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Regulation of cadherin adhesion complexes and their modulators : effects on synapse formation and plasticity Globa, Andrea Kathleen 2017

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REGULATION OF CADHERIN ADHESION COMPLEXES AND THEIR MODULATORS: EFFECTS ON SYNAPSE FORMATION AND PLASTICITY by  Andrea Kathleen Globa  B.Sc., The University of Winnipeg, 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  October 2017  © Andrea Kathleen Globa, 2017 ii  Abstract Synapses of the central nervous system are specialized points of cell-cell contact that transmit signals from one neuron to another in an efficient manner. A fundamental property of synapses is that they can be altered in response to activity, a process called “synaptic plasticity.” Synaptic activity can cause lasting increases in synaptic strength (long-term potentiation, or ‘LTP’), and decreases in synapse strength (long-term depression, or ‘LTD’).  The cell adhesion molecules ‘cadherins’ and their intracellular binding partners β-catenin and δ-catenin are key mediators of synaptic plasticity. The disruption of the cadherin adhesion complex impairs LTP, and increased cadherin stability at synaptic membranes impairs LTD. The role of cadherins in synaptic plasticity has been well studied in the hippocampus, and cadherins have been shown to influence spatial learning and memory. However, little is known about the importance of cadherins in other brain regions, and in other forms of learning. In the first half of this dissertation, I examine the role of the cadherin adhesion complex in cocaine-mediated plasticity in the ventral tegmental area (VTA) of the mesocorticolimbic dopamine circuit. I demonstrate that cadherins play an important role in activity- and cocaine-mediated plasticity in the VTA. Furthermore I find that increasing cadherin localization at the synaptic membranes of VTA dopamine cells impairs AMPA receptor trafficking, synaptic plasticity, and cocaine-mediated behavioural conditioning. Previous work has also shown that the cadherin adhesion complex protein, δ-catenin, can be modified through the addition of the fatty acid, palmitate, to cysteine residues in a process called palmitoylation. δ-catenin palmitoylation results in increased cadherin-δ-catenin interactions and increases in synapse strength. The palmitoylation of δ-catenin is mediated by the palmitoyl acyltransferase, zDHHC5. Although zDHHC5 has been shown to play an important role in synaptic plasticity, its role in neuronal development has not been examined. In the second half of this dissertation, I examine the role of zDHHC5 in dendrite outgrowth and synapse formation, finding that the palmitoylation function and proper localization of zDHHC5 at the plasma membrane of postsynaptic spines are important for the stability of dendritic spines and the formation of excitatory synapses.   iii  Lay Summary Synapses are specialized junctions where brain cells communicate. These connections encode information in the brain, and can be strengthened or weakened in response to activity. There are many proteins at synapses that regulate their function. One of these proteins is called cadherin. Cadherins play important roles in the strengthening and weakening of synapses, and contribute to the formation of spatial memories. However, these proteins have not been studied in brain regions important for other behaviours. In the first half of this dissertation, I examine the role of cadherins in a brain region that can regulate our experience of pleasure and reward, finding that cadherins are important for synaptic changes in response to cocaine. In the second half of this dissertation, I examine an enzyme that can act on cadherin adhesion complexes, called zDHHC5. I find that zDHHC5 plays a role in the formation of new synapses.   iv  Preface A portion of the content in Chapter 1.8, entitled “The zDHHC family of Palmitoyl acyl transferases,” is a review that has been published as:  Globa AK, Bamji SX. Protein Palmitoylation in the development and plasticity of neuronal connections. Current Opinion in Neurobiology. doi:10.1016/j.conb.2017.02.016  The manuscript was written and figures prepared by AKG under the supervision of SXB.  The work in Chapter 2, entitled “Cadherins mediate cocaine-induced synaptic plasticity and behavioural conditioning” has been published as:  Mills F1, Globa AK1, Liu S, Cowan CM, Mobasser M, Phillips AG, Borgland SL, Bamji SX. (2017) Cadherins mediate cocaine-induced synaptic plasticity and behavioral conditioning. Nature Neuroscience doi:10.1038/nn.4503   1These authors contributed equally to this work.  All experiments were conceived by FM, AKG and SXB, and all experiments were jointly conducted by FM and AKG, with the following exceptions: electrophysiological experiments were conducted by SL under the supervision of SLB, and tissue processing and data analysis from immunogold electron microscopy experiments was assisted by CMC and MM. Experiments by FM and AG were done in equal partnership, and equal intellectual contribution. FM, AKG and SXB wrote the manuscript.  The work in Chapter 3, entitled “zDHHC5-mediated palmitoylation promotes excitatory synapse formation” will be published as:  Globa AK, Matin N, Tang, V, Bamji SX. (2017) zDHHC5-mediated palmitoylation promotes excitatory synapse formation. (manuscript in preparation).  All experiments were conceived by AKG and SXB, and conducted by AKG with the following exceptions: NM performed Western blots to validate zDHHC5 antibody in brain lysates, and to examine expression of E648* mutant DHHC5 in Figure 3.4. VT assisted with immunostaining and analysis of synapse density in Figure 3.5.   Ethics Certificate Numbers:  The animal studies presented in this thesis were performed with ethical approval from the UBC Animal Care Committee (certificates #A10-0316 and #A14-0338)  v  Table of Contents Abstract .......................................................................................................................................... ii	Lay Summary ............................................................................................................................... iii	Preface ........................................................................................................................................... iv	Table of Contents .......................................................................................................................... v	List of Figures ............................................................................................................................. viii	List of Abbreviations ................................................................................................................... xi	Acknowledgements .................................................................................................................... xiii	Dedication ................................................................................................................................... xiv	Chapter 1: Introduction ............................................................................................................... 1	1.1	 The Mesocorticolimbic Dopamine Circuit. ....................................................................... 1	1.1.1	 Dopaminergic Neurons of the Ventral Tegmental Area ............................................. 2	1.1.2	 Afferent Projections onto VTA Dopamine Neurons .................................................. 3	1.1.3	 Efferent Projections from VTA Dopamine Neurons .................................................. 4	1.2	 Dopamine in Learning and Motivation .............................................................................. 5	1.3	 Synapse Structure and Function ......................................................................................... 7	1.3.1	 Structure of the Presynaptic Compartment ................................................................. 8	1.3.2	 Structure of the Postsynaptic Compartment ............................................................... 9	1.3.2.1	 NMDAR Receptors ............................................................................................ 10	1.3.2.2	 AMPA Receptors ............................................................................................... 11	1.3.3	 Synaptic Plasticity ..................................................................................................... 13	1.3.3.1	 Drug-Mediated Synaptic Plasticity in the Ventral Tegmental Area .................. 14	1.3.3.2	 Drug-mediated Synaptic Plasticity in the Nucleus Accumbens ........................ 17	1.4	 The Cadherin Adhesion Complex .................................................................................... 17	1.4.1	 Cadherin Binding Proteins ........................................................................................ 20	1.4.1.1	 β-catenin Structure and Function ....................................................................... 21	1.4.1.2	 β-catenin Degradation and Signalling ................................................................ 22	1.4.1.3	 δ-catenin Structure and Function ....................................................................... 24	1.4.2	 The Cadherin Adhesion Complex in Synapse Plasticity .......................................... 25	1.4.3	 The Cadherin Adhesion Complex in the Dysfunction of the Nervous System ........ 27	1.4.4	 Cadherin Adhesion Complex in Addiction ............................................................... 28	1.5	 The Structure and Function of the Hippocampal Circuit ................................................. 29	1.5.1	 Anatomy of the Hippocampus .................................................................................. 30	1.5.2	 Hippocampal Function .............................................................................................. 33	1.6	 Primary Hippocampal Cell Culture ................................................................................. 33	1.7	 Synapse Formation in Hippocampal Neurons ................................................................. 34	1.8	 The zDHHC Family of Palmitoyl Acyl Transferases ...................................................... 36	1.8.1	 Palmitoylation of Synaptic Proteins .......................................................................... 37	1.8.1.1	 zDHHC5-Mediated Palmitoylation of Synaptic Proteins .................................. 38	1.8.2	 Activity-Mediated Changes in zDHHC5 Localization and Function ....................... 39	1.8.3	 Palmitoylation in Synapse Formation ....................................................................... 40	1.8.4	 Palmitoylation in the Dysfunction of the Nervous System ....................................... 41	1.9	 Rationale and Hypothesis ................................................................................................ 42	vi  Chapter 2: Cadherins Mediate Cocaine-Induced Synaptic Plasticity and Behavioural Conditioning ................................................................................................................................ 44	2.1	 Introduction ...................................................................................................................... 44	2.2	 Materials and Methods ..................................................................................................... 47	2.2.1	 Animals ..................................................................................................................... 47	2.2.2	 Immunoblot Analysis ................................................................................................ 47	2.2.3	 Immunohistochemistry ............................................................................................. 48	2.2.4	 Electrophysiology ..................................................................................................... 49	2.2.5	 TH Immunocytochemistry ........................................................................................ 51	2.2.6	 Conditioned Place Preference ................................................................................... 51	2.2.7	 Electron Microscopy Sample Preparation ................................................................ 53	2.2.8	 Immunogold Electron Microscopy ........................................................................... 54	2.2.9	 Rotarod ...................................................................................................................... 57	2.2.10	 Context-Dependent Fear Conditioning ..................................................................... 57	2.2.11	 Locomotor Sensitization to Cocaine. ........................................................................ 57	2.2.12	 Food Consumption Testing ....................................................................................... 58	2.2.13	 Statistical Analysis .................................................................................................... 58	2.3	 Results .............................................................................................................................. 59	2.3.1	 Cadherins are Expressed in Dopaminergic Neurons and Required for LTP ............ 59	2.3.2	 Cadherins are Recruited to VTA Synapses During Cocaine CPP ............................ 62	2.3.3	 GluA1 is Recruited to VTA Synapses During Cocaine CPP .................................... 67	2.3.4	 Cadherin Stabilization at VTA Synapses Reduces Cocaine CPP ............................. 70	2.3.5	 Cadherin Stabilization at VTA Synapses Blocks Synaptic Plasticity ....................... 72	2.4	 Discussion ........................................................................................................................ 78	Chapter 3: zDHHC5-Mediated Palmitoylation Promotes Excitatory Synapse Formation . 87	3.1	 Introduction ...................................................................................................................... 87	3.2	 Materials and Methods ..................................................................................................... 89	3.2.1	 cDNA Constructs ...................................................................................................... 89	3.2.2	 Cell Cultures ............................................................................................................. 89	3.2.3	 Immunocytochemistry .............................................................................................. 90	3.2.4	 Immunogold EM ....................................................................................................... 91	3.2.5	 Fluorescence Recovery After Photobleaching .......................................................... 92	3.2.6	 Spine Turnover Analysis ........................................................................................... 93	3.2.7	 Immunoblot Assay .................................................................................................... 93	3.2.8	 Image Analysis and Quantification ........................................................................... 93	3.2.9	 Statistical Analysis .................................................................................................... 94	3.3	 Results .............................................................................................................................. 94	3.3.1	 zDHHC5 is Localized to Pre- and Postsynaptic Compartments of Hippocampal Synapses ................................................................................................................................ 94	3.3.2	 Membrane-Associated zDHHC5 Promotes Excitatory Synapse Formation via its PAT Function ........................................................................................................................ 95	3.3.3	 zDHHC5 does not Regulate Dendrite Length or Complexity ................................ 101	3.3.4	 zDHHC5 Promotes Excitatory Synapse Formation in vivo .................................... 102	3.3.5	 zDHHC5 E648* Mutation Affects Protein Localization and Mobility .................. 103	3.3.6	 zDHHC5 Localization at Dendritic Spines Promotes Spine Stability .................... 105	vii  3.4	 Discussion ...................................................................................................................... 106	Chapter 4: Conclusion .............................................................................................................. 111	4.1	 The Role of Cadherins in Plasticity in the Ventral Tegmental Area ............................. 111	4.2	 β-catenin and Wnt Signaling in Dopaminergic Cells .................................................... 113	4.3	 zDHHC5 in Hippocampal Synaptogenesis .................................................................... 114	4.4	 Methodological Strengths and Limitations .................................................................... 