missing LepRbs throughout development.    2.4.2 Role of leptin in reward-seeking    Several lines of evidence suggest that leptin modulates reward-seeking behavior. Intra-VTA leptin reduced the reward value of sucrose via a reduction in dopamine neuronal activity (Domingos et al., 2011), intra-VTA or i.c.v. leptin suppressed the rewarding effects  52 of  intracranial self-stimulation (Bruijnzeel et al., 2011), i.c.v. leptin attenuates acute food deprivation-induced relapse to drug seeking (Shalev et al., 2001)  and reverses conditioned place preference for sucrose (Figlewicz et al., 2001) or high fat foods (Bruijnzeel et al., 2011).  Furthermore, firing rate of putative dopamine neurons is decreased after i.v. leptin administration or bath application of leptin to midbrain slices (Hommel et al., 2006; Trinko et al., 2011).  Studies using leptin deficient ob/obmice have provided conflicting results. Fulton et al. (Fulton et al., 2006) reported that the baseline and electrically evoked release of dopamine in the nucleus accumbens was significantly reduced in ob/ob mice. In contrast, Rosenberry et al. (Roseberry et al., 2007) demonstrated that basal or cocaine-induced dopamine levels in the nucleus accumbens were increased in ob/ob mice when measured via microdialysis. This discrepancy is likely due to technical differences such as the use of in vivo microdialysis (Roseberry et al., 2007) compared to amperometry in forebrain slices (Fulton et al., 2006). Amphetamine-induced locomotion and sensitization was reduced in ob/ob mice compared to wildtype mice (Fulton et al., 2006). However, basal locomotor activity is lower in ob/ob mice compared to wildtype mice potentially confounding these results (Dauncey, 1986; Calcagnetti et al., 1987; Dauncey and Brown, 1987; Collin et al., 2000; Roseberry et al., 2007). Taken together, these studies suggest that leptin in the VTA reduces activity of dopamine neurons and modulates reward seeking behavior.  Our findings that leptin reduces excitatory synaptic transmission onto dopamine neurons are consistent with these studies, and suggest that in the constitutive presence of leptin, selective glutamatergic afferents to the VTA are tonically dampened.    53 2.4.3 Modulation of synaptic transmission   Leptin can modulate excitatory synaptic transmission in other brain regions.  For example, mice with mutations in LepRb (db/db mice or fa/fa rats) show a disrupted ability of hippocampal neurons to undergo both LTP and LTD (Li et al., 2002).  Leptin bath applied to hippocampal slices facilitates LTP (Wayner et al., 2004), likely due to leptin-induced forward trafficking on NMDARs to the synapse (Shanley et al., 2001).  Additionally, leptin is reported to preferentially enhance GluN2B-mediated NMDAR responses in cerebellar granule cells (Irving et al., 2006).   Thus, unlike postsynaptically mediated alteration of glutamatergic synapses in the hippocampus or cerebellum described above, leptin action in the VTA caused a presynaptic suppression of glutamate release.  Interestingly, leptin-induced synaptic depression was not due to a direct effect of leptin on dopamine neurons. Several lines of evidence support our conclusion that depression of AMPAR-mediated EPSCs by leptin was mediated presynaptically.  First, leptin depressed NMDAR-mediated EPSCs with a similar time course and maximum to that of AMPAR EPSCs, suggesting reduced availability of synaptic glutamate to activate either glutamate receptor subtype as opposed to a postsynaptic decrease in number or function of both receptors.  Secondly, decreased mEPSC frequency concomitant with unchanged mEPSC amplitude suggests that leptin reduced synaptic glutamate without affecting the number or function of AMPARs on dopaminergic neurons. Thirdly, leptin induced paired-pulse facilitation suggesting a decreased release probability. Finally, while postsynaptic inhibition of Jak2 or PI3K did not alter leptin-induced synaptic depression, bath application of AG490 or wortmannin abolished the effect of leptin, suggesting that LepRb activation does not occur directly on dopamine neurons.   54 2.4.4 LepRb expression in VTA    Several studies have demonstrated that there is high LepRb expression in the VTA using immunohistochemistry to LepRb protein in rats (Figlewicz et al., 2004); fluorescent in situ hybridization to LepRb mRNA colocalized with TH mRNA in rats (Hommel et al., 2006) and immunohistochemistry to leptin activated pSTAT3 colocalization with TH in mice (Fulton et al., 2006). However, in mice expressing EGFP in LepRb-containing neurons, only ~6% overlapped with TH expression in the VTA (Leshan et al., 2010).  Low promoter activity driving EGFP expression in the LepRb EGFP mice may account for some of this discrepancy, however it is clear that LepRb expression on dopamine neurons is less than originally described. Our results are consistent with the idea that leptin control of the mesolimbic dopamine system occurs indirectly.     Leptin acting at LepRb expressed on presynaptic glutamatergic terminals in the VTA may be one mechanism by which leptin inhibits excitatory synaptic transmission. Terminals expressing LepRb EGFP in the VTA include those from the lateral hypothalamus (LH), periaqueductal grey area (PAG) and hypothalamic preoptic area (Leshan et al., 2010).  While POA neurons are mainly inhibitory (Maeda and Mogenson, 1980; Luo and Aston-Jones, 2009), PAG neurons provide glutamatergic input to the VTA (Omelchenko and Sesack, 2010). Interestingly, activation of LepRb-expressing neurons in the LH modulates dopamine production and content in the VTA contributing to a decrease in incentive value of food (Leinninger et al., 2009). However, LepRb expression was mainly on LH GABAergic neurons. While it is possible that LepRb-containing LH  55 GABAergic terminals within the VTA synapse onto glutamatergic afferents to control glutamate release, this effect is unlikely to occur in our slices due to the presence of picrotoxin in all extracellular solutions.  Another possibility is that leptin activates LepRb on somata outside the VTA in our horizontal midbrain slices. These slices contain a significant portion of the posterior hypothalamic area, a region with dense LepRb expression (Elmquist et al., 1998). Therefore, it is feasible that leptin could activate receptors on cell bodies in these regions that could project directly or indirectly to the VTA thus inhibiting glutamate release. It should be noted that differences in leptin signaling and receptor expression levels might differ between young mice used in this study and adult animals used in other studies. However, location of receptor expression is not expected to change with age. Furthermore, others have reported consistent effects of leptin in the VTA of adult and 3 week old mice (Trinko et al., 2011).  Taken together, leptin likely activates LepRbs on glutamatergic inputs directly or indirectly projecting to the VTA.     2.4.5 Diversity of signaling pathways mediating changes in VTA neurons    Interestingly, activation of PI3K, but not ERK was required for leptin-induced synaptic depression in the VTA. Leptin-induced activation of Jak2 recruits insulin receptor substrate-1 (IRS-1) leading to PI3K activation. Inhibition of PI3K by the potent inhibitor wortmannin blocked leptin-induced synaptic depression only when applied in the bath solution. In hippocampal cultures, leptin caused a PI3K-dependent forward trafficking of postsynaptic GluA1 subunits (Moult et al., 2010).  This discrepancy is likely because in VTA, leptin inhibition of excitatory synaptic transmission is mediated presynaptically.   56 Leptin-induced inhibition of firing of VTA neurons was mediated by ERK signaling as bath application of the MEK inhibitor, U0126 abolished leptin-induced suppression of firing rate (Trinko et al., 2011). While these experiments did not test if leptin-induced suppression of firing was mediated by direct or indirect depolarization of dopamine neurons, it does suggest that leptin-modulation of firing rate is mediated by an independent mechanism to leptin-induced synaptic inhibition.   2.4.6 Upregulation of excitatory amino acid transporters    One interesting hypothesis for the mechanism underlying a leptin-induced synaptic inhibition is that leptin may increase glutamate clearance from the synapse by upregulating transporter function. Recent evidence demonstrated that leptin activation of Jak2 led to upregulation of Na+/K+-dependent excitatory amino acid transporters (EAATs), particularly EAAT2 and EAAT4 (Hosseinzadeh et al., 2011).  Consistent with this, PI3K/Akt signaling has been implicated in forward trafficking of both EAAT2 and EAAT4 (Bohmer et al., 2004; Wu et al., 2010).  Interestingly, in addition to astrocytic EAAT4 expression, EAAT4 is densely expressed on neurons within the VTA (Massie et al., 2008). In our experiments, however, the reduction in evoked EPSCs occurred more quickly than what is typically oberved for upregulation of EAATs. In addition, it is unlikely that glutamate transporters are efficient enough to clear glutamate from the synapse before binding postsynaptic receptors, which would be necessary to explain reduction in the frequency of mini EPSCs (Wu et al., 2010). Therefore, our data suggest that leptin reduces  57 the probability of glutamate release from terminals, rather than modulating glutamate clearance via upregulation of transporters.    2.4.7 Summary and conclusions   Increased excitatory synaptic transmission onto dopamine neurons is associated with learning of cues predicting food reward (Stuber et al., 2008b). A reduction in excitatory synaptic transmission may raise the threshold required for burst firing of dopamine neurons (Overton and Clark, 1997). Because burst firing has been implicated in promoting dopamine release and driving salience of food related cues, a reduction of salience to food-related cues may be a functional consequence of leptin-induced inhibition of glutamate release. This idea is consistent with studies demonstrating a leptin-induced reduction in hedonic food intake (Domingos et al., 2011) and reduced motivation to obtain food (Davis et al., 2011).  Because leptin’s action was selective to presynaptic glutamatergic inputs to the VTA, it is possible that circulating leptin may tonically dampen select inputs to the VTA, thereby reducing their influence to promote burst firing of dopamine neurons. Fasting decreases circulating leptin (Weigle et al., 1997), therefore one can speculate that tonic suppression of glutamatergic inputs to the VTA would be relieved in this situation resulting in heightened salience to food-related cues.     