116	4.5	 Future Directions ........................................................................................................... 118	4.5.1	 Role of Cadherin Adhesion Complexes in Drug-Mediated Plasticity in Mesolimbic Circuitry .............................................................................................................................. 119	4.5.2	 Presynaptic Targets of zDHHC5 Palmitoylation .................................................... 121	4.5.3	 Role of zDHHC5 in Drug-Mediated Plasticity in the Ventral Tegmental Area ..... 122	Bibliography .............................................................................................................................. 124	  viii  List of Figures Figure 1.1 Simplified Schematic of the Mesolimbic Dopamine System Circuitry in the Rodent Brain. ......................................................................................................................... 2	Figure 1.2 General Structure of a Glutamatergic Synapse. ............................................................ 8	Figure 1.3 Structure of AMPAR Subunits and Tetramers. .......................................................... 12	Figure 1.4 Cocaine-Mediated Synaptic Plasticity at Glutamatergic Inputs onto VTA Dopamine Neurons .................................................................................................................... 15	Figure 1.5 The Cadherin Family of Cell Adhesion Molecules .................................................... 19	Figure 1.6 Cadherin Interactions in Cis and Trans. ..................................................................... 20	Figure 1.7 β-catenin and Wnt Signaling ...................................................................................... 23	Figure 1.8 Basic Anatomy of the Rodent Hippocampus. ............................................................ 31	Figure 1.9 GABAergic Interneurons in the CA1 Region of the Hippocampus. .......................... 32	Figure 1.10 Phylogenetic Tree of the Mouse zDHHC Protein Family. ....................................... 37	Figure 2.1 Validation of Immunogold EM Reagents. .................................................................. 56	Figure 2.2 Cadherins are Expressed in Dopaminergic Neurons and are Essential for LTP in the VTA. ........................................................................................................................ 59	Figure 2.3 Quantification of Cadherin Expression in VTA Neurons. ......................................... 61	Figure 2.4 Cocaine-Induced CPP leads to Recruitment of Cadherin and GluA1 to Excitatory Synapses onto Dopaminergic Neurons in the VTA. ................................................ 63	Figure 2.5 Overall Levels of Cadherin and GluA1 at VTA Synapses are Unaffected by Acquisition and Extinction of CPP. ......................................................................... 65	ix  Figure 2.6 Time Spent in Cocaine-Conditioned Chamber is Correlated with Increased Cadherin localized to the Synaptic Membrane at both Pre- and Post-Synaptic Compartments of Excitatory Synapses onto Dopaminergic Neurons in the VTA. .......................... 65	Figure 2.7 Food CPP does not affect the Localization of Cadherin and GluA1 at VTA synapses. ................................................................................................................................. 67	Figure 2.8 No Changes in Localization of Cadherin at Synapses onto Non-Dopaminergic Neurons in the VTA Following Cocaine CPP. ........................................................ 69	Figure 2.9 Increased Cadherin and GluA1 Localization to the Synaptic Membrane at Individual Synapses onto Dopaminergic Neurons in the VTA following Cocaine CPP. ......... 69	Figure 2.10 Stabilization of Cadherin by β-catenin at Synapses in the VTA Reduces Cocaine-Induced CPP. ........................................................................................................... 71	Figure 2.11 No Changes in Food Consumption or Body Weight in DAT-Cre;β-catΔex3 mice. 72	Figure 2.12 Stabilization of Cadherin at Synapses in the VTA Prevents the Removal of GluA2-Containing AMPARs and Blocks the Insertion of GluA1-Containing AMPARs. .. 73	Figure 2.13 No Changes in DAT-Cre;β-catΔex3 Mice of Overall Expression of Wnt Targets in VTA Dopamine Neurons or Levels of Cadherin, GluA2 or GluA1 at VTA Synapses. ................................................................................................................. 74	Figure 2.14 Stabilization of Cadherin at Synapses in the VTA Blocks LTP by Retaining GluA2-Containing AMPARs and Preventing the Insertion of GluA2-lacking AMPARs. . 76	Figure 2.15 No Changes in DAT-Cre;β-catΔex3 Mice to Morphology or Density of VTA Synapses. ................................................................................................................. 78	Figure 2.16 Model of Changes in Cadherin and AMPAR Subunit Localization in Control and DAT-Cre;β-catΔex3 Mice during CPP. ................................................................... 80	x  Figure 3.1 zDHHC5 is Localized to Pre- and Postsynaptic Compartments of Hippocampal Synapses. ................................................................................................................. 95	Figure 3.2 Masking of Immunostaining Images using GFP Cell Fill from Transfected Neurons. ................................................................................................................................. 96	Figure 3.3 zDHHC5 Expression and Membrane Localization Affects Excitatory but not Inhibitory Synapse Density. .................................................................................... 97	Figure 3.4 Validation of E648* Mutant Expression and zDHHC5 Antibody Specificity. .......... 98	Figure 3.5 Excitatory Synapse Density measured by the Colocalization of Presynaptic Marker VGLUT1 and Postsynaptic Marker PSD-95. ........................................................ 100	Figure 3.6 zDHHC5 does not Affect Dendrite Outgrowth or Complexity. ............................... 101	Figure 3.7 zDHHC5 Expression Affects Excitatory but not Inhibitory Synapse Formation in vivo. ........................................................................................................................ 102	Figure 3.8 A zDHHC5 Truncation Mutant found in a Patient with Schizophrenia Results in Improper Localization and Increased Mobility of the Protein. .............................. 104	Figure 3.9 GFP-zDHHC5 Localization in Spines Promotes Spine Stability. ............................ 106	 xi  List of Abbreviations A/N   AMPAR/NMDAR ABHD   α/β-hydrolase domains ABP   actin binding protein AD   Alzheimer’s Disease AMPARs  α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid type receptors ANOVA  analysis of variance APC   adenomatous polyposis coli ARVCF  armadillo repeat gene deleted in Velo-Cardio-Facial syndrome CA   Cornu Ammonis CA1    Cornu Ammonis area 1 CA2   Cornu Ammonis area 2 Ca2+   calcium ion CA3    Cornu Ammonis area 3 CELSRs  cadherin EGF LAG seven-pass G-type receptors CK1   casein kinase 1α/δ  cLTP   chemical long-term potentiation CNVs   copy number variants CPP   conditioned place preference DAT-Cre;β-catΔex3 Slc6a3:Cre/+;Ctnnb1lox(ex3)/lox(ex3) mice DG    dentate gyrus DHHC  aspartate-histidine-histidine-cysteine motif DHPG   3,5-dihydrophenylglycine DIV   days in vitro Dsh   disheveled E   embryonic day EC   entorhinal cortex EC repeats  extracellular cadherin repeats EPSC   excitatory postsynaptic current EPSP   excitatory postsynaptic potential EPSP   excitatory postsynaptic potential Fz   frizzled GABA   γ-aminobutyric acid GABARs  GABA receptors GAD67  L-glutamic acid decarboxylase 67 GluA(1-4)  glutamate receptor subunits 1-4 GRIP1  glutamate receptor interacting protein 1 GSK3   glycogen synthase kinase 3 α/β HAV   histidine-alanine-valine motif Ih   large hyperpolarization-activated cation current IPSC   inhibitory postsynaptic current L-LTP  late phase LTP LIMK1  LIM Kinase 1 LRP5/6  lipoprotein receptor-related protein 5 or 6 xii  LTD   long-term depression LTP   long-term potentiation MAGUK  membrane-associated guanylate kinase Mg2+   magnesium ion mGluR  metabotropic glutamate receptor mPFC   medial prefrontal cortex MSNs   medium spiny neurons NAc    nucleus accumbens NASPM  1-Naphthyl acetyl spermine trihydrochloride NCAM  neural cell adhesion molecule NL1    neuroligin-1 NMDARs  N-methyl-D-aspartate type receptors O-LM   oriens-lacunosum moleculare PAT   palmitoyl-acyltransferase PDZ   PSD-95/Disc large/Zona-occludens PEST   proline-glutamic acid-serine-threonine motif PCR   polymerase chain reaction PFA   paraformaldehyde PICK1  protein interacting with C kinase-1 PKA   protein kinase A PKC    protein kinase C PPT   palmitoyl-protein thioesterases PSD   postsynaptic density PSD-95  postsynaptic density-95 RE   recycling endosome RNAi   ribonucleic acid interference ROI   region of interest RRP   readily releasable pool  S-SCAM  synaptic scaffolding molecule shRNA   short hairpin ribonucleic acid  SILAC  stable isotope labeling with amino acids in cell culture SNPs   single nucleotide polymorphisms SNVs   single nucleotide variants STD LTP  spike timing dependent long-term potentiation STDP   spike timing dependent plasticity STEP61   striatal enriched phosphatase 61 STREX  spliced stress-related exon SVs   synaptic vesicles SynCAM  synaptic cell adhesion molecule TH   tyrosine hydroxylase VGLU T2  vesicular glutamate transporter 2 VTA   ventral tegmental area xiii  Acknowledgements First of all, I would like to thank my Ph.D. supervisor, Dr. Shernaz Bamji for all of her guidance and support throughout my time in the lab. Shernaz - I can always count on you to push me to do my best work, whether that’s performing difficult experiments, writing clearly and concisely, or communicating my research at conferences. (Plus, you were also always there to “share” the white wine with me at parties…) I’m grateful to have had such an excellent mentor over the past 6 years, and I hope that we can keep in touch in the future.  I would also like to thank the members of my research advisory committee, Dr. Lynn Raymond, Dr. Liisa Galea and Dr. Tim O’Connor, for their time, consideration, and support throughout my Ph.D. I truly appreciate your commitment to my development as a scientist.   I would like to thank all the members of the Bamji lab who have been my second family here in Vancouver. I always knew that I could count on you all for a word of encouragement, help with experiments, and congratulatory or conciliatory beers (depending on which was more appropriate at the time). I would especially like to thank my current and past colleagues Stefano Brigidi, Jenya Pethoukov, Riki Dingwall, Jordan Shimell and Mahsan Mobasser, whom I hope to always call my friends.   I would especially like to thank my scientific “other half” Fergil Mills – I’m so glad I had the chance to learn so much about research (and share a lot of dance music!) with you. There certainly would be no ‘Ferndrea’ without you, thanks for making the lab so much fun.  As it always happens, there are many new faces in our lab – new friends who have joined our group as I’m preparing to leave. Bhavin, Matt and Nusrat – although our time together has been relatively short, I’m glad to know that I’m leaving the lab in the hands of such positive, wonderful people.   I would especially like to thank my parents, Rita and Ed; and my brother, Adam for all their love, thoughtfulness and support through not only the grad school process, but also in all areas of my life. I’m thankful to always have you in my corner. (Thank goodness for FaceTime!)  Finally, I cannot thank enough my wonderful fiancé Viktor for his endless patience, love and kindness during this process. I know that dealing with a stressed out Ph.D. student isn’t the easiest thing. I’m thankful that you’ve always helped to calm me down and given me some needed perspective during the hard times, and celebrated with me during the exciting moments. I look forward to our future together.  Funding for this work was provided by the CIHR Frederick Banting and Charles Best Graduate Scholarships, the UBC 4-year Fellowship, the Dorothy May Ladner Memorial Fellowship, and the College for Interdisciplinary Studies (CFIS).  xiv  Dedication   For my Oma and Opa. Thank you for always supporting my education.      1  Chapter 1: Introduction This thesis focuses on the regulation of cocaine mediated synaptic plasticity by cadherin adhesion complexes, and the regulation of synapse formation by the cadherin complex modifying protein, zDHHC5. The first project (described in Chapter 2) examines cocaine-mediated changes in synapse strength in the ventral tegmental area of the mesocorticolimbic dopamine circuit. In chapters 1.1-1.4, I will first describe the circuit, the role of dopamine in motivated behaviour, cocaine-mediated synaptic plasticity in this circuit, as well as introduce specific synaptic proteins relevant to the study. The second project (described in Chapter 3) examines the role of the palmitoyl acyl-transferase, zDHHC5, a regulator of cadherin stability at the synapse, in dendrite outgrowth and synaptogenesis in primary hippocampal cell culture. In chapters 1.5-1.8, I will therefore provide background for the hippocampal circuitry, our current understanding of synapse formation, as well as introduce the zDHHC family of palmitoyl acyl-transferases.   1.1 The Mesocorticolimbic Dopamine Circuit.  The mesocorticolimbic dopamine circuit consists of a large population of midbrain dopamine neurons, located within the ventral tegmental area (VTA), which project to their targets in several brain regions (Figure 1.1). This circuit has been shown to play an important role in working memory, attention, motivation and response to reward-predictive cues. Drugs of abuse cause an increase in dopamine release from the VTA dopamine neurons to their targets in the nucleus accumbens (NAc) and the medial prefrontal cortex (mPFC). For this reason, a great deal of research has focused on the excitatory and inhibitory inputs formed onto these VTA dopamine neurons, as changes in the strength of these inputs could result in increased dopamine 2  output from the VTA. I will focus my review on the dopaminergic neurons of the VTA as we are specifically focused on this region in this thesis.   Figure 1.1 Simplified Schematic of the Mesolimbic Dopamine System Circuitry in the Rodent Brain. The inputs to the nucleus accumbens (NAc) and ventral tegmental area (VTA) are highlighted (glutamatergic projections in blue, dopaminergic projections in red, GABAergic projections in orange, and orexinergic projections in green). While glutamatergic synapses excite postsynaptic cells and GABAergic synapses inhbit postsynaptic cells, dopamine exerts more complex modulatory effects on target neurons. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Neuroscience Kauer & Malenka, copyright 2007.  1.1.1 Dopaminergic Neurons of the Ventral Tegmental Area Dopaminergic neurons of the VTA are critical for motivated behaviours, and it is thought that the activity of these neurons is altered in response to drugs of abuse. The majority of VTA neurons are dopaminergic neurons (~65% of cells), but there are also γ-aminobutyric acid (GABA) containing and glutamate containing neurons (~30% and 5%, respectively) within the VTA (Margolis et al., 2006; Ungless and Grace, 2012; Steffensen et al., 1998; Yamaguchi et al., 2007; Hnasko et al., 2012). Dopamine neurons can fire either in a tonic, low frequency single 3  spike pattern, or in a phasic, bursting pattern where action potentials fire at high frequencies over several hundred milliseconds (Grace and Bunney, 1984a, 1984b). Indeed, research has shown that burst firing is critical for dopaminergic signaling. Burst firing results in significantly increased dopamine release at the synaptic cleft of dopaminergic outputs, however this is tightly controlled by dopamine reuptake mechanisms (Floresco et al., 2003). In contrast, tonic firing results in increased dopamine at extrasynaptic sites, which is less tightly regulated by reuptake (Floresco et al., 2003). Although the GABAergic neurons of the VTA are less well understood, a subset of these GABA neurons synapse onto VTA dopamine neurons, providing local inhibition of dopamine cell firing in response to aversive stimuli (Creed et al., 2014).   1.1.2 Afferent Projections onto VTA Dopamine Neurons The VTA dopamine neurons receive glutamatergic projections from a number of cortical brain regions, although the majority of inputs are from subcortical regions (Geisler et al., 2007). The subcortical inputs include the pedunculopontine nucleus, laterodorsal tegementum nucleus, the lateral habenula, periaqueductal grey, the bed nucleus of the stria terminalis, and the dorsal raphe nucleus (Geisler et al., 2007; Morales and Margolis, 2017). VTA dopamine neurons receive GABAergic inputs from the periaqueductal grey, dorsal raphe nucleus, lateral hypothalamus, and ventral pallidum (Morales and Margolis, 2017). Furthermore, VTA dopamine neurons receive GABAergic input from interneurons within the VTA. These interneurons have a strong affect on dopamine cell firing, and behaviour (Creed et al., 2014). Dopamine cell firing patterns are driven by the excitatory glutamatergic inputs and inhibitory GABAergic inputs onto VTA dopamine neurons, which coordinate neuronal activity. Burst firing is regulated by excitatory afferent inputs, specifically through Ca2+ influx via N-4  methyl-D-aspartate type receptors (NMDARs; (Grace and Bunney, 1984b; Johnson and North, 1992a; Komendantov et al., 2004; Zweifel et al., 2009; Paladini and Roeper, 2014).2017-10-12 5:26 PM Furthermore, the excitability of dopamine neurons is significantly reduced by GABAA receptor activation (Johnson and North, 1992a; Tan et al., 2012; van Zessen et al., 2012).    1.1.3 Efferent Projections from VTA Dopamine Neurons   The VTA dopamine neurons project to the NAc, the mPFC, and the amygdala (Beier et al., 2015; Swanson, 1982). Interestingly, more recent examination of the VTA dopaminergic cell population indicates that these neurons are not a homogeneous group. Indeed, VTA dopamine cells have differing properties depending the target brain regions onto which they project (Margolis et al., 2006; Lammel et al., 2008, 2011, 2012; Baimel et al., 2017). These VTA dopamine cell subpopulations receive inputs from different brain regions and their activity has differential effects on behaviour (Lammel et al., 2012). Specifically, inputs from the laterodorsal tegmentum synapse onto dopamine neurons that project to the NAc lateral shell, and drive a type of behavioural conditioning where spatial context is paired with a rewarding stimulus, called conditioned place preference (see chapter 2.2.6 for more detail on conditioned place preference methods). The inputs from lateral habenula synapse onto dopamine neurons that project to the medial prefrontal cortex and the rostromedial tegmental nucleus, and drive behavioural conditioning where spatial context is paired with an aversive stimulus, called conditioned place aversion (Lammel et al., 2012).  Recent work has shown that VTA dopamine neurons also respond to orexin and dynorphin differentially, depending on their projection targets (Baimel et al., 2017). While VTA dopamine neurons that project to the NAc lateral and medial shell exhibited increased firing in 5  response to orexin A, dopamine neurons projecting to the basolateral amygdala were unaffected. Furthermore, dynorphin selectively reduced firing in VTA dopamine neurons that project to the NAc medial shell and basolateral amygdala, but did not affect dopamine neurons projecting to the NAc lateral shell (Baimel et al., 2017). It seems that orexin A and dynorphin may tune dopamine responses in a target-specific manner. Indeed, much more work needs to be done to tease apart the subtle differences between dopaminergic cell subpopulations.  1.2 Dopamine in Learning and Motivation The precise role of dopamine in motivated behaviour is still not clearly understood. It was initially thought that dopamine release signaled reward, and that dopamine directly encoded the pleasurable perception of rewarding stimuli. Indeed, the “dopamine reward hypothesis” was proposed to explain the finding that rats would lever press for electrical self-stimulation of the medial forebrain bundle (Bielajew and Shizgal, 1986; Olds and Milner, 1954), which contains projections from the VTA. This theory suggested that the dopamine output in the NAc encoded the motivational and pleasurable characteristics of rewarding natural stimuli (Wise, 1978). However, this hypothesis has been largely abandoned due to a growing body of evidence indicating that dopamine synthesis or dopamine receptor activation is not necessary for the hedonic impact, or “liking”, of natural rewards or drugs of abuse (Berridge et al., 1989; Brauer and De Wit, 1997; Gawin, 1986; Leyton et al., 2005; Nann-Vernotica et al., 2001). Moreover, the concentration of dopamine in the NAc actually drops significantly during reward consumption (Hamid et al., 2016). Several alternative theories have been proposed to determine the connection between dopamine and motivation.  6  First, the “incentive salience hypothesis” proposes that the function of dopamine is to give motivational value (i.e. incentive) to reward-predicting stimuli, which makes these stimuli more likely to attract attention and drive approach behaviours (Berridge, 2007; Robinson and Berridge, 1993). Given that addictive drugs increase dopamine transmission, the incentive salience hypothesis speculates that addiction may result from repeated use of addictive drugs because the individual becomes hyper-sensitized to the act of drug-taking and its associated cues (Robinson and Berridge, 1993).  The second hypothesis highlights the role of dopamine in the process of learning. The “reward prediction error hypothesis” is based on the finding that after sensory stimuli are paired with a reward (i.e. auditory tone precedes arrival of food reward), the phasic activation of dopamine neurons and resulting dopamine release shifts from the time of reward delivery (food reward) to cue onset (auditory tone) (Day et al., 2007; Schultz et al., 1997). This is called positive prediction error. In contrast, the activity of dopaminergic neurons is greatly reduced when an expected reward is omitted (i.e. auditory tone, but food reward not received), called a negative prediction error (Schultz et al., 1997). Together, this suggests that dopaminergic signals can encode unexpected changes in reward delivery (Collins and Frank, 2016).   It has also been suggested that dopamine plays an important role in the willingness to work to obtain rewards, and allocate appropriate behavioural resources and motivation to pursue rewards (Hernandez et al., 2010; Salamone and Correa, 2002; Salamone et al., 2001). There is experimental support for each of these alternative hypotheses, however the integration of these approaches seems to best reflect dopamine’s role in the function of the NAc (Hamid et al., 2016; Syed et al., 2016).  7  Many researchers have studied the role of dopamine in the NAc, and how the release of dopamine from VTA neurons onto medium spiny neurons of the NAc can drive motivated behaviour. However, dopamine release plays an important role in the function of other brain regions as well. For instance, dopamine release in the prefrontal cortex has been linked to normal working memory function (Durstewitz and Seamans, 2002; Seamans et al., 1998), and dopamine in the dorsal striatum is critical for proper motor function (Palmiter, 2008). However, the role of dopamine in these brain regions is beyond the scope of this thesis.  1.3 Synapse Structure and Function Neurons within the mesocorticolimbic circuit and indeed, throughout the brain communicate with each other through specialized structures called synapses. Synapses are specialized points of cell-cell contact that are able to rapidly transmit information from the presynaptic neuron to the postsynaptic neuron. These points of cell communication are characterized by the neurotransmitter that they use to transduce a signal from one cell to another. Some neurotransmitters cause the postsynaptic cell to be more likely to fire an action potential (e.g. glutamate), others make the postsynaptic cell less likely to fire an action potential (e.g. GABA), and some neurotransmitters are neuromodulatory, having different effects depending on the receptor with which they interact (e.g. dopamine, serotonin). Glutamatergic synapses are the major class of excitatory synapses found in the mammalian brain, consisting of a presynaptic compartment in close apposition to a postsynaptic membrane localized to a spiny protrusion emerging from the cell’s dendrite (Figure 1.2). In the subsequent subchapters I will focus on the structure, function and plasticity of excitatory glutamatergic synapses.  8   Figure 1.2 General Structure of a Glutamatergic Synapse. Glutamatergic synapses are usually formed where a presynaptic bouton along an axon (top) contacts a postsynaptic dendritic spine that protrudes from a dendrite (bottom). The presynaptic compartment contains synaptic vesicles that contain the neurotransmitter glutamate. Synaptic vesicles can fuse with the plasma membrane allowing the release of glutamate across the synaptic cleft. Glutamate binds to postsynaptic receptors such as AMPA and NMDA receptors that are primarily localized to the postsynaptic density (PSD). Binding of glutamate allows the postsynaptic receptors to alter their conformation, opening the channel pore and allowing the influx of ions. This influx of ions depolarizes the postsynaptic neuron. Many cell adhesion molecules are localized to both pre- and postsynaptic sites (Image made by Andrea Globa).  1.3.1 Structure of the Presynaptic Compartment The presynaptic compartment releases the neurotransmitter glutamate through the membrane fusion of synaptic vesicles (SVs). The presynaptic compartment is composed of many neurotransmitter-filled SVs and the active zone. The active zone is characterized by an electron-dense matric of proteins, known as the cytoskeletal matrix or presynaptic grid, and the active zone plasma membrane (Ziv and Garner, 2004). The fusion of SVs with the plasma membrane in response to action potentials occurs at the active zone.    The movement of SVs is regulated by the synaptic vesicle cycle, where the steps include SV docking, priming, fusion and recycling (Südhof and Rizo, 2011). SVs move through the cytoskeletal matrix and are docked at the active zone plasma membrane. Following docking, primed SVs will fuse with the plasma membrane upon depolarization and calcium influx, releasing their contents into the synaptic cleft. Endocytic recycling occurs when plasma presynaptic postsynaptic 		synaptic vesicle Cell adhesion  molecules NMDA receptor AMPA receptor 9  membrane outside of the active zone is endocytosed, so it can be recycled for reuse (Cingolani and Goda, 2008; Murthy and De Camilli, 2003).   The presynaptic terminal actin cytoskeleton is extensive (Drenckhahn et al., 1984; Landis et al., 1988), and has been proposed to regulate and maintain the SV pools, in part by serving as a scaffold to prevent vesicle movement and in part by directing the transfer of SVs (Cingolani and Goda, 2008). Cell adhesion molecules such as cadherins, neurexins and synaptic cell adhesion molecules (synCAMs) are often localized adjacent to the active zone, and can work with the actin cytoskeleton to recruit SVs to the plasma membrane active zone (Brigidi and Bamji, 2011; Sun and Bamji, 2011).  1.3.2 Structure of the Postsynaptic Compartment The postsynaptic compartment of glutamatergic synapses is usually localized to dendritic spines, which are tiny protrusions along the dendrite. The postsynaptic compartment is designed to receive signals from presynaptic glutamate release, and transduce these signals using glutamate receptors at the postsynaptic membrane. Beneath the postsynaptic membrane is the postsynaptic density (PSD), which is a specialized membrane microdomain that is directly apposed to the presynaptic active zone across the synaptic cleft. The PSD is made up of an electron-dense matrix of protein, including cell adhesion proteins, cytoskeletal proteins, scaffolding proteins, glutamate receptors, G-proteins and their modulators, and signal transduction molecules (Boeckers, 2006). Post-synaptic density-95 (PSD-95) is one of the most abundant PSD proteins; indeed, the presence of PSD-95 is often used as a marker for the presence of excitatory synapses. This scaffolding protein is one of the membrane-associated guanylate kinase (MAGUK) family 10  proteins (Sheng and Hoogenraad, 2007). Given that PSD-95 has a several domains through which it can associate with other proteins, it is not surprising that this protein plays an important role in synapse assembly. PSD-95 also plays an important role in synapse function, as it anchors glutamate receptors within the membrane through both direct and indirect interactions (Sheng and Hoogenraad, 2007). Indeed, the overexpression of PSD-95 in cultured hippocampal neurons enhances glutamate receptor clustering and increases the number of postsynaptic spines (El-Husseini et al., 2000), while knockdown reduces the clustering of receptors at postsynaptic compartments (Elias et al., 2006).  One of the most important functions of the PSD is the clustering and transport of membrane-bound glutamate receptors. The two main types of glutamate receptor at the postsynaptic membrane that mediate fast excitatory glutamatergic transmission are N-methyl-D-aspartate type receptors (NMDARs), and α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid type receptors (AMPARs).  1.3.2.1 NMDAR Receptors NMDARs are a major class of receptor that is important for excitatory glutamatergic transmission at hippocampal synapses. Although NMDARs do not mediate a significant portion of fast basal synaptic transmission, these receptors are critical for synaptic plasticity. The NMDARs have several unique features, including a voltage-sensitive block where extracellular Mg2+ ions block the channel pore under basal conditions, high Ca2+ permeability, and slow ‘activation/deactivation’ kinetics. NMDARs are also unique in that they are both ligand-gated and voltage-gated. Both glutamate and the co-agonist glycine are required to open the receptor, which provides an additional control over NMDARs. Calcium influx through synaptic NMDARs 11  is most commonly associated with the downstream expression of pro-survival genes and synaptic plasticity (Hunt and Castillo, 2012; Lonze and Ginty, 2002). NMDARs are necessary for burst firing of dopamine neurons (Zweifel et al., 2009), and for strengthening of excitatory synapses formed onto VTA dopamine neurons. In mice where the obligatory GluN1 subunit of NMDARs is knocked out in dopamine neurons, burst firing and phasic dopamine release is impaired (Zweifel et al., 2009). Furthermore, the increases in synaptic strength normally observed after cocaine exposure are abolished in these mice (Engblom et al., 2008), although the behavioral effects are less clear (Engblom et al., 2008; Zweifel et al., 2009).  1.3.2.2 AMPA Receptors AMPARs mediate the primary depolarization at glutamatergic synapses in the brain, and play a critical role in synaptic plasticity. Their role in cocaine-mediated plasticity in the VTA will be discussed in Chapter 1.3.3.1, and further studied in Chapter 2. Most AMPARs are calcium-impermeable, although this is dependent on the composition of AMPAR subunits, and modifications that can be introduced by alternative splicing and RNA editing (Greger and Esteban, 2007). AMPARs are assembled from 4 subunits, GluA1 – GluA4, and form tetramers with unique properties depending on the composition of the tetramers. At hippocampal synapses, AMPARs are predominantly GluA1/2 heterodimers, with a small fraction of GluA2/3 heterodimers (Lu et al., 2009; Wenthold et al., 1996). GluA2/3 and GluA1 specific antibodies show strong staining in dopaminergic cells within the VTA (Chen et al., 2001; Petralia and Wenthold, 1992). A study examining the mRNA expression of AMPAR subunits found that GluA2 was the most strongly expressed subunit in the VTA, followed by GluA1, with GluA3 and GluA4 being expressed least strongly (Sato et al., 1993). Furthermore, cultured VTA 12  dopamine cells also have a substantial number of GluA1/2 heteromers, some GluA2/3 heteromers, and few GluA2-lacking receptors at the cell surface under basal conditions (Gao and Wolf, 2007).  The presence or absence of the GluA2 subunit has important effects on the biophysical properties of AMPARs, such that GluA2-containing AMPARs are calcium impermeable, and therefore have a linear current-voltage relationship, whereas GluA2-lacking receptors are calcium permeable, and therefore have an inwardly rectifying current-voltage relationship (Anggono and Huganir, 2012). Interestingly, the mRNA transcript encoding the GluA2 subunit undergoes RNA editing which introduces a glutamine to arginine point mutation at the pore of the receptor. The presence of the arginine residue is responsible for the unique properties of GluA2-containing AMPARs (Hume et al., 1991; Swanson et al., 1996; Verdoorn et al., 1991). GluA2-lacking receptors play an important role in synaptic plasticity at excitatory synapses forming onto DA neurons within the VTA (Saal et al., 2003; Ungless et al., 2001), which will be discussed in chapter 1.3.3.1.  