Here, we propose a novel mechanism for leptin signaling in the VTA, which is, to our knowledge, the first report of a presynaptic inhibition of excitatory synaptic transmission induced by leptin.  Leptin-induced synaptic inhibition in the VTA is dependent on activation  58 of PI3K of non-dopaminergic neurons.  Taken together, this study provides new insight for how leptin modulates the mesolimbic dopamine system to suppress reward-seeking behaviors.                      59 Chapter 3: Activation of PLC-coupled dopamine receptors in the lateral orbitofrontal cortex alleviates cognitive impairment and potentiates excitatory synaptic transmission in juvenile rats  3.1 Introduction    The OFC is implicated in associative learning and cognitive flexibility. OFC neurons hold value-related information ‘online’ to guide future decision-making, and thus estimate the likelihood of specific outcomes to guide future responses (Schoenbaum et al., 2011). Dopaminergic signaling in the OFC has been implicated in various behaviors including impulsivity, risky decision-making, reversal-learning and responding on progressive ratio schedules of reinforcement (Winstanley et al., 2005; Zeeb et al., 2010; Cetin et al., 2004; Stopper et al. 2014 ).  OFC neurons receive dopaminergic inputs from the midbrain and express both excitatory Gαs coupled dopamine D1Rs and inhibitory Gαi/o coupled D2Rs (Simon et al., 2011; Berger et al., 1976).      While some studies to date have assessed the effect of dopamine on OFC function in adults, little is known about dopamine signaling in the OFC of prepubescent juvenile rodents, despite prevalent administration of monoaminergic drugs for treatment of ADHD in children (McCarthy et al., 2012; (Brinker et al., 2007),.  Indeed, very few studies have demonstrated differential effects of monoaminergic drugs on impulsivity, a hallmark symptom of ADHD, in juvenile vs. adult rodents (Bizot et al., 2007; Garske et al., 2013) and none have explored the mechanism behind these dopamine-mediated effects in the OFC.  The density of dopaminergic innervation to the PFC increases during development and adolescence until approximately postnatal day 60 (P60) (Kalsbeek et al., 1988). Parallel increases in D1R and  60 D2R expression across development have also been demonstrated (Garske et al., 2013; Tarazi and Baldessarini, 2000).  Therefore, we hypothesize that there are age-dependent differences in dopaminergic signalling in the OFC.     In the PFC, activation of NMDARs underlies the sustained firing of prefrontal pyramidal cell networks upon cessation of external inputs, which likely promotes efficient prefrontal function to drive decision-making (O'Donnell, 2003).  In the medial prefrontal cortex (mPFC), dopamine can modulate this network activity (Durstewitz et al., 2000). In experiments from prepubescent mPFC brain slices, dopamine D1Rs typically enhance NMDAR responses, while D2Rs attenuate both AMPAR and NMDAR-mediated responses (Seamans et al., 2001a; Wang and O'Donnell, 2001; Gonzalez-Islas and Hablitz, 2003; Chen et al., 2004a; Wirkner et al., 2004; Beazely et al., 2006). D1R agonists enhance cellular excitability in mPFC slices from adult rats, whereas D2R agonists reduce pyramidal cell excitability (Tseng and O'Donnell, 2004). Dopamine modulation of mPFC neurons has been well characterized. However, no studies to date have explored how dopamine signaling alters excitatory synaptic transmission in the OFC. Given that mPFC and OFC functions are clearly dissociated (Buckley et al., 2009; McAlonan and Brown, 2003; St Onge and Floresco, 2010) and exhibit differential responses to administration of psychostimulants (Crombag et al., 2005; Homayoun and Moghaddam, 2006), a clearer understanding of how dopamine modulates synaptic transmission in the OFC of juvenile or adult animals is warranted.  Superficial layers of rat PFC receive dopaminergic input (Séguéla et al., 1988; Lindvall et al., 1974), therefore we have examined the effects of D1R or D2R agonists on NMDAR- 61 mediated EPSCs in layer II–III pyramidal cells from distinct OFC subregions of juvenile and adult rats.    3.2 Methods 3.2.1 Subjects   All animals were juvenile (P21-30) or adult male wistar rats (P60-70), provided by Charles River and housed in groups of 2-6.  Rats were maintained on a 12:12 hour light:dark schedule (lights on at 7:00am), and given food and water ad libitum, with the exception of during behavioral experiments.  All experimental protocols were in accordance with the Canadian Council on Animal Care.   3.2.2 Slice preparation   Rats were anesthetized with isoflurane, decapitated, and brains were rapidly extracted into ice-cold sucrose solution containing (in mM):  75 sucrose, 87 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 7 MgCl2, 0.95 CaCl2, 1-1.5 ascorbic acid. Sagittal brain sections containing lateral, medial, or ventral OFC (Paxinos and Watson, 2007) were cut at 300 µm on a vibratome (Leica, Nussloch, Germany).  Slices were transferred to 250 mL aCSF containing (in mM) 126 NaCl, 1.6 KCl, 1.1 NaH2PO4, 1.4 MgCl2, 26 NaHCO3, 11 glucose, 2.4 CaCl2, and incubated for a minimum of 60 minutes at 31.4-33˚C prior to recording.  All solutions were continuously saturated with 95% O2/5% CO2.    62 3.2.3 Electrophysiology    Slices were placed in the recording chamber and perfused with aCSF with the addition of picrotoxin (100 µM) to block GABAA receptor-mediated inhibitory postsynaptic currents.  Cells were visualized on an upright microscope using ‘Dodt-type’ gradient contrast infrared optics (Dodt et al., 2002).  Whole-cell voltage clamp recordings were made using a Multiclamp 700B amplifier (Molecular devices, Union City, California).  Recording electrodes (3-5 MΩ) were filled with (in mM): 120 cesium methanesulfonate, 20 HEPES, 0.4 EGTA, 5 TEA-Cl, 2 MgCl2, 2.5 MgATP, 0.5 NaGTP, and 5.4 biocytin (in some experiments), with a pH of 7.2-7.3 and 280-295 mOsm.  To stimulate local presynaptic terminals, a tungsten bipolar stimulating electrode was placed 100-300 µm adjacent to the recording electrode and used to evoke EPSCs at 0.1 Hz. Series resistance (6-20 MΩ) and input resistance were monitored on-line with a 5-mV depolarizing step (50 ms) given just after every afferent stimulus. Recordings exhibiting a >20% change in series resistance were discarded.  Cells were held at +40 mV and peak amplitudes of NMDAR currents measured 20 ms after the stimulation artefact, a time point where AMPAR currents have decayed. In experiments measuring AMPAR-mediated EPSCs, cells were voltage clamped at -70 mV.  EPSCs were filtered at 2 kHz, digitized at 10 kHz and collected on-line using pCLAMP 10 software.   3.2.4 Immunohistochemistry   Immediately after recording, some slices were fixed in 4% PFA for 24-48 hours, then transferred to phosphate buffered saline (PBS; pH 7.3). Slices were then washed at 4 °C in  63 0.2% Triton and 0.2% bovine serum albumin dissolved in PBS.  Cells were labeled with streptavidin-conjugated Texas Red at a concentration of 6.5 µL/mL and incubated overnight at 4 °C. Cells were visualized on an upright fluorescent microscope at 100 and 400x magnification to confirm location and pyramidal cell morphology.    3.2.5 Intracranial cannulations   Rats were anesthetized with isofluorane, injected with subcutaneous ketoprofen (10 mg/kg) and saline (2-3 mL, 0.9%), and secured in the stereotaxic frame.  Bilateral 23-gauge cannula (made in-house) were implanted into the OFC and secured with 3-4 jeweller’s screws and dental acrylic. Stereotaxic coordinates for adults were (in mm) AP: +3.5 (from Bregma), ML: ±3.2, and DV: -4.3 (from dura). Juvenile placements were AP: +3.3, ML: ±3.1, DV: -2.5.  All rats were implanted and then given one day of recovery before placing them back with their original cage mates  (grouped 4-6 for juvenile and 2 for adults). Rats were cannulated on P19 for juveniles and P60-70 for adults. Upon completion of behavioral testing, all rats were transcardially perfused with 4% paraformaldehyde, and 40 µm sections mounted on gelatin coated slides were stained using cresyl violet to confirm cannula placements in lateral OFC (Fig. 15).  All behavioral data from cannulations in brain regions outside the OFC (juvenile n=5, adult n=1) were not included in the analysis.    3.2.6 Behavioral training    All place reversal training was performed in a plus maze using previously established protocols (Kim and Ragozzino, 2005; Ragozzino and Choi, 2004).  The maze was  64 constructed of opaque black acrylic with each of the four arms 55 cm long, 10 cm wide, and 15 cm in height. An 8 mL ceramic crucible served as a food well and was secured at the end of each arm. The maze was placed on a table that was 72 cm in height. Extra-maze visual cues were placed throughout the room. For juvenile rats, two visual cues were placed adjacent to one of two arms and were kept consistent throughout the experiment.  Each arm was arbitrarily labeled North, South, East, or West and remained constant throughout behavioral experiments.     Rats were pre-exposed to Froot Loops™ cereal (Kelloggs, Battle Creek, MI) in their home cage 1-2 days before habituation training.  Habituation consisted of 3-6 days where each rat learned to consume ¼ to ½ piece of cereal from each of the four arms. This continued until each animal ate from all four arms a minimum of 5 times in 15 minutes or less. The last day of habituation consisted of a ‘block’ phase where a black acrylic block was placed into one of the arms, therefore transforming the maze into a T shape.  The rat was placed in the stem (start) arm and allowed to consume cereal at the other two baited ‘choice’ arms. This continued for seven trials, where one trial consisted of the rat consuming both cereal pieces in each of the two arms.     The following day (acquisition) consisted of training the rats on a place-discrimination task. Using the same T-maze setup, only the North choice arm was baited.  The rat was placed in either the East or West start arm and the responses toward the North (baited) arm or South (unbaited) arm were recorded. This comprised one trial. In between trials, the rat was placed on a cage top adjacent to the maze. Start arms were pseudo- 65 randomized such that the same start arm was never used for more than 3 consecutive trials.  After every fourth trial the maze was turned 90° and wiped out with a sponge sprayed with 2% Virkon (Dupont, Mississauga, ON) to minimize the use of intra-maze cues in learning the response strategy.  Testing continued until each rat reached a criterion of ten consecutive correct responses. No treatment was given to any animals on acquisition training.     Test day two (reversal) was similar to acquisition except that the reinforced arm was now the South arm instead of the North. Each rat was pseudo-randomized into two groups so the mean trials to criterion on the acquisition phase were matched.  Prior to testing, each animal received an infusion of SKF83959 (20 ng/side) or vehicle (10% DMSO in saline).  A 30-gauge stainless steel injector was inserted into each guide so that it extended 1 mm below the cannula tip. For both groups total injection volume was 500 nL/side at an infusion rate of 100 nL/min. Injectors were left in place for an additional 2 minutes, then the animal was placed back in its home cage for an additional ten minutes before the start of testing.      Errors committed during the reversal phase were analyzed to assess whether the drug treatment altered perseveration of a previously learned place discrimination or regression to a previous strategy once perseveration had ceased (Ragozzino and Choi, 2004; Kim and Ragozzino, 2005; Baker et al., 2011). Trials were grouped into blocks of 4 and then incorrect trials were counted and analyzed. Perseverative errors consisted of 3 or more incorrect trials per block. Once rats made less than three incorrect trials per block, all incorrect trials were labeled regressive errors, indicating the rat was regressing back to its previously learned  66 strategy during acquisition, and served as an index to measure how well the rat could maintain its choice of a newly learned strategy.    3.2.7 Drugs   Agonists SKF38393 (10 µM, Tocris, Ellisville, MO), quinpirole (10 µM; Sigma, St. Louis), SKF83959 (1 µM, Tocris, Ellisville, MO), and dopamine (0.1 or 10 µM dissolved in 75 µM sodium metabisulfite, Sigma, St. Louis) were bath applied for 5 minutes after establishing 10 minutes of stable baseline recordings. In some experiments, the antagonists SCH39166 (1 µM; Tocris, Ellisville, MO) or sulpiride ( (-)sulpiride: 500 nM; Tocris, Ellisville, MO, or (+/-)sulpiride: 1 µM;  Sigma, St. Louis, MO) were bath applied for 5 minutes prior to and during application of SKF38393 or quinpirole.  PKA inhibitor (PKI; 20 µM; Tocris, Ellisville, MO), or PLC inhibitor U73122 (1 µM; Tocris, Ellisville, MO) was dissolved in internal solution.    3.2.8 Statistical analysis   All electrophysiology data are expressed as mean percent change in baseline levels ± s.e.m .  In all experiments comparing baseline versus the effect of drug treatment, a student’s paired t test was used to measure between an averaged 5 minute time-point from the baseline (averaged from minutes 6 through 10) vs an averaged 5 minute time point 1 minute past application of agonists. Example traces were constructed from an average of 12 sweeps (2 minutes) taken before and after drug application. Stimulus artefacts were removed for clarity.  For electrophysiology experiments, “n” refers to the number of cells recorded from at least  67 three rats expressed as (n = cells/rats). A power analysis indicates that to detect a minimum 10% change in effect with a statistical power level of 0.8, we required a sample size of at least four cells per group. Unless otherwise indicated, data met the assumptions of equal variances. When comparing effects between juvenile and adults, unpaired t-tests were used to compare the maximal effect of any treatment.  For multiple group comparisons a one-way ANOVA with a Newman Keul’s post hoc tests, or a two-way ANOVA with planned comparisons (using Bonferroni post hoc tests) was used. Significance was set at p<0.05 for all experiments.   3.3 Results 3.3.1 Subregion-specific potentiation of NMDAR EPSCs    To determine if D1R or D2R activation differentially modulated NMDAR-mediated EPSCs in the OFC of juvenile vs. adult rats, we recorded layer II/III pyramidal cells of 3 major OFC subregions: lateral, ventral, and medial (Figure 6A, C, E). Application of the D1R agonist, SKF38393 (5 min, 10 µM (Gonzalez-Islas and Hablitz, 2001)), to the lateral OFC significantly potentiated evoked NMDAR EPSCs of juvenile pyramidal neurons (14 ± 5% increase from baseline; n=8 cells/7 rats, p < 0.05, paired t-test t=2.6, df =7; Fig. 6A, B).  In contrast, SKF38393 did not significantly potentiate NMDAR EPSCs in ventral (3 ± 3% reduction from baseline; n = 7/6, p > 0.05, paired t-test t = 1.0, df = 6; Fig. 6C, D) or medial (5 ± 5 % reduction from baseline vs; n = 8/4, p > 0.05, paired t-test t= 1.0, df = 7; Fig. 6E, F) OFC subregions.  Application of 1 µM SKF38393 did not significantly potentiate NMDAR in any OFC neurons and 100 µM SKF38393 caused a non-selective response in all subregions (data not shown). We also tested if the full agonist SKF81297 (5 min, 10 µM)  68 altered NMDAR EPSCs of juvenile rat lateral OFC neurons. However, we observed no significant effect of SKF81297 (2 ± 1 % reduction from baseline; n = 4/3,  p > 0.05, paired t-test, t= 1.3, df = 3), likely due to its ability to directly block the pore of NMDAR receptors, thus confounding a potential increase (Cui et al., 2006). Taken together, these data suggest that there is regional selectivity of D1R-mediated potentiation of NMDARs in the juvenile OFC.     To confirm that the potentiation of NMDARs by SKF38393 was due to activation of D1Rs, we bath applied SCH39166, a selective D1R antagonist (1 µM; 10 min (Yanovsky et al., 2011)) prior to and during application of SKF38393 (10µM; 5 min). SCH39166 alone had no effect on evoked EPSCs (3 ± 3% reduction from baseline; n = 7/4, F(2,6)=1.5, p > 0.05, one-way ANOVA; Fig. 7AB). In the presence of the D1R antagonist, SKF38393 did not potentiate NMDAR currents of juvenile lateral OFC neurons (4 ± 5% increase from baseline; n = 7/4, F(2,6)=1.5, p > 0.05, one-way ANOVA; Fig. 7AB).      To elucidate the signaling mechanism behind the D1R-mediated increase in NMDAR currents, we blocked protein kinase A (PKA) signaling with intracellular application of a PKA inhibitor (PKI; 20 µM (Lopshire and Nicol, 1998)).  PKI abolished D1R-mediated potentiation of NMDAR currents (6 ± 3% reduction from baseline; n = 8/5, p > 0.05, paired t-test, t= 1.9, df = 7; Fig. 7 C, D), illustrating a role for postsynaptic PKA signaling.      D1R mRNA in OFC neurons increases from pre-adolescence through adulthood  69 (Garske et al., 2013). Therefore, we investigated whether 10 µM SKF38393 would further potentiate NMDARs of adult OFC pyramidal neurons in a subregion-selective manner.  SKF38393 potentiated NMDAR EPSCs in adult lateral OFC pyramidal neurons (17 ± 9% increase from baseline; n = 6/4, p < 0.05, paired t-test t= 1.9, df = 5, Fig. 8A, B) with a similar efficacy to that of juveniles (F(1,26)=0.09; p > 0.05, 2-way ANOVA, Fig.9D).  Furthermore, potentiation of NMDAR EPSCs in adult OFC neurons was also regionally selective, as SKF38393 did not potentiate NMDAR EPSCs in either ventral (0.5 ± 2% increase from baseline; n = 6/4, p > 0.05, paired t-test t = 0.16, df = 5; Fig. 8C, D) or medial OFC (8 ± 7 % increase from baseline; n = 7/5, p > 0.05, paired t-test t = 1.2, df = 6; Fig. 8E, F).  3.3.2 D2 receptor-mediated suppression of EPSCs  To determine whether D2R activation modulates NMDAR EPSCs in the lateral OFC, we bath applied the D2R agonist, quinpirole (10 µM; 5 min (Kotecha et al., 2002)).  In lateral OFC pyramidal neurons from both juvenile and adult animals, quinpirole significantly inhibited NMDAR EPSCs (10 ± 4% reduction from baseline; n = 8/6, p < 0.05, paired t-test, t = 2.5, df = 7, Fig. 9A; Adult, 17 ± 5% reduction from baseline; n = 8/6, p < 0.05, paired t-test, t= 3.3, df = 7; Fig. 9C). The effect of quinpirole on NMDAR EPSCs was not significantly different between age groups (F (1,26)=0.09, p > 0.05, 2-way ANOVA; Fig. 9D). The D2R antagonist,  (-/-) sulpiride (500 nM), blocked the quinpirole-mediated suppression of NMDAR EPSCs of lateral OFC neurons (2 ± 2% reduction from baseline; n = 5/5, F (2,4)=0.49, p > 0.05, 2-way ANOVA; Fig. 9B). Sulpiride alone had no effect on evoked EPSCs (n = 5/5, F (2,4)=0.49, p > 0.05, 2-way ANOVA; Fig. 9B).  Taken together, these  70 results indicate that D1R activation potentiates NMDAR EPSCs, while D2R activation inhibits NMDAR EPSCs in lateral OFC pyramidal neurons of both juvenile and adult rats.   We also investigated the effects of D1R or D2R activation on AMPA receptor-mediated EPSCs in juvenile later OFC neurons. Application of SKF38393 did not change the amplitude of AMPAR currents (2 ± 4% increase from baseline; n=6/4, p> 0.05, paired t-test, t=0.48 df=5; Fig. 10A,B).  Similarly, quinpirole was unable to modulate AMPAR EPSCs (2 ± 4% increase from baseline; n=8/3, p> 0.05, paired t-test, t=0.58 df=7; Fig. 10C,D). Because no effects were seen on evoked AMPAR responses, this likely indicates that the bidirectional modulation of NMDARs by D1R and D2R agonists occurs through a postsynaptic mechanism, rather than modulation of presynaptic glutamate release.     To test the possibility of dopamine being co-released with evoked glutamate, we bath applied the D1R agonist with a D2R antagonist in lateral OFC slices from juvenile rats, with the hypothesis that evoked endogenous dopamine may reduce D1R-mediated responses by acting at D2Rs. SKF38393 in the presence of sulpiride significantly potentiated NMDAR EPSCs (13 ± 5% increase from baseline; n = 6/5; F (2,5)=6.43, p < 0.05, 2-way ANOVA), but this was not significantly different from application of SKF38393 alone (t-test, t = 0.07, df  = 12; p > 0.05). Sulpiride alone had no effect on evoked EPSCs (1 ± 2% reduction from baseline; n = 6/5; F(2,5)=6.43, p > 0.05, 2-way ANOVA).  These data suggest that endogenous dopamine is not evoked with afferent stimulation to significantly alter D1R-mediated effects at NMDARs.     71 3.3.3 Age-specific effects of D1:D2R co-activation  Previous studies have demonstrated that D1Rs and D2Rs act cooperatively to potentiate evoked firing in the striatum (Hopf et al., 2003; Seif et al., 2011).  Therefore, we tested if co-activation of D1R and D2R receptors modified NMDAR EPSCs of lateral OFC neurons in either juvenile or adult animals.  Interestingly, co-application of quinpirole and SKF38393 potentiated NMDAR EPSCs in lateral OFC pyramidal neurons of juvenile rats (32 ± 10 % increase from baseline; n = 8/5, p < 0.05, paired t-test, t= 2.9, df = 7; Fig. 11A). This effect was similar in magnitude to application of SKF38393 alone (p>0.05, unpaired t-test, t=1.3, df=11; Fig. 11A). In contrast, D1R:D2R cooperation was absent in the lateral OFC of adult rats (0.5 ± 7% reduction from baseline; n = 5/4, p > 0.05; paired t-test, t= 0.9, df = 4, Fig. 11B).   