Figure 1.3 Structure of AMPAR Subunits and Tetramers. Structure of AMPAR subunits (left). AMPAR subunits have an extracellular N-terminal region, four transmembrane domains, and a short or long intracellular C-terminal tail (depending on subtype). Within the second transmembrane domain lies the ‘Q/R editing site’ – this single amino acid can be modified from a glutamine (Q) to arginine (R) by 13  RNA editing, preventing Ca2+ ions from passing through the AMPAR. Tetrameric structure of the AMPAR (right).  The channel pore region of the AMPAR is formed by the second transmembrane domain and positions the Q/R site at the pore to determine ion selectivity of the receptor. The ligand binding site is formed between the extracellular domains. Adapted with permission from Shepherd & Huganir, 2007.  AMPAR subunit composition can also affect trafficking, synaptic delivery and localization of AMPARs along the postsynaptic membrane, through differences in the subunits’ C-terminal tails. The cytosolic C-terminal tails are either long (as in GluA1 and GluA4), or short (as in GluA2 and GluA3), and can regulate the synaptic delivery of AMPARs (Köhler et al., 1994; Shi et al., 2001). Indeed, long-tailed (ie. GluA1-containing) AMPARs are important for activity-dependent insertion of AMPARs to hippocampal synapses during synaptic plasticity (Hayashi et al., 2000; Shi et al., 1999). In contrast, short-tailed (ie. GluA2/3) AMPARs are present at synapses and appear to be constitutively recycled under basal conditions (Song et al., 1998; Lüscher et al., 1999; Noel et al., 1999; Shi et al., 2001). However internalization of GluA2-containing AMPARs is necessary for synaptic weakening in long-term depression (Lüthi et al., 1999).  1.3.3 Synaptic Plasticity The strength of synaptic connections is continually altered in response to previous neural activity, meaning that synapses are incredibly plastic in nature. Indeed, synaptic plasticity is critical for the development of the CNS, as well as higher brain functions such as learning and memory. Synaptic connections can be strengthened through a process called long-term potentiation (LTP), and they can be weakened through long-term depression (LTD). There are several types of LTP and LTD that rely on the activation of NMDARs, metabotropic glutamate receptors (mGluRs), or endocannabinoid receptors. These forms of synaptic plasticity have been 14  most extensively studied in the hippocampus. Synaptic plasticity in the mesocorticolimbic circuit in response to addictive drugs will be discussed in detail in the subsequent sections.  1.3.3.1 Drug-Mediated Synaptic Plasticity in the Ventral Tegmental Area Drugs of abuse have been shown to cause an increase in dopamine release from dopaminergic neurons in the VTA. Indeed, the excitatory synapses forming onto VTA dopamine neurons are key sites that can regulate the activity of this circuit (Figure 1.4). Changes in the trafficking of AMPARs at these synapses have been identified in rodents after exposure to drugs of abuse (Wolf and Tseng, 2012). Initial electrophysiological studies identified an increase in the AMPAR/NMDAR (A/N) ratio after non-contingent (i.e. intraperitoneal, i.p.) cocaine administration (Ungless et al., 2001), with subsequent studies finding that this increase in A/N ratio was specific to drugs of abuse such as cocaine, amphetamine, morphine, nicotine, and ethanol, but not observed with therapeutic drugs such as the specific serotonin reuptake inhibitor, fluoxetine (Faleiro et al., 2003; Saal et al., 2003). The changes in response at excitatory synapses formed onto VTA dopamine neurons can be seen within 3-5 hours of cocaine administration (Argilli et al., 2008). This increase in the A/N ratio was NMDAR-dependent, as the administration of the NMDAR antagonist MK-801 along with cocaine blocked the increase in A/N ratio (Ungless et al., 2001). Indeed, the cocaine-induced increase in A/N ratio was seen as a form of LTP, as LTP was occluded in brain slices from mice that had been given cocaine (Ungless et al., 2001; Argilli et al., 2008; Mameli et al., 2011). Interestingly, the magnitude of the increase in A/N ratio was found to be similar whether rodents were given chronic cocaine injections or a single injection (Borgland et al., 2004).  15   Figure 1.4 Cocaine-Mediated Synaptic Plasticity at Glutamatergic Inputs onto VTA Dopamine Neurons Under basal conditions, the AMPARs localized to excitatory synapses onto VTA dopamine neurons are Ca2+ impermeable, GluA1/2 heteromers. After exposure to cocaine, GluA1/2 heteromers are removed from these synapses and Ca2+ permeable GluA1 homomers are inserted (Ungless et al., 2001; Mameli et al., 2007; Argilli et al., 2008) (Image made by Andrea Globa).  Although the increase in A/N ratio observed in the VTA after cocaine administration was initially attributed to an increase in the number of AMPARs inserted at synapses with no change in synaptic NMDARs (Ungless et al., 2001), further studies were able to refine this model. The increased A/N ratio was actually due to the replacement of low conductance GluA2-containing AMPARs (which are Ca2+ impermeable) with higher conductance GluA1 homomer type AMPARs (which are Ca2+ permeable; Argilli et al., 2008; Bellone and Lüscher, 2006; Mameli et al., 2007). This cocaine-induced switch in AMPAR type is dependent on the interaction between the PSD-95/Discs large/Zona-occludens (PDZ) domain containing protein, protein interacting with C kinase-1 (PICK1), and GluA2 subunits (Bellone and Lüscher, 2006), indicating that protein kinase C mediated phosphorylation of GluA2 subunits likely drives the endocytosis of GluA2-containing AMPARs during this type of plasticity (Anggono and Huganir, 2012; Hanley, 2008). Glutamatergic Input Na+ Na+ Ca2+ Basal Conditions Cocaine Exposure GluA1/2 heteromer GluA1  homomer dopamine neuron 16  It was initially thought that the increased A/N ratio was a transient effect, as this increase could be observed 5 days but not 10 days after non-contingent cocaine injections (Ungless et al., 2001; Borgland et al., 2004). However, the A/N ratio was much more persistently elevated when rats self-administered cocaine as compared to previous studies, where non-contingent i.p. injections were used (Chen et al., 2008). Rats that self-administered cocaine showed an increased A/N ratio compared to saline controls for up to 3 months after the last cocaine self-administration session. Importantly, rats that self administered for sucrose rewards did not show this lasting increase, nor did cocaine-infused ‘yoked controls’, which received a similar dosage and pattern of cocaine infusions as the self-administration group, but cocaine infusion was not contingent on lever pressing. These results suggested the increase in A/N ratio is not strictly due to the pharmacological effects of cocaine, but instead could be a form of associative learning involving cues that signal drug delivery (Chen et al., 2008).  The reversal of cocaine-mediated GluA1 homomer insertion relies on a form of LTD that is mediated by the metabotropic glutamate receptor mGluR1. The activation of mGluR1 results in the removal of GluA1 homomers from excitatory synapses onto VTA dopamine neurons, and the insertion of newly synthesized Ca2+ impermeable GluA2-containing AMPARs localized to VTA synapses (Bellone and Lüscher, 2006; Mameli et al., 2007). This form of plasticity can be induced by the mGluR agonist 3,5-dihydrophenylglycine (DHPG) or low-frequency stimulation (Bellone and Lüscher, 2005, 2006). Indeed, if mGluR function is increased in vivo by i.p. injection of a positive modulator, the cocaine-induced increase in GluA1 homomers at VTA synapses is significantly reduced (Mameli et al., 2009). Furthermore, the activation of mGluR1 in the VTA reduces cocaine-induced plasticity observed ‘downstream’ in the NAc.  17  1.3.3.2 Drug-mediated Synaptic Plasticity in the Nucleus Accumbens  Drug-mediated plasticity in the NAc has also been an important focus of study. An increase in the number of Ca2+ permeable GluA1 homomer AMPARs at synapses onto medium spiny neurons (MSNs) in the NAc has been observed after cocaine self-administration. It is thought that this increase in AMPARs might contribute to the incubation of craving observed in this model of addiction (Conrad et al., 2008; Mameli et al., 2009; McCutcheon et al., 2011a, 2011b). Similar to the VTA, mGluR activation in the NAc can also reduce the number of GluA1 homomers present at synapses within the NAc after extended access cocaine self administration (McCutcheon et al., 2011b). Interestingly, behavioural sensitization studies have shown that increased locomotor response to cocaine is correlated with increased surface expression of both GluA1/2 heteromers in NAc MSNs, suggesting that synaptic changes in the NAc may contribute to drug-mediated behaviours (Boudreau and Wolf, 2005; Boudreau et al., 2007; McCutcheon et al., 2011a).  1.4 The Cadherin Adhesion Complex Cadherins are transmembrane cell adhesion proteins with a significant extracellular domain. Indeed, this superfamily of proteins is characterized by the unique extracellular cadherin (EC) repeats, which are important for calcium binding. The number of EC repeats varies between members of the cadherin superfamily (Takeichi, 2007). Although the classical family of cadherins has been most widely studied, there are other subfamilies within cadherin superfamily of proteins. These include the desmocollins, desmogleins, Drosophila Flamingo/mammalian cadherin EGF LAG seven-pass G-type receptors (CELSRs) and protocadherins (Figure 1.5;  Brasch et al., 2012; Takeichi, 2007).  18  Classic cadherins are characterized by 5 extracellular cadherin repeats (EC1-5) within the extracellular domain, and interact with the actin cytoskeleton through their associations with intracellular binding proteins α-, β-, δ-, and p120-catenin (Arikkath and Reichardt, 2008; Dalva et al., 2007). Classic cadherins form calcium-dependent trans interactions with cadherins on opposing membranes, as well as cis interactions with neighboring cadherin proteins (Figure 1.6) (Shapiro et al., 1995; Boggon et al., 2002; Harrison et al., 2011). These interactions are usually homophilic, but some studies suggest that neuronal (N-) and retinal (R-) cadherin can form cis heterodimers in cultured cells (Shan et al., 2000).  N-cadherin is the most strongly expressed classical cadherin in the central nervous system (Missler et al., 2012; Uchida et al., 1996). N-cadherin is found at pre- and postsynaptic terminals, and has been shown to play a role in synapse formation and synaptic plasticity (Arikkath and Reichardt, 2008; Missler et al., 2012). Here, I will review the role of cadherin binding proteins β-catenin and δ-catenin, and discuss the role of cadherin adhesion complexes in synapse plasticity. The role of cadherins in synapse formation will be highlighted in chapter 1.7, where synapse formation is discussed in the literature review for Chapter 3. 19   Figure 1.5 The Cadherin Family of Cell Adhesion Molecules Representative examples of the domain structures of different cadherin subfamilies. All cadherin family members share one common structural motif: they all have at least one ‘extracellular cadherin’ (EC) domain (purple ovals, numbered from the most distal to membrane proximal). Other common features include EGF-repeats (green rectangles), laminin A G domains (blue diamonds), and flamingo boxes (pink oval). Some cadherins also have a prodomain (grey) which is cleaved when the protein reaches the cell surface. Adapted from Trends in Cell Biology, 22(6) Brasch, Harrison, Honig, & Shapiro. Thinking outside the cell: how cadherins drive adhesion. Pages 299-310. Copyright (2012), with permission from Elsevier.  20   Figure 1.6 Cadherin Interactions in Cis and Trans. X-ray crystallography studies of cadherin interactions showing trans and cis interactions between classical cadherins. The five EC repeats of classical cadherins form a curved structure that is stabilized when three calcium ions bind to sites between each of the EC repeats (Boggon et al., 2002; Harrison et al., 2011). Cadherin trans interactions are mediated by a ‘strand-swap’ interface, where the N-terminal strand from the EC1 domain is exchanged with a partner molecule on the opposing membrane. A conserved tryptophan residue interacts with a hydrophobic His-Ala-Val pocket on the opposing strand, forming a pair of ‘ball-and-socket’ joints (Shapiro et al., 1995; Boggon et al., 2002; Harrison et al., 2011). Classical cadherins also form cis interactions, which may mediate the formation of an ordered lattice structure. The cis interactions occur between the concave side of the EC1 domain, which interacts with the convex side of the EC2/3 domain. Specifically, conserved valine residue 81 is critical for this cis interaction, and in N-cadherin, valine residue 174 is also important for strong cis interactions (Harrison et al., 2011). Adapted with permission from (Wu et al., 2010).   1.4.1 Cadherin Binding Proteins Classic cadherins have a strongly conserved intracellular domain, with a juxtamembrane domain capable of binding p120-catenin and δ-catenin, and an extended β-catenin binding motif. Juxtamembrane interactions with catenin proteins regulate the cis clustering of cadherins as well as interactions with secondary binding partners (Ide et al., 1999; Jones et al., 2002; Silverman et al., 2007; Misra et al., 2010). β-catenin interactions prevent the endocytosis of cadherin adhesion complexes, resulting in increased cadherin stability at the synaptic membrane (Tai et al., 2007).  21  1.4.1.1 β-catenin Structure and Function The intracellular protein β-catenin is a major binding partner of all classical cadherins. β-catenin binds to the C-terminal tail of cadherin through its 12 armadillo repeats (Hülsken et al., 1994; Pai et al., 1996). Although β-catenin does not directly bind to the N-cadherin endocytic motif, β-catenin binding does reduce the efficiency of N-cadherin endocytosis and as a result stabilizes cadherin at synaptic membranes (Tai et al., 2007). Furthermore, the β-catenin binding site includes a proline-glutamic acid-serine-threonine (PEST) sequence that facilitates rapid protein turnover when exposed. Therefore, the binding of β-catenin to cadherin may also block cadherin proteolysis (Huber and Weis, 2001). In addition to the armadillo repeats, β-catenin has a PDZ binding domain near the C-terminal tail (Perego et al., 2000) and an α-catenin binding domain near the N-terminal tail (Aberle et al., 1997; Pokutta and Weis, 2000). Although β-catenin was initially thought to simultaneously bind to both cadherin and α-catenin to form a link between the cadherin adhesion complex and the actin cytoskeleton, in vitro studies examining protein-protein interactions show that α-catenin does not simultaneously interact with the cadherin-β-catenin complex and actin (Yamada et al., 2005). This suggests that the cadherin adhesion complex likely interacts with the underlying actin cytoskeleton in a more dynamic way. Indeed, it is suggested that α-catenin monomers bind more strongly to cadherin-β-catenin complexes, and α-catenin homodimers interact more strongly with the actin cytoskeleton (Drees et al., 2005).  β-catenin plays an important role in synapse formation through its interactions with cadherins, with the actin cytoskeleton and with other binding partners. The loss of β-catenin reduces the quantal response from AMPARs and the activity-dependent scaling of synaptic strength, which suggests that β-catenin may also play a role in AMPAR trafficking at synapses 22  (Okuda et al., 2007). Furthermore, the expression of a form of β-catenin that is resistant to degradation results in increased cadherin localization at synaptic membranes, and impairments in LTD (Mills et al., 2014).   1.4.1.2 β-catenin Degradation and Signalling Although this thesis will primarily examine the role of β-catenin at the synapse as part of the cadherin adhesion complex (see Chapter 2), it is important to note that β-catenin is also a key member of the canonical Wnt signaling pathway. Wnt signaling influences gene expression and plays a critical role in development (Clevers, 2006; Clevers and Nusse, 2012).  Normally, β-catenin levels in the cytoplasm are tightly controlled by a ‘destruction complex’ composed of the scaffolding protein Axin2, disheveled (Dsh), adenomatous polyposis coli (APC), casein kinase 1α/δ (CK1) and glycogen synthase kinase 3 α/β (GSK3) (Aberle et al., 1997; Gao et al., 2002). CK1 and GSK3 are constitutively active serine/threonine kinases that phosphorylate β-catenin at N-terminal serine and threonine residues (Yost et al., 1996; Polakis, 2002). This targets β-catenin for ubiquitinylation by the E3 ubiquitin ligase protein β-TrCP (Hart et al., 1999) and subsequent breakdown in the proteasome. Wnt signaling modulates the activity of the destruction complex. Wnt ligands interact with receptors Frizzled (Fz) and the low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), triggering LRP5/6 phosphorylation (He et al., 2004; Tamai et al., 2004). Although it was previously thought that Wnt ligand binding caused the dissociation of the destruction complex, more recent work suggests that after Wnt ligand binding, the intact destruction complex becomes strongly associated with the phosphorylated LRP5/6 (Clevers and Nusse, 2012; Li et al., 2012a). Although the destruction complex is still able to sequester and phosphorylate β-catenin, the ubiquitinylation by β-TrCP is inhibited (Li et 23  al., 2012a). As a result the destruction complex becomes ‘saturated’ with β-catenin, and any newly synthesized β-catenin accumulates in the cytoplasm (Figure 1.7). When the level of cytoplasmic β-catenin is increased, it is translocated to the nucleus where it can interact with TCF/LEF transcription factors and regulate gene transcription (Behrens et al., 1996; Molenaar et al., 1996).   Figure 1.7 β-catenin and Wnt Signaling  In the absence of Wnt, the ‘destruction complex’ is localized in the cytoplasm where it is positioned to bind, phosphorylate and ubiquitinate β-catenin. Wnt binding induces the association of the complex with phosphorylated LRP. The complex can still capture and phosphorylate β-catenin but ubiquitination is blocked. As a result, newly synthesized β-catenin accumulates. Reprinted from Cell, Vol 149, Clevers & Nusse, Wnt/ β-catenin signaling and disease, p. 1192-1205 Copyright (2012), with permission from Elsevier.   Although β-catenin-TCF signaling is essential during development (Logan and Nusse, 2004; Clevers, 2006) and is implicated in cancer (Polakis, 2000), its role in the mature nervous system is not well understood. However, β-catenin does seem to play a role in activity-dependent gene regulation in neurons. Synaptic activity promotes the calpain-mediated cleavage of β-catenin, resulting in the loss of the N-terminal region that is normally phosphorylated by GSK3 and CK1. This truncated form of β-catenin translocates to the nucleus and induces TCF-mediated gene transcription of immediate early genes such as Fosl1 (Abe and Takeichi, 2007). Interestingly, this truncated form of β-catenin lacks its N-terminal phosphorylation sites, 24  suggesting that mechanisms other than the ‘destruction complex’-mediated phosphorylation and ubiquitinylation likely contribute to β-catenin regulation in neurons.   1.4.1.3 δ-catenin Structure and Function δ-catenin is one of the p120-catenin family proteins. Members of the p120-catenin family include p120-catenin, armadillo repeat gene deleted in Velo-Cardio-Facial syndrome (ARVCF), δ-catenin, and the plakophilins (Anastasiadis and Reynolds, 2000). At the N-terminus, δ-catenin contains a conserved coiled-coil domain that may be important for recruiting other interacting proteins to the cadherin adhesion complex. At the C-terminus, δ-catenin contains a PDZ-binding motif which allows δ-catenin to interact with PDZ-domain containing scaffold proteins such as synaptic scaffolding molecule (S-SCAM; Ide et al., 1999), PSD-95 (Jones et al., 2002), and glutamate receptor interacting protein 1/actin binding protein (GRIP1/ABP; Misra et al., 2010; Silverman et al., 2007). Similar to β-catenin, δ-catenin contains conserved Armadillo repeats, which allow δ-catenin to interact with cadherin (Ishiyama et al., 2010). However, δ-catenin interacts with the cadherin juxtamembrane domain (Ishiyama et al., 2010; Tanaka et al., 2012). As the cadherin juxtamembrane domain is essential for cadherin cis clustering (Shapiro et al., 1995; Yap et al., 1998), this suggests that δ-catenin influences synapse function through the regulation of cadherin cis interactions.   δ-catenin is strongly localized to dendrites and is specifically enriched in dendritic spines (Kosik et al., 2005), where it links cadherin to the actin cytoskeleton through interactions with cortactin (Martinez et al., 2003), Rho-family GTPases Rac1 and cdc42 (Abu-Elneel et al., 2008), and p190RhoGEF, a RhoA guanine nucleotide exchange factor (Kim et al., 2008). δ-catenin is able to shuttle between the postsynaptic membrane and the cytosol. Indeed, membrane-bound 25  cadherins compete with the cytosolic p190RhoGEF for δ-catenin interactions (Kim et al., 2008), suggesting that δ-catenin might have different functions when localized at synaptic membranes compared to the cytosol. Phosphorylation of δ-catenin by Cdk5 drives it away from synaptic membranes (Poore et al., 2010), and palmitoylation by zDHHC5 drives δ-catenin to postsynaptic spines (Brigidi et al., 2014, 2015). Palmitoylation will be further discussed in Chapter 1.8.    1.4.2 The Cadherin Adhesion Complex in Synapse Plasticity Many studies have demonstrated that the cadherin adhesion complex plays a critical role in synaptic plasticity in a well-studied region of the brain called the hippocampus (hippocampal circuitry is discussed in Chapter 1.5). The stability of cadherin adhesion complexes localized at the synaptic membrane is altered in response to synaptic activity. Furthermore, N-cadherin interacts with AMPAR subunits GluA1, GluA2 and GluA4 (Nuriya and Huganir, 2006; Saglietti et al., 2007) which may indicate that cadherins influence synaptic strength through their effects on AMPAR stability at synaptic membranes. Synaptic activity drives changes in cadherin stability at the membrane. Under basal conditions, cadherin undergoes constant turnover and is rapidly endocytosed, however after NMDAR activation, the internalization of N-cadherin is greatly reduced (Tai et al., 2007). This is a result of decreased β-catenin phosphorylation, resulting in increased N-cadherin-β-catenin interactions (Tai et al., 2007). These increased interactions with β-catenin prevent N-cadherin endocytosis. However, activity also promotes the formation of the cis-dimeric conformation of N-cadherin, which helps to stabilize cadherin at the synaptic membrane (Tanaka et al., 2000). Interestingly, cis clustering seems to occur independent of β-catenin, suggesting that interactions with multiple proteins may act to stabilize cadherin at synaptic membranes (Tanaka et al., 2000). 26  It is possible that the activity-mediated increase in cis clustering is mediated by interactions with p120-catenin or δ-catenin at the juxtamembrane domain (Yap et al., 1998), however this has not been shown conclusively. After synaptic activity, N-cadherin protein expression is also increased, resulting in an increase in the number of N-cadherin puncta at synaptic sites (Bozdagi et al., 2000). Indeed, there are impairments in the late phase of LTP (L-LTP) if N-cadherin interactions or protein synthesis are blocked. The overall increase in membrane associated cadherin in response to activity results in increased size and stability of synapses and dendritic spines in vitro (Okamura et al., 2004; Tanaka et al., 2000; Yam et al., 2013). N-cadherin is also required to stabilize dendritic spines whose formation is induced by LTP in organotypic hippocampal slice cultures (Mendez et al., 2010), furthermore acute slices from N-cadherin conditional knockout mice do not exhibit lasting changes in spine size and excitatory postsynaptic potentials that are often associated with LTP in wildtype slices (Bozdagi et al., 2010). Cadherin-mediated changes in synaptic strength are dependent on increased N-cadherin transsynaptic adhesion. Indeed, treatment with antibodies that bind to the cadherin extracellular domain or treatment with an HAV peptide that blocks trans cadherin interactions disrupts the induction of LTP in hippocampal brain slices, but does not affect the strength of synapses that have already been potentiated previously (Tang et al., 1998).   Cadherins have been strongly implicated in LTP, however their role in LTD is less well characterized. The conditional knockout of N-cadherin in hippocampal neurons results in impairments in LTP, with no effect on LTD (Bozdagi et al., 2010). However, LTD was impaired in a mouse model where cadherins were aberrantly stabilized at the synaptic membrane by the expression of a mutant β-catenin which was resistant to protein degradation (Mills et al., 2014), 27  indicating that cadherin endocytosis is likely required for LTD. Interestingly, these studies showed no impact of either the loss (Bozdagi et al., 2010) or stabilization (Mills et al., 2014) of cadherin adhesion complexes on gross hippocampal structure, synapse and spine density, or basal synaptic neurotransmission.   1.4.3 The Cadherin Adhesion Complex in the Dysfunction of the Nervous System Given the important role of cadherin adhesion complexes in the development and function of synapses, one would expect that mutations within cadherin and catenin genes could potentially cause widespread dysfunction in the nervous system. Indeed, de novo copy number variants (CNVs) and single nucleotide polymorphisms (SNPs) affecting classic cadherins 8, 9, 10, and 13, and protocadherin-10 have been identified in patients with neurodevelopmental disorders such as autism spectrum disorders (ASDs) (Friedman et al., 2015). Furthermore, the CTNND2 gene which encodes δ-catenin is deleted in cri-du-chat syndrome, a disorder that causes severe intellectual disability and speech impairment (Israely et al., 2004). Several genome-wide allelic association studies have also identified SNPs in the GPI-anchored cadherin-13, which are associated with neurodevelopmental disorders such as ADHD (Rivero et al., 2013), schizophrenia (Børglum et al., 2014) and ASD (Sanders et al., 2011). Missense SNPs in N-cadherin have also been identified in patients with obsessive-compulsive disorder and Tourette’s syndrome, although it is still unknown how these SNPs might affect the functioning of N-cadherin (Moya et al., 2013).  Studies have linked aberrant N-cadherin-β-catenin signaling to Alzheimer’s disease (AD) in some cases. A study examining human AD brains found that they had significantly reduced N-cadherin protein levels compared to age matched controls (Ando et al., 2011). It has also been 28  suggested that a γ-secretase cleaved fragment of the N-cadherin C-terminal domain might accelerate the synaptic dysfunction caused by β-amyloid (Andreyeva et al., 2012). Furthermore, studies have suggested that aberrant activation of β-catenin-TCF-regulated gene expression occurs in neurological diseases such as Alzheimer’s disease and bipolar disorder (Chong et al., 2005; De Ferrari and Inestrosa, 2000; Gould and Manji, 2002). These data indicate that changes in cadherin adhesion complexes often occur in conditions of neuronal dysfunction. It remains to be seen whether these variations in cadherin and catenin genes and expression levels are causal factors in neurological disorders.  1.4.4 Cadherin Adhesion Complex in Addiction Variations in cell adhesion molecules have broadly been implicated as risk factors for different forms of substance abuse (Uhl and Drgonova, 2014). Indeed, cadherin adhesion complex protein variants have also been detected in studies examining individuals with substance abuse issues. SNPs in cadherins 11 and 12 were also identified as risk factors for alcohol abuse (Lydall et al., 2011). A genome-wide allelic association study of mutations associated with methamphetamine dependence identified polymorphisms in cell adhesion genes, including cadherin-13, as risk factors for addiction (Uhl et al., 2008a). SNPs in the cadherin-binding proteins α- and δ-catenin were identified in individuals at risk for substance abuse (Uhl et al., 2008b). Furthermore, genome-wide association studies have also identified a gene variation in the 8th intron of cadherin-13 that predicted an increased sensitivity to the positive effects of cocaine (Hart et al., 2012). Interestingly, changes in β-catenin and cadherin expression levels have been observed in animal models after exposure to drugs of abuse. Studies examining changes in mRNA levels in 29  the primate brain after self-administration of cocaine found a significant increase in β-catenin expression in the nucleus accumbens (Freeman et al., 2001). In rats, cocaine exposure in utero causes an increase in the mRNA levels of β-catenin, N-cadherin, and cadherins 3, 6, 7 and 11 in the cortex (Novikova et al., 2005). β-catenin protein expression is significantly increased in the rat cortex in utero (Novikova et al., 2005), or in adult mouse nucleus accumbens and caudoputamen after cocaine exposure (Zhang et al., 2002). Though β-catenin protein levels increase overall, the amount of β-catenin in the nucleus remains unaltered (Novikova et al., 2005), suggesting that the increased β-catenin likely contributes to cadherin adhesion rather than Wnt signaling. Unfortunately, N-cadherin protein levels have not been examined after cocaine exposure.  Together, these data suggest that the cadherin adhesion complex may play a role in synaptic changes observed in response to drugs of abuse. Although a great deal of work has been done to determine the role of cadherins in synaptic plasticity in the hippocampus, little is known about the role of cadherins in other brain regions such as the mesocorticolimbic dopamine circuit. In Chapter 2, we examine the role of cadherins in synapse plasticity within the VTA.  1.5 The Structure and Function of the Hippocampal Circuit In the previous sections, I discussed motivated learning, which is regulated by synaptic plasticity in the mesolimbic circuitry of the brain. I will now review the hippocampal circuitry, synapse formation and synaptic function in the hippocampus as Chapter 3 involves the study of dendrite outgrowth and synapse formation in cultured hippocampal neurons.  The hippocampus is a distinct anatomical structure within the medial temporal lobe in the human brain. This structure has been the focus of study by neurobiologists for many years 30  because of its role in the consolidation of memories. This brain region exhibits synaptic plasticity in response to activity, and it is hypothesized that this plasticity might mediate the formation of hippocampal-dependent memories (Neves et al., 2008). For these reasons, a great deal of research has focused on detailed examinations of the circuitry and synapses of the hippocampus. The study of synapse formation and plasticity using this brain region has allowed us a much greater understanding of the cellular and molecular mechanisms that mediate learning and memory.  1.5.1 Anatomy of the Hippocampus The hippocampus is typically depicted as a trisynaptic loop, with three major subfields: the dentate gyrus (DG), Cornu Ammonis (CA) area 3 (CA3), and CA area 1 (CA1). The hippocampal circuit receives its main inputs from layer II neurons of the lateral and medial entorhinal cortex (EC). These cortical neurons input the hippocampus via the perforant path and synapse onto the dendrites of granule cells within the DG. Inputs from EC provide polymodal sensory information to the hippocampal circuit. The granule cells of the dentate gyrus project their axons to the pyramidal cells of CA3, and the CA3 pyramidal cells send their excitatory projections to the pyramidal cells in area CA1 through the Shaffer collaterals (Figure 1.8). The CA1 neurons project back to the EC, as well as to other cortical regions (Neves et al., 2008).  31   Figure 1.8 Basic Anatomy of the Rodent Hippocampus.  The hippocampal trisynaptic loop wiring diagram. The major input to the hippocampal circuit comes from the perforant path from the entorhinal cortex (EC) layer II neurons. Axons from the perforant path form excitatory connections with dendrites from the dentate gyrus (DG) granule neurons. These granule neurons send their excitatory axonal projections, called mossy fibres, to innervate the apical dendrites of the CA3 pyramidal neurons. CA3 neurons project to the CA1 neurons through the excitatory Schaffer collateral axonal tract. The CA1 neurons send excitatory projections to distinct neuronal populations in the EC. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Neuroscience Neves, Cooke & Bliss, copyright 2008.  Given that the hippocampal circuit exhibits a simple laminar pattern and consistent circuitry, this structure has commonly been studied in electrophysiological experiments. Indeed, the most well characterized synapses in the brain are where the Shaffer collaterals synapse onto the apical dendrites of the CA1 pyramidal neurons (Kerchner and Nicoll, 2008). Transverse hippocampal slice preparations are particularly useful for the study of synaptic transmission using extracellular recording techniques. Specific pathways such as the Shaffer collaterals can be selectively stimulated, and evoked responses can be measured in CA1. Furthermore, pharmacological agents can be washed on and off to block or activate specific receptors, which 32  provides more information about the importance of receptor subtypes in synaptic plasticity (Neves et al., 2008).   