D1R:D2R cooperativity in striatum can involve coupling to phospholipase C (PLC) (Seif et al., 2011; Rashid et al., 2007). Therefore, we tested if co-activation of D1Rs and D2Rs required PLC to potentiate NMDARs in juvenile lateral OFC. Co-application of SKF38393 and quinpirole did not potentiate NMDAR EPSCs in the presence of intracellularly applied PLC inhibitor, U73122 (2 ± 4% increase from baseline; n = 11/8, p > 0.05; paired t-test, t = 0.5, df = 10; Fig. 11C), suggesting that co-activation of D1Rs and D2Rs couple to PLC to potentiate NMDARs in lateral OFC neurons.  U73122 did not alter evoked NMDAR EPSCs with co-application of SKF38393 and quinpirole in adults (0.9 ± 6% reduction from baseline; n = 6/5, p > 0.05, paired t-test, t= 0.1, df = 5; Fig. 11D).     72 Next, we tested if D1R:D2R cooperativity in juvenile lateral OFC required intracellular calcium to potentiate NMDARs.  Intracellular application of BAPTA (10 mM), a calcium chelator, inhibited D1R:D2R-mediated potentiation of NMDAR EPSCs (9 ± 4% increase from baseline; n = 7/5, p > 0.05, paired t-test, t= 2.1, df = 6, Fig. 11C), suggesting that increased intracellular calcium concentration is necessary for D1R:D2R-mediated potentiation of NMDAR EPSCs in lateral OFC neurons.  Taken together, individually applied D1R or D2R agonists have opposing actions at excitatory synapses of lateral OFC pyramidal neurons. However, when co-applied D1R and D2R agonists may act cooperatively to potentiate NMDARs in a PLC-dependent manner only in pyramidal neurons of juvenile lateral OFC.   There are several possible explanations for D1R:D2R-mediated cooperativity requiring PLC activation.  Firstly, postsynaptic D1R and D2R activation may induce a PLC-dependent endocannabinoid-mediated suppression of presynaptic GABA release, as has been demonstrated in the striatum (Seif et al., 2011; Gerdeman et al., 2002; Kreitzer and Malenka, 2005). This would likely result in a shift from inhibition to excitation potentially enhancing postsynaptic NMDAR currents. However, the inclusion of picrotoxin in our solutions precludes this hypothesis as GABAergic activity is blocked. Secondly, atypical PLC-coupled D1Rs (Pollack, 2004; Yu et al., 1996) may be present in juvenile lateral OFC pyramidal neurons. However, it is unclear why these receptors would be selectively targeted with SKF38393 in the presence of quinpirole as we have demonstrated that SKF38393 alone activates PKA-coupled D1Rs. A third possibility is that SKF38393 and quinpirole selectively activate D1R:D2R heterodimers that couple to PLC (Hasbi et al., 2010; O'Dowd et al., 2012).  73 Therefore, we tested if SKF83959, a biased agonist that selectively targets PLC coupled D1R:D2R heterodimers (Hasbi et al., 2009), could mimic D1R:D2R cooperativity in juvenile lateral OFC.   3.3.4 Age-specific effects of a PLC-coupled dopamine receptor agonist  In juvenile rats, SKF83959 (1 µM) potentiated NMDAR EPSCs in lateral OFC pyramidal neurons (14 ± 6% increase from baseline; n = 7/6, p < 0.05, paired t-test, t = 2.7, df = 6; Fig. 12A). PLC signaling was required for SKF83959-induced potentiation of NMDAR EPSCs, because when U73122 was included in the internal solution, SKF83959-mediated potentiation of NMDAR EPSCs was blocked (2 ± 3% reduction from baseline; n = 6/4, p > 0.05, paired t-test, t = 0.7, df = 5; Fig. 12A, SKF83959 vs SKF83959 +U73122: p<0.05, unpaired t-test, t=2.4, df=10; Fig. 12A).  To investigate if the potentiation we observed through SKF83959 was specific to dopamine receptors, we first co-applied the D1 antagonist SCH39166 prior to SKF83959, which abolished the ability of SKF83959 to potentiate NMDAR currents (5 ± 6% increase from baseline; n = 8/4, F(3,24)=0.38, p > 0.05, 1-way ANOVA; Fig. 12C).  Next, we co-applied the D2 antagonist sulpiride with SKF83959. Similarly, the effect of SKF83959 was blocked (1 ± 2% reduction from baseline; n = 6/5, F(3,24)=0.38, p > 0.05, 1-way ANOVA; Fig. 12C), indicating that potentiation of NMDAR EPSCs through SKF83959 requires activation of both D1 and D2 receptors.    Next, we tested if SKF83959 modulated NMDAR EPSCs in adult lateral OFC pyramidal neurons. Bath application of SKF83959 (1 and 10 µM) did not significantly increase evoked NMDAR EPSCs in adult neurons (1 µM: 2 ± 4% increase from baseline; n =  74 8/4, vs 10 µM: 4 ± 14% increase from baseline; n=5/5, unpaired t-test, t= 0.03, df = 7; Fig. 12B). Taken together, these data suggest that SKF83959, an agonist which targets D1R:D2R heterodimers (Hasbi et al., 2009), potentiates NMDARs in a PLC-dependent manner only in juvenile lateral OFC.   3.3.5 Bidirectional effects of dopamine in young vs adult OFC   Because targeting D1R:D2R with co-application of selective agonists yielded a cooperative potentiation of NMDAR EPSCs in juvenile lateral OFC neurons, we wanted to determine if the endogenous ligand for D1Rs and D2Rs, dopamine, modulated NMDAR EPSCs in a similar manner. In lateral OFC pyramidal neurons of both juveniles and adults, dopamine caused an early phase suppression of NMDAR EPSCs during bath application at 0.1 µM (juvenile: 11 ±  5% reduction from baseline, n=6/3 vs. adult: 12 ± 6% reduction from baseline, n=4/4; main-effect of time, F(2,16)=5.1, p<0.01, 2-way ANOVA). Posthoc tests revealed that the effects of dopamine were not significantly different between age groups (juvenile: 11± 5% reduction from baseline; n= 6/3 vs. adult: 12 ± 6% reduction from baseline; n=4/4, p>0.05, t=0.07 df=8, Fig. 13A). In contrast, there was a significant time x age interaction on NMDAR EPSCs with 10 µM dopamine application (F(2,20)=7.1, p<0.01, 2-way ANOVA; Fig. 13B).  Posthoc analyses revealed that this concentration caused an early phase suppression of EPSCs in adult lateral OFC neurons (17 ± 7% reduction from baseline, n=5/4, t=3.3, df=1, p<0.01; Fig. 13B) but no effect in juveniles (1 ± 5% reduction from baseline, n=7/5, t=0.19, df=1, p>0.05; Fig. 13B). Subsequently, 10 µM continued to cause a late-phase suppression in adult OFC neurons, but caused a potentiation in EPSCs in juvenile  75 neurons (adult: 13 ± 5% reduction from baseline, t=2.5, df=2, p<0.05  vs  juvenile: 12 ± 5% increase from baseline, t=2.7, df=2, p<0.05, Fig. 13B). Analysis of effect sizes revealed a significant concentration x age interaction (F(1,18)=5.0, p<0.05, 2-way ANOVA; Fig13C), and significant differences in the effects of age in the 10 µM concentration (t=3.3, df=1, p<0.01; Fig. 13B).  These data suggest that higher concentrations of dopamine have differential effects on the modulation of NMDAR currents in the lateral OFC of juvenile and adult rats.  To determine if the age-dependent effects of dopamine on lateral OFC pyramidal neurons was due to activation of PLC-coupled dopamine receptors in juvenile, but not adult rats, we applied U73122 via the patch pipette. Similar to what we observed with 0.1 µM dopamine alone, inclusion of U73122 revealed a significant main effect of time on the ability of dopamine to modulate EPSCs (F(2,16)=10.2, p<0.01, 2-way ANOVA; Fig. 13D). Post hoc analyses revealed an initial suppression of NMDAR responses (20 ± 5% reduction from baseline, n=5/3, t=2.6, df=2; Fig. 13D), which was not apparent approximately 7 minutes after dopamine application (11 ± 8% reduction from baseline, t=1.4, df=2, p>0.05; Fig. 13D). Interestingly, inhibition of PLC signaling through U73122 now reversed the previously excitatory effects of dopamine at 10 µM, which now caused an inhibition of evoked EPSCs (29 ± 10% reduction from baseline, n= 5/3 t=3.7, df=2, p<0.01), which persisted into the washout period (29 ± 11% reduction from baseline, t=2.5, df=2, p<0.05; Fig. 13D).  Taken together, these data suggest that high concentrations of dopamine target PLC-coupled dopamine receptors in juvenile lateral OFC.     76 3.3.6 Amelioration of reversal learning deficits in juvenile rats  Our in vitro data thus far points to a novel pathway by which dopamine acts in the lateral OFC of juvenile rats.  We hypothesized that activation of this pathway could modulate OFC-dependent behaviors selectively in juvenile, but not adult rats.  To investigate this idea, we trained both juvenile and adult rats on a reversal-learning task using a place discrimination/reversal paradigm (Ragozzino and Choi, 2004; Kim and Ragozzino, 2005). OFC lesions or administration of dopamine antagonists to this region impair performance on this task (Mizoguchi et al., 2010; Calaminus and Hauber, 2008). Because SKF83959 potentiated NMDAR EPSCs via PLC in juveniles, but not adults, we hypothesized that intra-lateral OFC administration would have age-dependent effects on reversal learning.  Juvenile rats learned the place discrimination at the same rate as adults (n=15 [juvenile] vs. 16 [adult], p > 0.05, t = 0.9, df = 29, unpaired t-test; Fig. 14A). Analysis of the reversal phase revealed a main-effect of drug treatment (F(1,27)=5.9, p<0.05) and age (F(1,27)=5.2, p<0.05, 2-way ANOVA; Fig. 14B).  Posthoc analyses revealed that vehicle-treated juveniles took significantly more trials to reach criterion on the reversal day compared to adults (juveniles: 77 ± 4 trials, n = 7 vs. adult: 56 ± 7 trials, n = 8; t=2.7, df=1, p < 0.05; Fig. 14B).  This effect was abolished by SKF83959 in the lateral OFC of juvenile rats (juveniles: 55 ± 5 trials, n = 7 vs. adult: 52 ± 6 trials, n = 8; t=0.46, df=1, p>0.05; Fig. 14B), thus facilitating their ability to reverse to the new place as efficiently as adults (F1,27=5.2, p>0.05). There were no significant differences due to age or drug treatment on the time required to complete each trial (F1,20=0.7, Fig.14D), suggesting that there was no impairment of locomotor activity.    77 Impairments in reversal learning can be characterized by either perseveration on previously learned strategies (perseverative errors), or an inability to maintain or reliably execute a newly learned response pattern (regressive errors) (Ragozzino and Choi, 2004; Kim and Ragozzino, 2005).  Adult rodents made the same amount of perseverative errors as juvenile rodents (F1,27 = 0.2, p > 0.05 Fig. 14C), and significantly less regressive errors than juveniles in the vehicle group (p < 0.01,  F1,27=8.3 Fig. 14C). While there was no significant effect of SKF83959 on regressive or perseverative errors in adults, SKF83959 significantly reduced regressive errors in juvenile rats (F1,27 = 5.1, p < 0.05), consistent with findings from our in vitro experiments, and suggesting that activation of  PLC-coupled dopamine receptors in the lateral OFC can ameliorate the ability to  maintain newly learned place discrimination.  