Figure 1.9 GABAergic Interneurons in the CA1 Region of the Hippocampus. Pyramidal cells are shown in blue. GABA interneurons that synapse onto pyramidal cells are shown in orange, and include axo-axonic cells, parvalbumin (PV) positive basket cells, and cholecystokinin (CCK) positive basket cells. GABA interneurons that mainly innervate other interneurons are shown in pink. Other interneurons shown in red, their axons are in purple and main synaptic terminations are shown in yellow. Some interneurons target the perisomatic region of pyramidal cells, and some have longer range projections to other hippocampal subregions, and some project to brain regions beyond the hippocampus (Klausberger and Somogyi, 2008). Reprinted with permission from AAAS.  The hippocampus also contains several populations of inhibitory GABAergic cells, which tune and synchronize the firing of pyramidal cells (Figure 1.9; Somogyi and Klausberger, 2005). The GABAergic cells within the hippocampus are incredibly heterogeneous, with approximately 21 subtypes (Klausberger and Somogyi, 2008). These include the basket cells and axo-axonic “chandelier” cells that control spike initiation, and the bistratified cells and oriens-lacunosum 33  moleculare (O-LM) interneurons that aid in synaptic integration. The interneuron specific cells form inhibitory synapses onto other interneurons (Klausberger and Somogyi, 2008; Chamberland and Topolnik, 2012). These connections add an additional layer of control to hippocampal activity.  1.5.2 Hippocampal Function The function of the hippocampus is integral to the consolidation of conscious memories, factual information or autobiographical events, also known as explicit memory. Indeed, lesions to the hippocampus in humans specifically impair the formation of new explicit memories, causing anterograde amnesia (Scoville and Milner, 1957). It is hypothesized that plasticity of synaptic connections in the hippocampus mediates the formation of this type of memory. Studies examining the function of the hippocampus in animal learning have shown that blocking or transiently knocking out postsynaptic receptors using an inducible mouse model not only impairs the strengthening of synapses, but also impairs learning and recall on a water maze task (Morris et al., 1986; Shimizu et al., 2000).   1.6 Primary Hippocampal Cell Culture Hippocampal neurons have been studied in vitro in dissociated cell cultures, a method which we use in the research described in Chapter 3. Importantly, the synapses formed in dissociated hippocampal cultures possess many phenotypic properties of in vivo neurons, can be maintained for weeks to months, and are amenable to molecular manipulation such as the overexpression of specific proteins or the use of RNA interference (RNAi) to knock down protein expression (Banker and Cowan, 1977; Fletcher and Banker, 1989; Neves et al., 2008). 34  For instance, neurons in dissociated hippocampal culture establish axonal and dendritic polarity and form synaptic connections with other neurons in culture over a well-characterized time course (Dotti et al., 1988; Bartlett and Banker, 1984).  Although the majority of neurons in primary hippocampal cell culture are glutamatergic pyramidal cells, GABAergic cells make up approximately 6% of neurons in hippocampal culture (Benson et al., 1994). These GABAergic cells are morphologically distinct, as they have fewer primary dendrites with longer segments, and these dendrites do not have postsynaptic spines (Benson et al., 1994). The GABA cells found in culture have been identified as basket cells based on morphology (Benson et al., 1994), although given the great diversity of GABA cells present in the hippocampus (Klausberger and Somogyi, 2008), it is likely that other GABA cell types are also represented in culture.   1.7 Synapse Formation in Hippocampal Neurons Synaptogenesis is critical for the proper functioning of the central nervous system. This process can be defined as the assembly of many pre- and postsynaptic proteins into the highly specialized structure of the synapse (as described in Chapter 1.3). In order for synapses to function properly, SVs, receptors, active zone cytoskeletal proteins, PSD scaffolding proteins, and cell adhesion molecules must move to sites of physical contact between the presynaptic axon and postsynaptic dendrite (McAllister, 2007). Presynaptic proteins are thought to be trafficked to presynaptic sites by piccolo transport vesicles, and synaptic vesicle transport vesicles (Sabo et al., 2006; Zhai et al., 2001), which are transported by motor proteins such as kinesin-1 and KIF1a (McAllister, 2007). Many postsynaptic proteins are present in dendrites before synapse formation. Some evidence suggests that NMDARs and AMPARs are transported to postsynaptic 35  sites in discrete packets (Washbourne et al., 2002, 2004), although other evidence suggests that NMDARs are recruited to postsynaptic sites from a diffuse pool of receptors (Bresler et al., 2004). Furthermore, changes to the actin cytoskeleton can drive spine and synapse formation (Zito et al., 2004). For a synapse to form, the presynaptic axon must contact the postsynaptic dendrite. Although most of these contacts are short-lived, a small subset of contacts become stabilized and nascent synapses can form. Netrin-1, a chemoattractive secreted protein that can direct axon guidance, is also localized in dendrites and its receptor, DCC, is present in both axons and dendrites during synaptogenesis. Interestingly, Netrin-1 also contributes to excitatory synapse formation in cultured cortical neurons (Goldman et al., 2013).  Cell adhesion molecules can stabilize these nascent synaptic contacts. Presynaptic neuroligins and postsynaptic neurexins are considered “synaptogenic” adhesion molecules, as they promote synapse formation (Craig et al., 2006). Indeed, neuroligin-1 can promote synapse formation in response to synaptic activity, in part through its effect on the actin cytoskeleton (Gutiérrez et al., 2009). However, other cell adhesion proteins contribute to synapse formation. Although their expression is not sufficient to induce synaptogenesis on their own, classical cadherins also play a significant role in modulating synapse formation. Cadherins are among the first proteins that are localized at opposing pre- and postsynaptic contact sites (Benson and Tanaka, 1998), and synapse formation is impaired in neurons that express Neuroligin-1 (NL1) but lack N-cadherin (Aiga et al., 2011; Stan et al., 2010). Furthermore, cleavage of the N-cadherin prodomain in neurons, which produces a mature form of N-cadherin that can participate in transsynaptic adhesion, occurs at the onset of synaptogenesis (7-10 DIV) allowing cadherin adhesion to influence the formation of synaptic contacts (Latefi et al., 2009). Studies in zebrafish have shown that the cleavage of the N-cadherin prodomain can regulate the rate of 36  synaptogenesis (Latefi et al., 2009), and that the overexpression of wildtype N-cadherin accelerates the process of synaptogenesis. In contrast, the expression of a mutant N-cadherin with a prodomain that cannot be cleaved results in delayed synaptogenesis and fewer synapses being formed (Latefi et al., 2009; Reinés et al., 2012). Immunoglobulin superfamily members such as neural cell adhesion molecules (NCAM) and synaptic cell-adhesion molecules (SynCAM) have also been shown to aid in synaptic target specificity (Craig et al., 2006).   1.8 The zDHHC Family of Palmitoyl Acyl Transferases  Protein palmitoylation is the most common form of protein S-acylation in eukaryotic cells and involves the reversible addition of the fatty acid, palmitate, to cysteine residues of the substrate protein. This lipid modification is mediated by a family of multi-pass transmembrane proteins containing a conserved aspartate-histidine-histidine-cysteine (DHHC) motif required for its palmitoyl acyl-transferase (PAT) activity (Bartels et al., 1999; Roth et al., 2002). The DHHC catalytic motif is located within a cysteine-rich, zinc finger–like domain, resulting in the current, standard ‘zDHHC’ nomenclature. To date, 23 mammalian zDHHC proteins have been identified with the majority of them being validated as having PAT function in yeast (Ohno et al., 2012) and in mammalian cells (Fukata et al., 2004). As palmitoylation is a reversible modification, the enzymes responsible for depalmitoylation are also of great interest to researchers. All palmitoyl-protein thioesterases (PPTs) identified to date contain α/β-hydrolase domains (ABHD proteins; Lord et al., 2013); however, the search for other families of enzymes with PPT activity continues.  37   Figure 1.10 Phylogenetic Tree of the Mouse zDHHC Protein Family.  (A) Schematic structural representation of zDHHC5, which has 4 transmembrane domains and the conserved cysteine rich domain and DHHC motif on the cytoplasmic loop of the protein. zDHHC structures diverge substantially at the N- and C-termini. zDHHC5 contains a PDZ domain-binding motif at the C-terminus. (B) Phylogenetic tree of the mouse zDHHC protein family. The tree is based on the alignment of the zDHHC-CDR core domains. Note that some DHHC clone numbers initially collected in (Fukata et al., 2004) are different from the current standard nomenclature, indicated in parentheses. zDHHC genes that have been implicated in neurological diseases indicated in yellow. Adapted from Macmillan Publishers Ltd: Nature Reviews Neuroscience, Fukata & Fukata, copyright 2010.   1.8.1 Palmitoylation of Synaptic Proteins Many proteins found in neurons are substrates for palmitoylation. Indeed, over 40% of all known synaptic proteins are palmitoylated, with 419 of the 1028 known synaptic proteins being identified as substrates for palmitoylation (Sanders et al., 2015). Many synaptic proteins exhibit a rapid rate of palmitate turnover (Brigidi et al., 2014, 2015; Thomas et al., 2012; El-Husseini et al., 2002), suggesting that zDHHC proteins must be expressed in axons and dendrites to mediated these fast changes in palmitoylation state. To date, zDHHC17 is the only PAT observed in axons (Ohyama et al., 2007; Stowers and Isacoff, 2007), while zDHHCs 1, 2, 5, 8, and 12 have been detected in dendrites (Oku et al., 2013; Noritake et al., 2009; Woolfrey et al., 2015; Brigidi et al., 2015; Thomas et al., 2012).  38  zDHHCs 2, 5 and 8 are all prominently expressed at the plasma membrane and appear to localize to a subset of synapses. Whereas Thomas and colleagues (2012) reported that zDHHC8 but not zDHHC5 is highly localized to synapses (Thomas et al., 2012), Brigidi and colleagues (2015) observed that zDHHC5 is localized to ~80% of excitatory and ~47% of inhibitory synapses (Brigidi et al., 2015). This discrepancy may be due to culture conditions and the overall activity of neurons in the culture. zDHHCs 2, 5 and 8 colocalize with the transferrin receptor, (TfR; Brigidi et al., 2015; Thomas et al., 2012; Woolfrey et al., 2015), indicating that they are trafficked to and from the postsynaptic membrane on recycling endosomes (RE; Thomas et al., 2012; Brigidi et al., 2015; Woolfrey et al., 2015; Fukata et al., 2013). zDHHC12 colocalizes with the golgi marker, giantin, in dendrites indicating its localization at golgi outposts (Dejanovic et al., 2014). In contrast, zDHHC3 is stably localized to the membrane of the Golgi, and does not exhibit activity-dependent changes in localization (Noritake et al., 2009). This thesis will focus on the function of zDHHC5 in hippocampal neurons.  1.8.1.1 zDHHC5-Mediated Palmitoylation of Synaptic Proteins zDHHC5 is localized to postsynaptic compartments, and has been identified as a PAT for synaptic proteins including GRIP1b, flotillin-2, the spliced stress-related exon (STREX) variant of large conductance calcium- and voltage- activated potassium (BK) channel, somatostatin receptor 5, and δ-catenin (Brigidi et al., 2014; Kokkola et al., 2011; Li et al., 2012b; Thomas et al., 2012; Tian et al., 2010). Interestingly, zDHHC5 interacts with PSD-95 through it’s PDZ-binding motif (Li et al., 2010), but does not palmitoylate PSD-95. Although biochemical fractionation studies have shown that the majority of zDHHC5 protein in the mouse brain is localized to postsynaptic compartments (Li et al., 2010), a stable isotope labeling with amino 39  acids in cell culture (SILAC) study identified several proteins involved in protein sorting and vesicle trafficking as potential targets of zDHHC5 palmitoylation when comparing differences in palmitoylation in wildtype compared to DHHC5 gene-trapped neural stem cell cultures (Li et al., 2012b). These included syntaxin 4a, syntaxin 7, Vamp2, Vamp3, and Rab3a. However, further experimental evidence is needed to determine whether zDHHC5 is localized presynaptically, and whether zDHHC5-mediated palmitoylation of vesicle trafficking proteins has any effect on synapse function.  1.8.2 Activity-Mediated Changes in zDHHC5 Localization and Function Brigidi and colleagues (2015) have demonstrated that the subcellular trafficking of zDHHC5 can also be regulated by synaptic activity. Under basal conditions, zDHHC5 is localized to the plasma membrane and interacts with both PSD-95 and Fyn kinase through zDHHC5’s C-terminal PDZ binding motif and polyproline repeats, respectively (Brigidi et al., 2015). zDHHC5 is maintained at the plasma membrane through Fyn kinase-mediated phosphorylation of tyrosine residue 533, which lies within the endocytic motif of zDHHC5. Increasing neuronal activity using an established chemical long-term potentiation (cLTP) protocol results in the rapid dissociation of the zDHHC5/Fyn/PSD-95 complex, resulting in the dephosphorylation of zDHHC5 and its translocation from dendritic spines to shafts on REs. Although it is still unclear which phosphatase dephosphorylates zDHHC5 thereby enabling its association with endocytic proteins, one candidate includes striatal enriched phosphatase (STEP) 61, a phosphatase known to dephosphorylate both Fyn (Nguyen et al., 2002) as well as its substrates (Kurup et al., 2010), in an activity-dependent manner (Jang et al., 2015). The dynamic, activity-regulated localization of zDHHC5 suggests this enzyme may differentially palmitoylate 40  substrates upon fluctuation in synaptic activity. However, it is not known how zDHHC5-mediated palmitoylation of neuronal proteins might affect the development of neural circuits.   1.8.3 Palmitoylation in Synapse Formation The palmitoylation of synaptic proteins has been shown to play an important role in synapse formation. Indeed, the PDZ domain protein PSD-95 is a critical component of the postsynaptic scaffold and a substrate for zDHHC2- and zDHHC3-mediated palmitoylation (Noritake et al., 2009). PSD-95 has been shown to contribute to synaptic assembly in part through the recruitment of AMPARs (El-Husseini et al., 2000). The clustering of PSD-95 is palmitoylation-dependent, such that the expression of a palmitoylation-deficient PSD-95 acts as a dominant negative to impair synapse formation (El-Husseini et al., 2000). A brain-specific splice variant of the small GTPase, Cdc42, is also a substrate for palmitoylation (Kang et al., 2008). Interestingly, the palmitoylated form of Cdc42 is localized to dendritic spines and contributes to synaptogenesis (Kang et al., 2008; Saneyoshi et al., 2010). Palmitoylation of the actin binding protein, LIM Kinase 1 (LIMK1), has recently been shown to play an important role in mediating the formation of synaptic connections (George et al., 2015). Both LIMK1 and 2 phosphorylate and inactivate members of the ADF/cofilin family of actin binding and filament severing proteins (Hotulainen and Hoogenraad, 2010); however, only LIMK1 has been shown to be a substrate for palmitoylation (George et al., 2015). LIMK1 palmitoylation results in its recruitment to dendritic spines where it acts to stabilize spines and promote the formation of synapses. Palmitoylation-mediated recruitment of LIMK1 to dendritic spines is required for the phosphorylation and activation of LIMK1 by its upstream regulator p21-activated kinase (PAK), that is itself enriched in spines. LIMK1 activation, in turn, mediates 41  the phosphorylation and inactivation of cofilin, resulting in a net increase in actin polymerization and the enlargement and stabilization of dendritic spines (George et al., 2015). Disrupting LIMK1 palmitoylation abolishes activity-induced spine enlargement consistent with the importance of actin polymerization in this process. Given the known changes in spine number and size in response to synaptic activity, it is tempting to speculate that synaptic activity may drive the palmitoylation of LIMK1 and its recruitment to postsynaptic spines. However, further work is required to test this hypothesis, as well as determine zDHHC enzyme(s) responsible for the palmitoylation of LIMK1.    