Taken together, these data demonstrate that juvenile rats take longer to reach criterion on a place reversal task because they have difficulty maintaining the new association, rather than an inability to initially suppress the previously learned response.  Furthermore, microinfusion of SKF83959 into the lateral OFC of juvenile rodents reduces regressive errors and improves performance on a reversal-learning task.   3.4 Discussion  Here, we demonstrate that in lateral OFC pyramidal neurons of juvenile but not adult rats, D1R and D2Rs cooperatively potentiate NMDARs by a PLC-mediated mechanism. In contrast, selective activation of D1Rs or D2Rs had opposing effects on NMDAR EPSCs in juveniles and adults. Finally, administration of SKF83959 to the lateral OFC, an agonist which targets PLC-coupled D1R:D2R heterodimers, ameliorated juvenile performance on a  78 reversal learning task. Taken together, these data suggest that D1R:D2R heterodimers may provide a novel therapeutic target for treatment of cognitive flexibility disorders in children.    D1R activation alone was sufficient to modestly potentiate NMDAR EPSCs in juvenile or adult rats. The D1R group is composed of the D1 and D5 receptor subtypes. They are preferentially coupled to Gαs proteins that stimulate adenylyl cyclase and PKA-dependent pathways. Consistent with this, SKF38393-potentiation of NMDARs was blocked with intracellular application of a PKA inhibitor or bath application of SCH39166, suggesting a postsynaptic D1R activation. Furthermore, these effects were transient, consistent with reported effects of SKF38393 in other cortical brain regions (Gonzalez-Islas and Hablitz, 2003).    3.4.1 Regional differences in D1R-mediated effects on NMDARs  Dopamine terminals in the mPFC and the OFC form symmetrical contacts onto dendritic shafts and spines (Séguéla et al., 1988) that also receive excitatory input (Carr and Sesack, 1996). Here, we provide functional evidence that dopamine can gate excitatory inputs to the OFC. Notably, D1R- mediated potentiation of NMDARs was restricted to the lateral OFC region in both juvenile and adult rats. Regional differences in dopamine receptor expression, in particular D1R expression, might explain why D1R-mediated increases in NMDAR currents are restricted to lateral OFC.  Although functional heterogeneity of the OFC has been described in primates and humans, subregion differences have been little explored in rodents.  One study demonstrated that medial OFC did not play a critical role in conditioned cue-induced reinstatement of cocaine seeking, whereas the lateral OFC was  79 involved in the long-term storage, retrieval, or utilization of stimulus-reward associations (Fuchs et al., 2004).   3.4.2 Mechanisms behind D1:D2 cooperativity in juvenile OFC  Interestingly, we observed a cooperative potentiation of NMDAR EPSCs when SKF38393 and quinpirole were co-applied. This effect only occurred in juvenile rats and was dependent on PLC activation and a rise in internal calcium, suggesting coupling through Gαq.  Cooperative actions of D1Rs and D2Rs coupling to PLC on spike firing have been demonstrated in the striatum (Hopf et al., 2003; Seif et al., 2011). Earlier reports described atypical D1Rs that couple to Gαq as well as Gαs. However, in our study, co-activation of D1R and D2R coupling to Gαq is more likely to have occurred because the effect of D1R activation alone on NMDARs was abolished with intracellular administration of a PKA inhibitor. This is consistent with other experiments demonstrating D1R activation alone does not couple to the Gαq pathway. Furthermore, the concentration of SKF38393 used here has not been previously demonstrated to recruit the Gαq pathway (Undie and Friedman, 1990).  FRET and co-immunoprecipitation studies suggest that D1Rs and D2Rs can heterodimerize in the striatum (So et al., 2005; Dziedzicka-Wasylewska et al., 2006), NAc (Perreault et al., 2011) and frontal cortex (So et al., 2009; Pei et al., 2010). D1R and D2R mRNA are present in layer II/III pyramidal neurons of the mPFC (Santana et al., 2009), and co-localization on cell bodies in medial PFC slices has been demonstrated (Vincent et al., 1995; Lee et al., 2004). Furthermore, stimulation of D1R:D2R or D5R:D2R heterodimers can activate PLC and increase internal calcium concentrations (So et al., 2009).  Consistent with this, and our data demonstrating co-activation of D1 and D2 receptors, targeting D1R:D2R heterodimers with  80 SKF83959 (Hasbi et al., 2009) potentiated NMDAR EPSCs in a PLC-dependent manner only in juvenile rats.  Because SKF38393 has similar affinity for  D1 and D5 receptors (Seeman and Van Tol, 1994), and D5R:D2R heterodimers also exist in the cortex (So et al., 2009) we cannot exclude the possibility that D5R:D2R heterodimers are being targeted in our experiments. Previous reports have suggested that the agonist SKF83959 has the ability to bind to DAT and other receptors in addition to putative D1:D2 heterodimers. However, non-specific effects from this agonist appear at higher concentrations than what was used in our experiments (Fang et al., 2013; Guo et al., 2013).     Alternatively, crosstalk between co-expression of D1Rs and D2Rs on the same neuron may underlie potentiated responses on juvenile OFC neurons.  Co-application of SKF38393 and quinpirole presumably targets both canonical, AC-coupled receptors in addition to those that couple to PLC.  Antagonistic activity at AC likely renders a net null-effect on our NMDA responses.  Potentiation in the response in juvenile animals is through activation of PLC. In isolated striatal neurons, D2R activation can induce PLC production through Gβγ subunit activation (Hernandez-Lopez et al., 2000).  Thus, additive PLC signals from D2R- Gβγ  and Gq-coupled D1 receptors could be an alternative mechanism behind our effect.      Another potential mediator of D1 and D2 crosstalk is calcyon, which can increase the affinity state for D1Rs (Lidow et al., 2001). D1 receptor activation also mobilizes additional calcyon trafficking to cell membranes.  Interestingly, internal calcium concentrations increase via calcyon signaling when D2 receptors are stimulated (Frégeau et  81 al., 2013). Therefore, the increased affinity state of the D1 receptor, combined with calcyon-mediated increases in internal calcium through D2, might also be another explanation for our effects of D1 and D2 agonist co-application.    3.4.3 Age-related discrepancies in response to dopamine   Dopamine also had differential effects on NMDARs in the lateral OFC of juvenile and adult rats.  Higher concentrations of dopamine caused an excitatory effect at NMDARs in the OFC of juvenile rats, yet an inhibitory response in adults. NMDAR EPSC potentiation by 10 µM, but not 1 µM, dopamine in juvenile rats was blocked by inhibiting PLC-signaling. These data suggest that higher concentrations of dopamine target PLC-coupled D1R:D2R heterodimers in juvenile rats. NMDAR activation in the PFC is implicated for maintenance of ‘up-states’ in activated cortical cell networks (Kroener et al., 2009). Small changes in NMDAR currents could presumably influence the state of the network, because the voltage difference between ‘down’ and ‘up’ states may only be as small as 10 mV (Lewis and O'Donnell, 2000). Thus, potentiation of NMDAR currents by either D1R or D1R:D2R heterodimers may promote an up-state in cortical networks.  3.4.4 Summary and conclusions  Previous reports have indicated juvenile rats have lower baseline dopamine signaling compared to adults due to sparse dopamine terminals (Kalsbeek et al., 1988) and lower D1R expression (Garske et al., 2013).  Reduced basal dopamine may contribute to age-related impairments in reversal learning (Groman et al., 2013) as demonstrated in our experiments.   82 Thus, SKF83959-mediated potentiation of NMDAR currents via activation of PLC-coupled D1:D2 heterodimers may improve cognitive flexibility of juvenile rats. Indeed, intra-lateral OFC administration of SKF83959 improved performance of juveniles on a reversal-learning task. While SKF83959 did not alter adult performance on this task, SKF83959 decreased regressive errors of juvenile rats suggesting that SKF83959 may improve the ability to maintain newly learned associations.   In this set of experiments we did not infuse the agonist during acquisition, therefore we cannot rule out if the facilitation in performance is due to changes in the initial learning of place-reward associations. Additionally, it is possible that SKF83959 could impair the retrieval of the initial place discrimination during acquisition, thus reducing the total number of trials to criterion (Fuchs et al., 2004). However, this would be reflected as a reduction in perseverative errors if rats could no longer recall the original site of food reinforcement, and not regressive errors, as we observed. Taken together, the current data suggest that juvenile rats have a greater ability flexibly alter their behavior while reversing to a new place discrimination after administration of SKF83959.    Consistent with these results, methylphenidate, a DAT blocker, reduced impulsive responding in juvenile but not adult rats (Bizot et al., 2007). Thus, it is feasible that higher dopamine concentrations induced by methylphenidate may potentiate excitatory synaptic transmission through PLC activation to mediate its behavioral effect selectively in juvenile rats.    83 In summary, our results demonstrate that D1Rs and D2Rs act cooperatively to potentiate NMDARs in the lateral OFC of juvenile rats. While dopamine inhibits NMDAR EPSCs in adult animals, activation of putative D1R:D2R heterodimers in the lateral OFC of juvenile rats potentiate NMDAR EPSCs and may facilitate the shift of cortical networks to an up-state.  Furthermore, activation of D1R:D2R heterodimers in juvenile rats improved cognitive flexibility by facilitating the choice of a newly developed stimulus-response association.  Thus, biased agonists targeting PLC-coupled D1R:D2R heterodimers may provide clinical utility in treatment of  cognitive deficit disorders in children.    84 Chapter 4: General discussion   This thesis investigates plasticity of excitatory synapses in brain regions that contribute to goal-directed behavior. First, we determine how leptin signaling reduces the strength of excitatory synaptic transmission onto dopamine neurons in the VTA.  Subsequently, we examine the orbitofrontal cortex, a projection target of the VTA. Here, we characterize the effects of dopamine on excitatory synaptic transmission and differences between adult and juvenile rats. This discussion will review the main findings, limitations, and future applications of the current results.  