1.8.4 Palmitoylation in the Dysfunction of the Nervous System As certain zDHHCs mediate activity-dependent changes in palmitoylation, it is unsurprising that these zDHHCs are critical for synaptic function. Indeed, zDHHC5 has been implicated in neurodevelopmental and neuropsychiatric disorders. Mice that are homozygous for a hypomorphic allele of the ZDHHC5 gene (zDHHC5 gene-trapped mice; zDHHC5-gt) show significant impairments on the acquisition of hippocampal-dependent contextual fear memories (Li et al., 2010). Genome-wide allelic association studies have reported an association between single nucleotide variants (SNVs) in the region of chromosome 11 where zDHHC5 is located have been linked with increased prevalence of bipolar disorder and schizophrenia (Fallin et al., 2004; Schizophrenia Working Group of the Psychiatric Genomics Consortium, 2014). Furthermore, a de novo nonsense mutation in the zDHHC5 gene resulting in a truncation of the C-terminal tail was recently identified in a schizophrenic patient (Fromer et al., 2014), which further supports the importance of zDHHC5 in normal cognitive functioning. 42  Several other zDHHC proteins including zDHHCs 7, 8, 9, 12, 13, 15, 17, and 21 have been implicated in neurological disorders such as Alzheimer’s disease, X-linked intellectual disability, and Huntington’s Disease (Bhattacharyya et al., 2013; Hornemann, 2015; Huang et al., 2004; Korycka et al., 2012; Mizumaru et al., 2009; Mukai et al., 2008; Yanai et al., 2006). Indeed, the generation of transgenic knockout mouse lines for zDHHC proteins will aid in the modeling of these neurological diseases, and further elucidate the role of palmitoylation in normal cognitive function. In Chapter 3, we examine the role of zDHHC5 in dendrite outgrowth, and synapse formation.   1.9 Rationale and Hypothesis Synapse formation and plasticity are critical to the development of the brain, and the encoding of experiences in neural circuits. In this thesis, I examine the role of cadherin adhesion complexes on cocaine-mediated plasticity, and the role of palmitoyl acyltransferase zDHHC5 in dendrite outgrowth and synaptogenesis.  Cadherin adhesion complexes have been shown to be critical for synaptic plasticity in the hippocampus, however, little is known about the role of cadherins in other brain circuits. My first objective was to examine changes in cadherin localization at synapses in the mesocorticolimbic reward circuit. We examined changes in cadherin localization in the VTA after wildtype mice developed a preference for cocaine, and used a transgenic mouse model to manipulate cadherin adhesion in this circuit to determine whether changes in cadherin function can affect cocaine-mediated behavioural conditioning. This work is of particular importance because mutations in cadherins and their binding partners have been identified in populations 43  with increased risk for substance abuse (Uhl et al., 2008a, 2008b; Lydall et al., 2011; Hart et al., 2012).  zDHHC5 is an enzyme that is localized at dendritic spines and can act on the cadherin adhesion complex protein, δ-catenin. Indeed, previous work has shown that zDHHC5 undergoes activity-mediated changes in localization that bring the enzyme in closer proximity to δ-catenin (Brigidi et al., 2015), where zDHHC5 can palmitoylate δ-catenin and drive increases in synaptic strength through δ-catenin’s increased interaction with N-cadherin at postsynaptic spines (Brigidi et al., 2014). Although several targets of zDHHC5 palmitoylation have been identified (Kokkola et al., 2011; Li et al., 2012b; Thomas et al., 2012; Brigidi et al., 2014), the role of zDHHC5 in neuronal development has not been determined. My second objective was to examine the role of palmitoyl acyltranserase zDHHC5 in the development of synapses. In Chapter 3, I examine zDHHC5’s role in dendrite outgrowth and complexity, and in excitatory and inhibitory synapse formation.     44  Chapter 2: Cadherins Mediate Cocaine-Induced Synaptic Plasticity and Behavioural Conditioning Drugs of abuse alter synaptic connections in the reward circuitry of the brain, which leads to long-lasting behavioral changes that underlie addiction. Here we show that cadherin adhesion molecules play a critical role in mediating synaptic plasticity and behavioral changes driven by cocaine. We demonstrate that cadherin is essential for long-term potentiation in the ventral tegmental area and is recruited to the synaptic membranes of excitatory synapses onto dopaminergic neurons following cocaine-mediated behavioral conditioning. Furthermore, we show that stabilization of cadherin at the membrane of these synapses blocks cocaine-induced synaptic plasticity, leading to a reduction in conditioned place preference induced by cocaine. Our findings identify cadherins and associated molecules as targets of interest for understanding pathological plasticity associated with addiction As stated in the preface, all work and experiments presented in this chapter were done in equal partnership by Andrea Globa and Fergil Mills, who are co-first authors on the paper publishing these results.  2.1 Introduction Drugs of abuse induce widespread alterations to the neural circuits that mediate reward learning in the brain. Cocaine exposure drives the strengthening of excitatory inputs onto dopaminergic neurons of the VTA (Lüscher, 2013; Mameli and Lüscher, 2011; Saal et al., 2003; Ungless et al., 2001), and causes increased release of dopamine from the VTA onto corticolimbic structures, including the nucleus accumbens (NAc), the prefrontal cortex (PFC) and the dorsal 45  striatum (Nestler, 2005; Pierce and Kumaresan, 2006). Drug-evoked synaptic plasticity in the VTA is believed to underlie behavioural changes that lead to addiction. The potentiation of excitatory inputs to dopaminergic neurons is increased following associative learning of reward-predicting cues (Stuber et al., 2008), and intact glutamatergic synapse function in the VTA is required for the formation of cocaine-induced conditioned place preference (CPP) (Harris and Aston-Jones, 2003), indicating that plasticity at these synapses may contribute to the learned association between environmental cues and the rewarding effects of cocaine. Electrophysiological studies have shown that cocaine-induced potentiation of VTA synapses is mediated by the insertion of Ca2+-permeable, GluA2-lacking AMPA receptors (AMPARs) to the synaptic membrane (Argilli et al., 2008; Mameli et al., 2011). In order to determine how drugs of abuse alter synapses within the reward circuit and cause behavioural changes underlying addiction, it is important to further understand the molecular mechanisms that mediate drug-induced plasticity at synapses in the VTA. Cadherin adhesion molecules have been shown to play a critical role in synaptic plasticity underlying different forms of learning and memory. Cadherins mediate adhesion at synapses through homophilic trans interactions across the synaptic membrane and associate with AMPARs through direct and indirect interactions with both GluA1 and GluA2 AMPAR subunits (Nuriya and Huganir, 2006; Saglietti et al., 2007; Silverman et al., 2007). In the hippocampus, cadherins are essential for long-term potentiation (LTP) and long-term depression (LTD). Following enhanced activity, cadherins are increasingly localized to the synaptic membrane, leading to increased synapse stability and the stabilization of AMPAR at the synaptic membrane (Bozdagi et al., 2010; Brigidi et al., 2014; Mendez et al., 2010; Tanaka et al., 2000). In contrast, during LTD the internalization of cadherin from the synaptic membrane is required for the 46  removal of AMPARs from the postsynaptic membrane (Tai et al., 2007). Disruption of trans-synaptic cadherin interaction in the hippocampus has been shown to abolish the acquisition of context-dependent memory formation (Schrick et al., 2007), while aberrant increases in cadherin stability at the membrane leads to impaired in behavioural flexibility on hippocampal-dependent tasks (Mills et al., 2014).  Because they regulate AMPAR trafficking and stability at synapses, cadherins are strong candidate molecules for mediating plasticity in the VTA underlying behavioural changes driven by drugs of abuse. In the context of addiction, genome-wide association studies have identified mutations in cadherin adhesion complex proteins as risk factors for substance abuse (Liu et al., 2006). However, very little is known about the expression of cadherins in the VTA, and their potential function in synaptic plasticity in this region has not been examined.  Here we show that cadherin plays a key role in synaptic plasticity in the VTA and behavioural changes driven by cocaine. We demonstrate that cadherins are widely expressed in dopaminergic neurons and are essential for long-term potentiation (LTP) of synapses in the VTA. We further demonstrate that recruitment of cadherin to excitatory inputs onto dopaminergic neurons is correlated with cocaine-mediated conditioned place preference (CPP). Finally, we demonstrate that stabilization of cadherin at the synaptic membrane of synapses onto dopaminergic neurons can completely block cocaine-induced changes in AMPAR localization and LTP, and greatly reduce behavioural conditioning driven by cocaine.   47  2.2  Materials and Methods 2.2.1 Animals Male C57BL/6 mice between 6-8 weeks old were used in all experiments, unless stated otherwise. For experiments examining the effects of cadherin stabilization of cocaine-induced plasticity and behavioral conditioning, we used mice Slc6a3:Cre/+;Ctnnb1lox(ex3)/lox(ex3) mice (termed DAT-Cre;β-catΔex3 mice for brevity), which are homozygous for a loxP-flanked exon 3 transgene (Harada et al., 1999) and express Cre recombinase in dopaminergic neurons (Bäckman et al., 2006). Littermates lacking the Slc6a3:Cre/+ transgene (+/+;Ctnnb1lox(ex3)/lox(ex3) mice) were used as controls. Mice were housed in reverse day/night cycle and given ad libitum access to food and water. Experimental procedures and animal housing conditions were approved by The UBC Animal Care Committee, and were in accordance with Canadian Council on Animal Care (CCAC) guidelines. All mice were housed with littermates in groups of two to five and were only used for one behavioral test each, unless otherwise noted.   2.2.2 Immunoblot Analysis Mice were killed by cervical dislocation, and their brains were quickly removed, and then sliced into 300 µm thick horizontal sections by vibratome. The VTA was dissected from these slices and homogenized in lysis buffer (20 mM Tris pH 7.4, 137 mM NaCl, 0.5% NP-40, 10% glycerol) with protease and phosphatase inhibitor tablets (Roche) and cleared by centrifugation at 14,000 x g for 40 min at 4°C. VTA lysates were separated by SDS-PAGE and probed with antibodies against N-cadherin (mouse, BD Transduction, CAT# 610920, predicted band size 130 kDa, 1:1,000), R-cadherin (rabbit, Novus, CAT# NBP2-27372, predicted band size 130 kDa 1:1,000), cadherin-7 (rabbit, Santa Cruz, CAT# sc-68422, predicted band size 90 kDa, 1:500), 48  cadherin-8 (rabbit, Abcam, CAT# ab97268, predicted band sizes 150 kDa precursor and 90 kDa protein, 1:1,000), and cadherin-11 (mouse, Invitrogen, CAT# 321700, predicted band size 110 kDa, 1:1,000). Proteins were visualized by chemiluminesence on a Bio-Rad Versadoc 4000 (Bio-Rad Laboratories (Canada) Ltd., Mississauga, ON). Immunoblot experiments were conducted twice to ensure reproducibility.  2.2.3 Immunohistochemistry  Mice were anesthetized with sodium pentobarbital (120 mg/kg), transcardially perfused by PBS followed by 4% paraformaldehyde (PFA) in PBS. Brains were removed and post-fixed in 4% PFA for two hours, then cryoprotected by saturation with 30% sucrose, frozen, and sliced into 20 µm thick coronal sections by cryostat. For immunolabelling of target proteins, sections were first placed in a blocking buffer containing 10% goat serum, 0.1% bovine serum albumin and 0.1% Triton-X-100 in PBS. Primary antibodies against DAT (dopamine transporter) (rat, Millipore, cat. no. MAB369, 1:500), N-cadherin (mouse, BD Transduction, cat. no. 610920, 1:250), R-cadherin (rabbit, Novus, cat. no. NBP2-27372, 1:250), cadherin-7 (rabbit, Santa Cruz, cat. no. sc-68422, 1:250), cadherin-8 (rabbit, Abcam, cat. no. ab97268, 1:250), cadherin-11 (rabbit, Santa Cruz, cat. no. sc-28643, 1:250), β-catenin (mouse, BD Transduction, cat. no. 610153, 1:250), Axin2, (rabbit, Abcam, cat. no. ab109307, 1:250), LEF1 (rabbit, Abcam, cat. no. ab137872, 1:250), c-Myc (rabbit, Cell Signaling, cat. no. 5605S, 1:250), c-Jun (rabbit, Cell Signaling, cat. no. 9165P, 1:250), and GAD67 (chicken, Abcam, cat. no. ab75712, 1:250) were diluted in this buffer, added to sections and incubated overnight at 4 °C. The following day, samples were washed three times with PBS, and secondary antibodies diluted in the blocking buffer were added to sections and incubated for 1-2 hours at room temperature. Slides were 49  washed again with PBS and stained with DAPI (0.5 µg/mL). Sections were mounted with ProLong Gold (Life Technologies, Carlsbad, CA), and were imaged on an Olympus Fluoview FV1000 confocal microscope using Fluoview software (Olympus, Melville, NY). Immunostaining experiments were conducted twice to ensure reproducibility. The brightness and contrast of entire images was judiciously adjusted using Photoshop (Adobe Systems Canada, Toronto, ON) following recommended, scientifically acceptable procedures, and no information was obscured or eliminated from the original images. Immunohistochemical experiments were repeated on brain slices from two mice per genotype to ensure reproducibility.   2.2.4 Electrophysiology  Electrophysiological recordings were taken from dopaminergic cells within the VTA. Horizontal slices of mouse midbrain were cut with a vibratome (Leica, Nussloch, Germany), and slices (250 µm) were equilibrated in artificial cerebrospinal fluid (aCSF) containing (in mM): 126 NaCl, 1.6 KCl, 1.1 NaH2PO4, 1.4 MgCl2, 2.4 CaCl2, 26 NaHCO3, 11 glucose (32°C-34°C) and saturated with 95% O2/5% CO2. Cells were visualized using infrared differential contrast video microscopy and whole-cell voltage-clamp recordings were made using a MultiClamp 700B amplifier (Axon Instruments, Union City, CA). Putative dopaminergic cells were identified by the presence of a large hyperpolarization-activated cation current, Ih (Lacey et al., 1990; Johnson and North, 1992b), fusiform shape and location in the lateral VTA proximal to the medial terminalis of the optic nucleus (Lammel et al., 2012). In this VTA subregion, Ih is a reliable predictor of dopamine neurons (Lammel et al., 2008, 2011). A subset of dopaminergic neurons was further confirmed by post-hoc immunostaining for tyrosine hydroxylase (TH) after recording. 99 of 131 post-hoc stained neurons were identified as TH positive. The remaining 32 50  were unable to be recovered. In the spike timing-dependent plasticity experiments, neurons were patch clamped in current-clamp mode with electrodes containing, (in mM): K-Methanesulfonate 125mM, KCl 5mM, HEPES 10mM, EGTA 0.2mM, MgCl2 2mM, 2.5 mg/ml MgATP, 0.25 mg/ml GTP, and 0.2% Biocytin, pH 7.2-7.4, 275-285 mOsm and picrotoxin (100 µM) in the external aCSF solution. Slices were preincubated in 200 µM HAV peptide, 200 µM scrambled control peptide, or vehicle. The spike-timing-dependent protocol for LTP induction was carried out as previously described (Liu et al., 2005). Briefly, the protocol consisted of 20 bursts of EPSP-spike pairs, with each burst consisting of 5 paired stimuli at 10 Hz (100 ms intervals), with an interburst interval of 5 s. Postsynaptic spikes were evoked by injection of depolarizing current pulses, with the onset of EPSPs preceding the peak of postsynaptic spikes by 5 ms. Evoked EPSPs were sampled at 0.1 Hz before and after LTP induction. In experiments where NASPM (1-Naphthyl acetyl spermine trihydrochloride, Tocris, Bristol, UK) was used, 100 µM of NASPM in aCSF was added to slices 25 min after STPD LTP induction to inhibit GluA2-lacking AMPA receptors. For the rest of experiments, electrodes (3–5MΩ) contained (in mM) 117 Cesium methanesulfonate, 20 HEPES, 0.4 EGTA, 2.8 NaCl, 5 TEA-Cl, 2.5 Mg-ATP and 0.25 Na-GTP (pH 7.2–7.3, 270–280 mOsm). To calculate AMPAR/NMDAR ratio, neurons were voltage clamped at 40 mV and an average of 30 EPSCs were measured before and after the application of D-2-amino-5-phosphonopentanoate (AP-5; 50 µM) for 5 minutes. NMDAR responses were calculated by subtracting the average response in the presence of AP-5 (AMPAR-mediated only) from that recorded in its absence. The peak of the AMPAR EPSC was divided by the peak of the NMDAR EPSC in order to compute the AMPAR:NMDAR ratio. Experiments measuring the I-V relationship were carried out in the presence of picrotoxin (100 µM) and when indicated in the presence of AP-5 (50 µM) to block GABAA and NMDA 51  receptors, respectively. The holding potentials were -70 mV, 0 mV and +40 mV. Synaptic currents were evoked by stimuli at 0.1 Hz, and the rectification index was calculated by dividing the gradient of the slope at negative potentials by the gradient of the slope at positive potentials. Excitatory and inhibitory transmission were recorded in cells voltage-clamped at −67 mV for mEPSCs and +10 for IPSCs in TTX (500 nM). AMPAR mEPSCs were selected based on their amplitude (>12 pA), decay time (<3 ms), and rise time (<1 ms) using the Mini60 MiniAnalysis program (Synaptosoft). Similarly, GABAA mIPSCs were selected for amplitude (>12 pA), rise time (<4 ms), and decay time (<10 ms).  