4.1 Net effects of leptin on firing activity of VTA dopamine neurons    In chapter 2, we demonstrate that leptin suppresses glutamatergic synaptic transmission through a presynaptic mechanism.  Leptin decreased glutamate release via action at presynaptic LepRb receptors and PI3K signaling. This is the first evidence suggesting that leptin signaling modulates synaptic transmission in the VTA.  Reductions in the efficiency of excitatory synaptic transmission may underlie reduced motivation to consume highly palatable food and other behaviors dependent upon the mesolimbic dopamine system.     Previous work has demonstrated that leptin can modestly reduce VTA DA neuronal firing rate in slices (Hommel et al., 2006; Trinko et al., 2011) or in vivo (Hommel et al.,  85 2006; Liu et al., 2011). Leptin suppression of spontaneous firing of dopamine neurons in slices was blocked by intracellular application of an ERK inhibitor, suggesting a postsynaptic action (Trinko et al., 2011).  The net effect of leptin-mediated reduction of glutamate release on dopaminergic firing patterns can only be speculated from the present experiments.  Leptin action at glutamatergic synapses might be input-specific, whereby less glutamate release from excitatory afferents from discrete brain regions might reduce the ability of DA neurons to burst fire. Bursting activity is regulated by glutamatergic inputs from distal brain regions like the PFC, laterodorsal tegmentum, and pedunculopontine tegmental nucleus (Gariano and Groves, 1988; Sesack and Carr, 2002; Floresco et al., 2003; Lodge and Grace, 2006). Alternatively, the ability of leptin to reduce glutamatergic inputs could also decrease tonic firing activity of dopamine neurons. However, tonic activity is predominantly controlled by GABAergic afferents and intrinsic conductances expressed on dopamine neurons (Floresco et al., 2003; Komendantov et al., 2004).  Yet, excitatory inputs from the lateral mesopontine tegmentum also contribute to the number of spontaneously active dopamine neurons, associated with tonic dopamine release in the NAc (Chen and Lodge, 2013).  Leptin is able to inhibit tonic and burst firing in vivo (Hommel et al., 2006; Liu et al., 2011), but it is yet to be determined if this is a result of leptin-induced suppression of in excitatory synaptic transmission.    4.2 Leptin concentrations in vivo versus in vitro    Many experiments demonstrating roles for leptin on cellular physiology  and behavioral outputs use acute application of supra-physiological doses. For example, 100  86 nM leptin (5 min application) decreased dopamine (Hommel et al., 2006; Trinko et al., 2011), or LH (Leinninger et al., 2011) neuronal firing.  Notably, leptin is tonically released from adipocytes with fasting plasma leptin levels at 1.5/2.3 ng/mL in rats/humans (Ahima et al., 1996; Uher et al., 2006), which increase to 4.8/3.2 ng/mL after mealtimes (Ahima et al., 1996; Uher et al., 2006), which are in the picomolar range, even at the highest concentrations. However, while leptin is transported across the blood brain barrier (Banks et al., 1999; Zlokovic et al., 2000; Banks and Farrell, 2003), only a fraction of serum concentrations reach leptin receptors in the brain (Burguera et al., 2000; Banks, 2001; Banks and Farrell, 2003). Elevated levels of serum leptin in obese animals are sometimes used to justify concentrations bath applied to slices from rodent brain, which can reach concentrations of approximately 40 ng/mL (2.5 nM) (Shanley et al., 2001; Caro et al., 1996). 10-fold higher concentrations of reagents are generally used in brain slice electrophysiology as only a fraction of the concentration of peptide will soak into the tissue to the recorded neuron.      In chapter 2, leptin suppressed evoked EPSCs onto dopamine neurons at a concentration (100 nM) known to modulate excitatory or inhibitory synaptic transmission in the hippocampus (Wayner et al., 2004; Solovyova et al., 2009; Caro et al., 1996). It is possible that this concentration of leptin could induce leptin receptor desensitization in our experiments. Chronically elevated leptin concentrations can desensitize signaling at LepRb via activation of transcription factor SOCS-3 which inhibits LepRb by disrupting auto-phosphorylation of JAK2 upon leptin binding (Bjorbaek et al., 1998a; Dunn et al., 2005; Mori et al., 2004).  However, it is unlikely that our results are due to receptor  87 desensitization, as the effects of leptin were stable and reversed through inactivation of JAK2.  In addition, induction of SOCS-3 takes approximately 1-2 hours to attenuate leptin signaling, longer than the 15-minute bath application used in our experiments (Croker et al., 2003; Dunn et al., 2005; Bjorbaek et al., 1998a).            Importantly, many studies examine the effects of acute application of leptin on cellular signaling, neuronal activity or synaptic transmission. However, in vivo leptin is tonically available to receptors. Acute application is important for assaying the functionality of leptin receptors in neurons, however future studies should be directed at determining how endogenous tonic signaling at leptin receptors influences cellular function. Knockdown of LepRb in the cells responsible for reducing glutamatergic responses should provide more insight into the particular locus of where leptin is acting.       4.3 Potential orbitofrontal contributions to leptin signaling     This thesis focused on leptin action in the VTA. However, human imaging studies have suggested that leptin signaling may also influence activation of the OFC, an important region required for decision-making about the relative value of food.  Obese humans have reduced OFC activation and respond more impulsively to images of palatable food. Impaired leptin signaling in these individuals might be one reason why the OFC and other prefrontal brain regions are not recruited, and thus are unable to regulate response inhibition (Batterink et al., 2010). Similarly, individuals with congenital leptin deficiency have reduced OFC activation in response to food cues, which is augmented by leptin  88 replacement therapy (Frank et al., 2011; Baicy et al., 2007). It is possible that leptin suppression of excitatory synaptic transmission onto dopamine neurons can alter dopamine concentration in the OFC. Importantly, modulation of dopamine signaling increases impulsive responding to palatable foods, or cues that predict them, in humans and rodents (Zeeb et al., 2010; Winstanley et al., 2010).  However, the exact circuitry responsible for these effects remains to be investigated.      LepRb-expressing dopamine neurons mainly project to the NAc and the amygdala (Fulton et al., 2006; Leshan et al., 2010; Liu et al., 2011). However, our data suggest that LepRb receptors implicated in reductions in excitatory synaptic transmission may be localized to glutamatergic terminals within the VTA.  Future directions using optogenetics to strategically target specific glutamatergic inputs to the VTA could determine which inputs to dopamine neurons are modulated by leptin. In addition, VTA dopamine neurons project to the OFC (Berger et al., 1976). We can speculate that leptin-induced suppression of synaptic strength of glutamatergic synapses onto dopamine neurons may modulate dopaminergic output to the OFC, which may be one mechanism for how leptin contributes to the assignment of reward value to palatable foods (Domingos et al., 2011).         4.4  Activation of PLC-coupled dopamine receptors and OFC dependent behavior   DAT blockers such as methylphenidate are often administered to children in order to alleviate symptoms of ADHD, but hinder cognitive performance and attention at higher doses (Tannock et al., 1995). In chapter 3, we found that infusion of an agonist into the  89 OFC that selectively activates PLC-coupled dopamine receptors facilitated cognitive flexibility only in juvenile rats.  We used a place reversal-learning task that is commonly employed to assess OFC function (Brigman et al., 2013; Burke et al., 2009; Calaminus and Hauber, 2008; Wilson et al., 2014). While more selective tasks for OFC function exist (Wilson et al., 2014), we were limited in the timeframe in which we could train and test juvenile rats, therefore we used a place reversal task as juveniles were able to learn it within 3-6 days. In addition, periadolescent rats are often unable to acquire more complex tasks in a similar timeframe as adults (Spear and Brake, 1983). Our results demonstrated that SKF8959 improved performance on the place reversal selectively in juvenile rats.  Thus, one can speculate that age-related reductions in impulsivity through the use of psychostimulants such as methylphenidate (Bizot et al., 2007) may be due to augmented OFC dopamine signaling through activation of PLC-coupled dopamine heterodimers in juvenile animals.   4.5 Experimental limitations     The experiments in this thesis focus on excitatory synaptic transmission in brain areas important for goal-directed behavior.  Whole-cell recordings in brain slices allow precise analysis of synaptic currents in a particular cell. However, the use of this data to interpret what occurs in a behaving animal is not without limitations, some of which are discussed below.     90 4.5.1 Parsing behavioral changes induced by leptin in VTA     In chapter 2, we determine the signaling pathway involved in reduced presynaptic glutamate signaling by leptin.  Other changes in the physiology of dopamine neurons occur through activation of different downstream pathways that are activated by leptin (Hommel et al., 2006; Liu et al., 2011; Trinko et al., 2011).  However, specific changes in behaviors associated with these effects were not examined in the present set of experiments. Administration of exogenous leptin is often used to assess changes in reward-related behavior by actions in the mesolimbic dopamine system (Domingos et al., 2011; (Krugel et al., 2003; Morton et al., 2009).  Yet this does not establish whether these behaviors are specifically mediated by changes at glutamatergic synapses in the VTA, as intra-VTA administration would likely target GABAergic and dopaminergic neurons as well as their respective inputs. Therefore, future studies aimed at determining if leptin induced suppression of synaptic transmission in the VTA indeed modulates ingestive behaviour.   4.5.2 Actions of dopamine receptor agonists on non-pyramidal cortical neurons    In Chapter 3, when we demonstrate that intra-OFC infusion of SKF83959, an agonist at PLC-coupled dopamine receptors, ameliorates reversal-learning deficits in juvenile rats.  Although we demonstrate that action of SKF83959 can potentiate NMDARs on OFC pyramidal cells via PLC signaling, we are unable to demonstrate if this action modulates behavior via recruitment of PLC-coupled dopamine receptor-mediated modulation of NMDARs on pyramidal neurons. Notably, inhibition of NMDARs in OFC pyramidal neurons impairs cognitive flexibility (Brigman et al., 2013) and activation of NMDARs  91 governs neuroplasticity associated with decision-making tasks (van Wingerden et al., 2012).  While it is possible that SKF83959 activated PLC-coupled dopamine receptors expressed on GABAergic interneurons in the OFC to improve reversal learning in juvenile rats, expression of dopamine receptors has not been reliably demonstrated in cortical interneurons (Vincent et al., 1995; Lee et al., 2004).   4.6 Future directions  4.6.1 Targeted deletion of leptin receptors in the VTA    The present experiments do not identify which population of leptin receptors contributes to reductions in evoked EPSCs. LepRb could be reducing glutamate release through expression on presynaptic glutamatergic terminals. Alternatively, reductions in evoked EPSCs could be the result of enhanced glutamate clearance through LepRb activation on astrocytes.  Knockdown of LepRb on glutamatergic inputs to the VTA may provide more insight into the circuitry leptin modulates.       This technique would also be useful for investigating how decreased glutamatergic inputs to VTA neurons might correspond to reductions in dopamine release and subsequent goal-directed behavior. Interestingly, deletion of LepRb expressed on VTA dopamine neurons reduces dopamine signaling in the amygdala, and produces an anxiogenic phenotype but has no effects on hedonic food consumption (Liu et al., 2011). Thus, LepRb expression on discrete cell types in the VTA might regulate distinct behavioral outputs.   Therefore, it is possible that presynaptic LepRbs capable of regulating glutamatergic  92 signaling in the VTA are the ones responsible for the motivational and hedonic effects of leptin.   4.6.2 Region-specific effects of D1:D2 co-activation in the OFC    In chapter 3, we demonstrated that potentiation of NMDAR currents by the canonical D1R agonist was selective to the lateral OFC subregion in juvenile and adult rats.  Regional differences in receptor expression or dopamine receptors coupling to NMDAR responses might contribute to the finding that D1R-mediated potentiation of NMDARs is selective to the LOFC. Further investigation behind subregion specificity of D1-mediated responses in the OFC is warranted, given that detailed characterizations of dopamine receptor expression is not specific to OFC-regions in the cortex (Gaspar et al., 1995; Khan et al., 1998; Muly et al., 1998; Tarazi and Baldessarini, 2000).  OFC subregions in rats receive differential levels of dopaminergic innervation––terminals in VOFC are much more sparse than LOFC or MOFC regions, which appear to receive relatively equal levels of innervation (Berger et al., 1976; Van Eden et al., 1987). Yet, it is unclear if patterns of dopamine receptor expression differ across OFC subregions or between cortical layers. Characterization of receptor expression within the entire OFC would allow us to predict which populations of cells might be differentially affected by dopamine, in addition to regions outside the LOFC that might also involve PLC-coupled dopamine receptor signaling.  Unfortunately, current antibodies for D1R or D2 receptors lack consistent selectivity. Therefore, genetic techniques are required to dissociate location of D1 or D2 receptors in the LOFC.  93 4.6.3 Investigation of physical interactions between  DAR subtypes       Additional experiments investigating dimerization of DARs in juvenile OFC neurons are needed to confirm that formation of a functional D1:D2R complex is responsible for our effects. Although D1:D2 heterodimers have been demonstrated in rat mPFC and post-mortem human tissue (Pei et al., 2010; Lee et al., 2004), it is unclear if they are expressed in pyramidal neurons of the rat OFC.  Our results describe functional data that support the expression of D1:D2 heterodimers in LOFC pyramidal neurons.   We cannot confirm the existence of heterodimers in the OFC without the use of other techniques, such as FRET.  However, this technique requires the selectivity of antibodies for D1 and D2 receptors or overexpression of tagged receptors in neurons (Fiorentini et al., 2008; Hasbi et al., 2010; Pei et al., 2010). Alternatively, an interfering peptide that inhibits a physical interaction between D1 and D2Rs (Pei et al., 2010) would provide evidence that D1:D2 heterodimers potentiate NMDARs in juvenile animals.    4.7 Conclusions     The research in this thesis examines the interactions between dopamine and glutamate, and describes how both leptin signaling and changes in development can alter synaptic plasticity in brain regions that control decision-making.  First, we demonstrated that glutamatergic synapses onto VTA dopamine neurons are supressed by presynaptic leptin receptor activation.  Additionally, we found that dopaminergic modulation of  94 NMDARs in the lateral OFC may underlie a novel mechanism behind age-dependent differences in cognitive function.     The OFC and VTA are crucial nodes within the mesocorticolimbic circuit that are essential for adaptive decision-making. Disruption in dopaminergic tone within the OFC is implicated in impulsive responding for high value rewards such as palatable food.  Therefore, it is equally critical to investigate the role of feeding peptides in the control synaptic transmission in the VTA, a prime source of these dopaminergic inputs, in addition to modifications in orbitofrontal synaptic communication by dopamine. The results in this thesis contribute to our growing understanding of the brain regions that underlie executive control over decision-making, and how they are affected by neuromodulators.  95 Figures                Figure 1. Leptin depresses AMPAR- and NMDAR-mediated synaptic transmission onto VTA dopamine neurons Evoked AMPAR (-70 mV) or NMDAR (+40 mV) EPSCs were recorded before, during, and after bath application of leptin (100 nM). (A) 15 minute bath application of leptin caused a long-lasting decrease of AMPAR-mediated EPSCs. (B) Example recording of leptin on evoked AMPAR EPSCs from a VTA dopamine neuron. Filled bar indicates presence of leptin in the bath. Inset, representative traces of AMPAR EPSCs before (black) and after leptin (grey).  Scale bars, 10 ms, 100 pA. (C) 15 minute bath application of leptin caused a lasting decrease in NMDAR-mediated EPSCs of VTA dopamine neurons. (D) Example recording of leptin on evoked NMDAR EPSCs from a dopamine neuron in the VTA. Filled bar indicates presence of leptin in the bath. Inset, representative traces of NMDAR EPSCs before (black) and after leptin (grey). (E) Bar graph of effect sizes of leptin on AMPA and NMDAR mediated EPSCs Scale bars: 20 ms, 100 pA. Stimulus artefacts have been removed for clarity.  Error bars indicate s.e.m.   96 A BCD EF G BaselineVehicle BaselineLeptin0123Leptin Vehicle *mEPSC frequency (Hz)0102030Leptin VehiclemEPSC peak amplitude (pA)BaselineLeptinBaseline (VEH)Vehicle0 10 20 30 40 50 600.00.51.0Cumulative Probability0 1 2 3 4 50.00.51.0Cumulative Probabilityinter-event interval (s) mEPSC peak amplitude (pA)*         97 Figure 2. Leptin-induced synaptic inhibition is mediated presynaptically AMPAR mEPSCs were recorded at -70 mV in the presence of picrotoxin, TTX and APV before and after a 15 min bath application of leptin (100 nM). (A) Frequency of mEPSCs events was significantly decreased after leptin application  compared to baseline (open bars, n=8, p<0.05). Vehicle treatment  did not alter mEPSC frequency compared to baseline (P > 0.05). (B) AMPAR mEPSCs amplitude was not significantly different before (open bar) or after (filled bars) bath application of leptin (right panel) or vehicle (left panel) (p > 0.05). (C) Example recordings of AMPAR mEPSCs before or after leptin (left side) or vehicle (right side) application. Scale bars: 100 ms, 20 pA.(D) Cumulative probability plots for inter-event interval for before (black) and after (grey) leptin application. A significant right-shift in the cumulative probability of mEPSC frequency was detected after leptin application compared to the baseline (p<0.001, Kolmogorov-Smirnov test). (E) Cumulative probability plots for amplitude for before (black) and after (grey) leptin application. No significant difference was detected in cumulative probability plots of mEPSC amplitude before or after leptin (p>0.05, Kolmogorov-Smirnov test). (F) Bar graph illustrating a significant increase in the paired pulse ratio of evoked EPSCs after leptin administration (*p<0.05). (G) Example recording of paired (50 ms interval) AMPAR EPSCs before (black) and after leptin (grey) application.  Scale bars: 20 ms, 50 pA. Stimulus artefact has been removed for clarity. Bars represent mean ± s.e.m.                      98                      Figure 3. Leptin-induced synaptic inhibition requires activation of JAK2 signaling Evoked AMPAR EPSCs were recorded before, during, and after bath application of leptin (100 nM) for 15 minutes.  Slices were preincubated in inhibitors for a minimum 20 minutes prior to and throughout the experiment.  Filled bars indicate the presence of leptin (100 nM) in the bath. (A) Bath application of AG490 (50 µM) abolished leptin-induced synaptic depression. (B) Example recording of AMPAR EPSCs from a representative dopamine neuron in the presence of AG490 and leptin.  Inset, example traces before (black) and after (grey) leptin application. (C) AG490 (50 µM) applied intracellularly did not inhibit leptin-induced synaptic depression.  (D) Example recording of AMPAR EPSCs before and after leptin application in the presence of intracellular AG-490. Inset, example traces before (black) and after (grey) leptin application. Scale bars: 20 ms, 100 pA.  Stimulus artefacts have been removed for clarity.  Error bars indicate s.e.m  99                             100  Figure 4. Leptin-induced synaptic inhibition requires PI3K activation Evoked AMPAR EPSCs were recorded before, during, and after bath application of leptin (100 nM) for 15 minutes.  Slices were preincubated in inhibitors for a minimum 20 minutes prior to and throughout the experiment.  Filled bars indicate the presence of leptin (100 nM) in the bath. (A) U0126 (10 µM) does not alter leptin-induced depression of AMPAR EPSCs of VTA dopamine neurons. (B) Example recording of AMPAR EPSCs from a representative dopamine neuron in the presence of U0126 and leptin. (C) Bath application of wortmannin (100 nM) abolished leptin-induced synaptic depression. (D) Example recording of AMPAR EPSCs from a representative dopamine neuron in the presence of wortmannin and leptin.  Inset, example traces before (black) and after (grey) leptin application. Scale bars: 20ms, 100 pA.(E) Wortmannin (100 nM) applied intracellularly did not inhibit leptin-induced synaptic depression. (F) Example recording of AMPAR EPSCs before and after leptin application in the presence of intracellular wortmannin. Inset, example traces before (black) and after (grey) leptin application. Scale bars: 20ms, 100 pA.  Stimulus artefacts have been removed for clarity.  Error bars indicate s.e.m.                      101           Figure 5. Putative mechanisms of leptin-induced synaptic depression onto VTA dopamine neurons Inhibition of Jak2, PI3K, but not MEK1/2 depresses presynaptic glutamate release onto VTA dopamine neurons.  Leptin may act via presynaptic LepRbs on glutamatergic terminals decreasing glutamate release or via PI3K-dependent upregulation of glutamate transporters (i.e. EAAT4), to increase clearance of glutamate from the synapse.     102                         103 Figure 6. SKF38393, a D1R agonist, potentiates NMDAR EPSCs in pyramidal neurons of the lateral OFC of juvenile rats (A) SKF38393 potentiated NMDAR EPSCs in pyramidal neurons in lateral OFC. Inset, approximate location of recorded cells. (B) Example time course of EPSC amplitudes from a single pyramidal neuron in lateral OFC in the presence of SKF38393 (filled bar). Inset, example traces from before (1) and after (2) SKF38393 application. (C) SKF38393 does not potentiate NMDAR EPSCs in ventral OFC pyramidal neurons.  Inset, approximate location of recorded cells.  (D) Example time course of EPSC amplitudes from a single pyramidal neuron in ventral OFC in the presence of SKF38393 (filled bar). Inset, example traces from before (1) and after (2) SKF38393 application.  (E) SKF38393 does not potentiate NMDAR EPSCs in medial OFC pyramidal neurons. Inset, approximate location of recorded cells. (F) Example time course of EPSC amplitudes from a single pyramidal neuron in medial OFC in the presence of SKF38393 (filled bar). Inset, example traces from before (1) and after (2) SKF38393 application. Error bars indicate s.e.m. Scale bars, 50 pA, 20 ms. Some points on sagittal maps are occluded.                  104                       Figure 7.  SKF38393 potentiates NMDAR EPSCs in lateral OFC of juvenile rats via D1Rs coupled to PKA signaling  (A) SCH39166 blocks NMDAR potentiation induced by SKF38393. (B) Example time course of EPSC amplitudes from a single pyramidal neuron in the presence of SCH39166 (open bar) and SKF38393 (filled bar). Inset, example traces from before (1) and after (2) SKF38393 and SCH39166 application. (C) Intracellular application of PKI blocks NMDAR-mediated potentiation induced by SKF38393. (D) Example time course of EPSC amplitudes from a single pyramidal neuron in the presence of SKF38393 and PKI.  Inset, example traces from before (1) and after (2) SKF38393 application. Error bars indicate s.e.m. Scale bars: 50 pA, 20 ms.   105                       106 Figure 8. SKF38393 potentiates NMDAR EPSCs of pyramidal neurons selectively in lateral OFC of adult rats (A) SKF38393 potentiated NMDAR EPSCs in pyramidal neurons in lateral OFC.  Inset, approximate location of recorded cells. (B) Example time course of EPSC amplitudes from a single pyramidal neuron in lateral OFC in the presence of SKF38393 (filled bar).  Inset, example traces from before (1) and after (2) SKF38393 application (C) SKF38393 does not potentiate NMDAR EPSCs in ventral OFC pyramidal neurons.  Inset, approximate location of recorded cells.  (D) Example time course of EPSC amplitudes from a single pyramidal neuron in ventral OFC in the presence of SKF38393 (filled bar).  Inset, example traces from before (1) and after (2) SKF38393 application. (E) SKF38393 does not potentiate NMDAR EPSCs in medial OFC pyramidal neurons. Inset, approximate location of recorded cells. (F) Example time course of EPSC amplitudes from a single pyramidal neuron in medial OFC in the presence of SKF38393 (filled bar). Inset, example traces from before (1) and after (2) SKF38393 application. Error bars indicate s.e.m.  Scale bars; 50 pA, 20 ms.                   107                    Figure 9. Quinpirole, a D2R agonist, inhibits NMDAR EPSCs of lateral OFC pyramidal neurons from adult or juvenile rats (A) Quinpirole decreased NMDAR EPSCs of lateral OFC neurons from juvenile rats. Inset, example traces from before (1) and after (2) quinpirole application. (B) Sulpiride (open bar) blocks quinpirole-mediated (filled bar) suppression of NMDAR EPSC amplitudes in juvenile lateral OFC neurons. Inset, example traces from before (1) and after (2) quinpirole + sulpiride application.  (C) Quinpirole decreased NMDAR EPSCs of lateral OFC neurons from adult rats.  Inset, example traces from before (1) and after (2) quinpirole application. Scale bars: 50 pA, 20 ms. (D) Effects of quinpirole or SKF38393 in lateral OFC neurons is not significantly different between juveniles and adults   108 0 10 20 30507510012515010 µM quinpiroleTime (min)AMPAR EPSCs (% baseline)AC0 10 20 30507510012515010 µM SKF38393Time (min)AMPAR EPSCs  (% baseline)1211221 230B10 20 30Time (min)1 2                        Figure 10. SKF38393 and quinpirole have no effect on AMPAR-mediated EPSCs in juvenile lateral OFC pyramidal neurons (A) SKF38393 does not change AMPAR EPSCs in lateral OFC neurons. Inset, example traces from before (1) and after (2) SKF38393 application. (B) Quinpirole does not change AMPAR EPSCs in lateral OFC neurons. Inset, example traces from before (1) and after (2) quinpirole application. Scale bars: 50 pA, 20 ms.   109                  Figure 11. Co-application of SKF38393 and quinpirole potentiates NMDAR EPSCs in juvenile but not adult lateral OFC pyramidal neurons (A) Application of SKF38393 (open circles) or SKF38393 + quinpirole (filled circles) potentiated NMDAR EPSCs of juvenile lateral OFC neurons.  Inset left, example traces from before (1) and after (2) SKF38393 application. Inset right, example traces from before (1) and after (2) SKF38393 + quinpirole application. (B) Application of SKF38393 + quinpirole did not potentiate NMDAR EPSCs in adult lateral OFC neurons. Inset, example traces from before (1) and after (2) SKF38393 + quinpirole application. (C) Intracellular application of U73122 (filled circles) or BAPTA (open circles) blocked potentiation of NMDAR EPSCs induced by coapplication of SKF38393 + quinpirole onto juvenile lateral OFC neurons. Inset left, example traces from before (1) and after (2) SKF38393 + quinpirole application with U73122. Inset right, example traces from before (1) and after (2) SKF38393 + quinpirole application with BAPTA. (D) Intracellular application of U73122 had no effect on NMDAR EPSCs before or after coapplication of SKF38393 + quinpirole onto adult lateral OFC neurons. Inset, example traces from before (1) and after (2) SKF38393 + quinpirole application with U73122. Error bars indicate s.e.m. Scale bars: 50 pA, 20 ms.    110                    Figure 12. SKF83959, a D1:D2 heterodimer agonist, potentiated NMDAR EPSCs of juvenile but not adult lateral OFC pyramidal neurons (A) SKF83959 potentiated NMDAR EPSCs of lateral OFC neurons from juvenile rats (filled circles). This effect was blocked by intracellular U73122 (open circles). Inset, example traces from before (1) and after (2) SKF83959 application. (B) Application of SKF83959 at 1 µM (filled circles) or 10 µM (open circles) did not potentiate NMDAR EPSCs in adult lateral OFC neurons. Inset, example traces from before (1) and after (2) 10 µM SKF83859 application. (C) In the presence of sulpiride (filled circles) or SCH39166 (open circles), NMDAR EPSCs are not potentiated by SKF83959 in juvenile lateral OFC neurons. Inset, example traces from before (1) and after (2) SKF83959 application. Error bars in indicate s.e.m. Scale bars; 50 pA, 20 ms.    111 A BDC112211221 µM U73122 (pipette)10 µM DA0.1 µM DAjuvenile0 10 20 305075100125150Time (min)NMDAR EPSCs  (% baseline)0 10 20 305075100125150Time (min)NMDAR EPSCs  (% baseline)0 10 20 305075100125150Time (min)NMDAR EPSCs  (% baseline)10 µM DA0.1 µM DAadult adultjuvenilejuvenile1122 2211                  Figure 13. Dopamine has differential effects on NMDAR EPSCs of juvenile or adult lateral OFC pyramidal neurons (A) 0.1 µM dopamine decreased NMDAR EPSCs of lateral OFC pyramidal neurons of either juvenile (filled circles) or adult rats (open circles). Inset, example traces from before (black) and 12 minutes after (grey) dopamine application from either juvenile (filled circles) or adult (open circles) rats. (B) In juvenile rats (filled circles), 10 µM dopamine potentiated NMDAR EPSCs. In contrast, 10 µM dopamine decreased NMDAR EPSCs in adult rats (open circles). Inset, example traces from before (black) and 12 minutes after (grey) dopamine application from either juvenile (filled circles) or adult (open circles) rats. (C) Effects of dopamine in lateral OFC neurons are age dependent (stats). (D) In juvenile rats, intracellular U73122 inhibited potentiation of NMDAR EPSCs induced by 10 µM dopamine (filled circles), but did not alter the effect of  0.1 µM dopamine (open circles) on NMDAR EPSCs. Error bars indicate s.e.m. Scale bars; 50 pA, 20ms. * p<0.05  112          .             Figure 14. Intra-lateral OFC SKF83959 improves juvenile performance on a reversal learning task (A) Acquisition of a place discrimination is not significantly different between juvenile (filled bars) and adult (open bars) rats (p > 0.05). (B) Juveniles (filled bars) require more trials to reach the reversal learning criterion than adult rats (open bars). SKF83959 does not alter reversal learning in adults, but improves performance in juveniles.   (C) Error analysis during reversal reveals no effect of SKF83959 on perseverative errors in juvenile (filled bars) or adult rats (open bars) (left panel). However, the increased regressive errors committed in juveniles compared to adults were ameliorated by SKF83959 (right panel).  (D) Average time required to complete each trial is not significantly different in any groups tested (p > 0.05).  Bars represent mean ± s.e.m.;  * p <0.05, ** p< 0.01.     113                      Figure 15.  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