2.2.5 TH Immunocytochemistry  Brain slices from patch-clamp recording were fixed overnight in cold 4% paraformaldehyde, rinsed in phosphate buffer solution (PBS), blocked in 10% normal donkey serum and incubated with monoclonal mouse anti-TH antibody (Sigma, Oakville ON, 1:1000 CAT# T2928) for 48 h at 4 °C. Secondary donkey anti-mouse fluorescein isothiocyanate antibody (Cedarlane, Burlington, ON, cat. no. NB120-6816, 1:500) was applied for 2 h at 4 °C. DyLight 594 streptavidin (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania; 1:200) was applied overnight at 4 °C. Slices were mounted using Fluoromount (Sigma, Oakville ON).  2.2.6 Conditioned Place Preference CPP was induced using a standard three-chambered apparatus, consisting of two conditioning compartments and a middle, connecting compartment (Stoelting Co., Wood Dale, IL). The two conditioning compartments had distinct wall patterns and floor textures to allow 52  mice to distinguish between them. Naïve mice were first allowed to habituate to the entire apparatus during a 30-minute session on day 1. Individual mice were removed from the experiment if they showed a strong baseline preference (>70%) for the conditioned chamber. On day 2, mice in the conditioned group received a 15 mg/kg injection of cocaine, and placed in a ‘conditioned’ chamber for 15 minutes. Mice were assigned to receive cocaine in one compartment or the other using an unbiased design. The following day, the mice received an equivalent volume of saline, and were placed in the opposite ‘non-conditioned’ compartment. This alternating pattern of conditioning was repeated three times (6 days total). On test day, place preference was assayed by giving each mouse with a priming injection of saline, placing them in the middle connecting compartment, and recording the amount of time spent in the two conditioning compartments over a 30 minute period. If mice underwent the extinction of CPP, the test day protocol was repeated each day until the drug group’s preference for the conditioned chamber had returned to habituation levels. In the CPP + homecage extinction experiment (‘CPP + HC’) mice underwent CPP as described above, and were then returned to their home cages without any re-exposure to the test apparatus for 6 days (the same duration of time as was required for extinction of CPP in the previous experiments). CPP using a palatable food reward (‘Food CPP’) was induced using similar apparatus and methodology. Mice were food restricted for one week before the start of testing, such their weight was maintained at 85% of their baseline weight, and were introduced to the palatable food 5 days before the start of testing so that they were familiar with the food. During conditioning 2-3 grams of palatable food (Bacon Softies™, VWR) was placed in the ‘conditioned’ chamber, and mice were given access to the food in this chamber for 30 minutes. The food pellet was weighed before and after the conditioning sessions so that the amount of food consumed could be determined. Mice received 53  no food reward in the unconditioned chamber during the 30-minute conditioning session. A control group received no food in either conditioning chamber. The alternating pattern of conditioning was repeated for 10 days before place preference was assayed on test day. All experiments were performed in dim lighting conditions, during the dark (wake) cycle. Experimenters were always blind to the genotype of the animal during testing, but not to the drug condition or food treatment group during cocaine-induced CPP or food CPP, respectively.  2.2.7 Electron Microscopy Sample Preparation  Mice were anesthetized with sodium pentobarbital (120 mg/kg), transcardially perfused by PBS followed by 4% paraformaldehyde (PFA) as described above, then post-fixed in 4% PFA overnight. Brains were then cut into 250 µm thick horizontal sections by vibratome. Small pieces of VTA tissue (<1mm in any dimension) were dissected from these slices and cryoprotected in 30% glycerol overnight at 4 °C. Samples were then plunge frozen in liquid ethane at -170 °C in an EM cryopreparation chamber (Leica), and transferred to a 1.5% Uranyl Acetate solution in 100% Methanol, kept at -90 °C in a Leica EM AFS for 30 hours. The temperature was increased to -45 °C over 11 hours. Next, samples were rinsed in 100% Methanol, and infiltrated with HM-20 acrylic resin (Electron Microscopy Sciences, Hatfield, PA) by increasing the resin to methanol ratio in 2-hour steps while maintaining the temperature at -45 °C. Samples were set up in capsules containing pure resin and polymerized under UV light for 24 hours at -45 °C, after which the temperature was slowly increased to 0 °C. Tissue sections were cut at 85 nm using a Diatome diamond knife and a Leica Ultramicrotome. Sections were collected on 300-mesh, formvar-coated Nickel grids.   54  2.2.8 Immunogold Electron Microscopy  Grids were rinsed with distilled water and subsequently immersed in a bead of TTBS with 0.1% Triton-X with 0.1% Sodium Borohydride and 50 mM glycine. The grids were then rinsed with TTBS with 0.1% Triton-X three times. Following this, nonspecific binding was blocked by immersing grids in a bead of 2% BSA in TTBS with 0.1% Triton-X for 10 minutes.  Primary antibodies against DAT (dopamine transporter) (rat, Millipore, CAT# MAB369), PSD-95 (rabbit, Frontier Institute, CAT# Af628), pan-cadherin (mouse, Sigma, CAT# C1821), GluA1 (rabbit, Millipore, CAT# ABN241), GluA2 (rabbit, NeuroMab/Antibodies Inc, CAT# 75-002), GAD67 (chicken, Abcam, CAT# ab75712), and VGLUT2 (guinea pig, Synaptic Systems, CAT#124014) were diluted in 2% BSA in TTBS with 0.1% Triton-X. Grids were immersed in 15 µl beads of diluted primary antibody overnight, at room temperature in a humidity chamber. The following day, grids were thoroughly rinsed by immersion in vials of TTBS with 0.1% Triton-X three times. Secondary antibodies were diluted in 2% BSA in TTBS with 0.1% Triton-X, and 0.05% Polyethyleneglycol (PEG) was added to prevent aggregation of gold beads. Grids were immersed in 15 µl beads of secondary antibody (Electron Microscopy Sciences, Hatfield, PA, goat-anti-rat 25 nm, cat. no. 25195, goat-anti-rabbit 15 nm, cat. no. 25112, goat-anti-rabbit 10 nm, cat. no. 25108, goat-anti-mouse 10 nm, cat. No. 25128, goat-anti-mouse 15 nm, cat. No. 25132, goat-anti-guinea pig 25 nm, cat. no. 25335, goat-anti-chicken 25 nm, cat. no. 25592) for 1.5 hours. Following this step, grids were repeatedly rinsed in TTBS with 0.1% Triton-X, and then rinsed in Milli-Q H2O and dried. Grids were then lightly counterstained with 2% uranyl acetate and Reynold's lead citrate. Images were collected at 98000X magnification on a Tecnai G2 Spirit transmission electron microscope (FEI Company, Eindhoven, Netherlands).  55  To analyze immunogold labeling, cell types were first identified by the presence of DAT, VGLUT2 or GAD67 markers, and synapse types were identified by the presence or absence of PSD-95 markers. The distance of all immungold-labelled cadherin from the synaptic membrane, or all immungold labelled GluA1 or GluA2 to the postsynaptic active zone membrane was measured. Due to the sizes of proteins and reagents involved (Mathiisen et al., 2006; see also Figure 2.1) the maximum cutoff of immunogold particles considered to be labelling target proteins at the synaptic membrane was 40 nm for cadherin, 30 nm for GluA1, and 35 nm for GluA2. The percentage of immungold particles localized to these regions was determined by [# immungold beads at target membrane]/[total # immunogold beads within 500 nm of target membrane at pre and post-synaptic compartments], and expressed as a percentage relative to saline-only controls which were processed and labelled in parallel. All images were acquired and analyzed blind to the genotype of each mouse.  56   Figure 2.1 Validation of Immunogold EM Reagents.  (a) Diagram of estimated sizes of immunogold reagents, and maximum distance from the synaptic membrane for immunogold particles recognizing target proteins. Empirical studies have shown that antibody-conjugated immunogold particles are localized to within 30 nm of target epitopes. Cadherin: The maximum distance for immungold particles recognizing cadherin at the synaptic membrane is ~40 nm. The primary antibody recognizes a target epitope in the C-terminal tail, and a conservative estimate for the size of this tail is ~10 nm in length based on crystal structures of cadherin C-terminal tail co-crystallized with β-catenin (Huber and Weis, 2001).  GluA1: The maximum distance for immungold particles recognizing GluA1 at the synaptic membrane is ~30 nm; the primary antibody recognizes an extracellular fragment.  GluA2: The maximum distance for immungold particles recognizing GluA2 at the synaptic membrane is ~35 nm; the primary antibody recognizes C-terminal tail fragment, and a conservative estimate of the size of this tail is ~5 nm, based on the length of the C-terminal tail sequence (Song and Huganir, 2002). (b-d) Top: Representative images of immunolabelling at synapses in the VTA. Bottom: Histogram of immunogold-labelling from the synaptic membrane.  (n= >100 synapses). The specificity of GluA1 (B), GluA2 (C) and PSD-95 (D) labelling at postsynaptic densities validates the use of these antibodies. (e) Top: PSD-95 and gephyrin immunolabelling at synapses in the VTA. Gephyrin immunostaining is restricted to symmetric synapses whereas PSD-95 staining is restricted to asymmetric synapses.  Bottom: The majority (>93%) of PSD-95 labelling was observed at asymmetric synapses that were not labelled by the inhibitory synapse marker, gephryin. This validates the identification of excitatory synapses through the use of PSD-95 labelling combined with asymmetric synapse morphology (n=4 mice, >150 synapses). (f) The dopamine transporter (DAT) antibody was validated by co-labeling with tyrosine hydroxlase (TH). Over 80% of cells in the VTA that were labelled with DAT also co-labelled for TH, indicating that the DAT antibody accurately identifies dopaminergic neurons (n=3 mice, >50 synapses). All scale bars = 100 nm. Data shown as mean ± SEM with individual mice (circles) overlaid. 57  2.2.9 Rotarod  Mice were trained on an accelerating rotarod apparatus (Mouse Rota-Rod, Ugo Basile) for five trials per day for two days. The rotarod accelerated from 4 to 400 rotations per minute over 5 minutes. Mice were scored on the latency of time until falling from the rotarod apparatus, with five-minute breaks between trials to allow for recovery. Experiments were performed during the dark (wake) cycle. Experimenters were blind to the genotype of the animal during testing and scoring. One cohort of mice completed the rotarod test, followed by context dependent fear conditioning.  2.2.10 Context-Dependent Fear Conditioning  Mice were placed in the conditioning chamber for 5 minutes, and after 3 minutes received an unconditioned foot shock stimulus (1 mA, 50 Hz) lasting 3 seconds. The next day mice were placed in the same chamber for 4 minutes, but did not receive an additional foot shock. Freezing behavior was determined by quantifying laser beam breaks in the conditioning chamber due to mouse activity, and total time % freezing was compared between groups. Experiments were performed during the dark (wake) cycle. Experimenters were blind to the genotype of the animal during testing and video scoring. One cohort of mice performed the context dependent fear conditioning experiment after completing the rotarod test.  2.2.11 Locomotor Sensitization to Cocaine.  During the habituation phase (day 1-2), mice were given an injection of saline (12 uL/g) and were placed in a 20 cm2 open field apparatus for 15 minutes. During cocaine sensitization testing (day 3-7), mice were instead given 15 mg/kg cocaine and placed in the apparatus. After a 58  10 day rest period, mice were given a 15 mg/kg ‘challenge’ dose of cocaine and re-introduced to the apparatus. During all phases of testing, mouse locomotion in the open field apparatus was recorded and then analyzed using Phenotracker software (TSE Systems, Hamburg, Germany). Experimenters were blind to the genotype of the animal during testing.  2.2.12 Food Consumption Testing  Mice were given pre-weighed pellets of low-fat (10% fat, Research Diets Inc, CAT# D12450B) or high-fat (60% fat, Research Diets Inc, CAT# D12492) food to consume over a 24-hour period. The food given was in excess of what the mouse could consume in this period, so they were not food restricted. Pellets were weighed after the 24-hour period so that the amount of food consumed could be calculated. Experimenters were blind to the genotype of the animal during testing.  2.2.13 Statistical Analysis  Unless otherwise noted, statistical analysis was done using unpaired Student’s t-test (two tailed) and two-way ANOVA. Data distribution was assumed to be normal but this was not formally tested. Correlative data examining the relationship between behavioural data and immunoEM data was analyzed using linear regression. Data from STDP electrophysiology experiments was analyzed by two way repeated measures (RM) ANOVA, and post-hoc analysis was done using Bonferroni’s test. Data from CPP experiments was also analyzed by two-way RM ANOVA with genotype as the between-subjects factor and time as the within-subjects factor. For comparisons between genotypes and within days, Bonferroni’s test was used. For comparisons to baseline within genotypes, Dunnett’s test was used. Correlative data examining 59  the relationship between pre- and postsynaptic cadherin localization was analyzed using linear regression. Results were considered significant when p<0.05. Analysis was performed using GraphPad Prism (GraphPad Software, San Diego, CA).  2.3 Results 2.3.1 Cadherins are Expressed in Dopaminergic Neurons and Required for LTP We first examined the expression of several classical cadherins and found that cadherins are widely expressed in both dopaminergic and non-dopaminergic neurons in the VTA (Figure 2.2, Figure 2.3). Nearly all dopaminergic cells were immunopositive for N-cadherin, R-cadherin, cadherin-7, cadherin-8, and cadherin-11, isoforms whose mRNA had previously been detected in this region (Hertel et al., 2008; Obst-Pernberg et al., 2001)  Figure 2.2 Cadherins are Expressed in Dopaminergic Neurons and are Essential for LTP in the VTA. (a) VTA neurons coimmunostained for cadherins (green), dopamine transporter (DAT; magenta) and DAPI (blue). Arrowheads indicate neurons positive for both cadherin and DAT; asterisks indicate neurons positive for cadherin but not DAT. Scale bar, 10 mm. (b) STD LTP in the VTA was abolished by treatment with a peptide containing an HAV motif that disrupts N-cadherin extracellular interactions (###P < 0.0001, significant interaction between peptide treatment and time, two-way repeated-measures ANOVA, F(78,624) = 4.037, *P < 0.05, Bonferroni’s test post hoc, n = 8 cells in 8 mice vehicle, 6 cells in 6 mice HAV, 5 cells in 5 mice scrambled peptide). Vehicle-only and scrambled peptide (HAV-S) had no effect on LTP. Data shown as mean ± s.e.m.  Normalized EPSP amplitude (%)Time (min)b # # #  Spike-Timing ProtocolVehicleHAV peptide (200 μM)HAV-S peptide (200 μM)*3 mV25 msVehicleHAV-SHAV212121DAT +DAPImerge* * * * *R-cadherinN-cadherin cadherin-11cadherin-7 cadherin-80204060801001201401601802000 5 10 15 20 25 30 35 40 45 50a21Figure-1 (Bamji)60  We next investigated the function of cadherins in activity-induced potentiation of excitatory synapses onto dopaminergic neurons in the VTA. We used an HAV antagonistic peptide that blocks cadherin interactions in trans to reduce cadherin stability at the synaptic membrane. Indeed, it has previously been shown that disrupting cadherin trans interactions, significantly attenuates cadherin membrane stability (Delva and Kowalczyk, 2009). Treatment of VTA slices with the HAV peptide abolished spike-timing-dependent (STD) LTP at excitatory synapses onto dopaminergic neurons (Figure 2.2b). STD LTP is mediated by the insertion of Ca2+-permeable GluA1 homomers similar to potentiation of VTA synapses induced by cocaine (Argilli et al., 2008; Engblom et al., 2008). As cadherin can stabilize AMPARs through its association with GluA1 and/or GluA2 subunits (Nuriya and Huganir, 2006; Saglietti et al., 2007; Silverman et al., 2007), this suggested that peptide treatment disrupted STD LTP by decreasing cadherin membrane stability and thus prevents the stabilization of newly-inserted GluA1 homomers at the synaptic membrane. We therefore next sought to investigate the relationship between cadherin localization and AMPAR trafficking in synaptic plasticity and behavioural conditioning driven by cocaine. 61   Figure 2.3 Quantification of Cadherin Expression in VTA Neurons. a) VTA inhibitory neurons co-immunostained for cadherins (green), GAD67 (magenta) and DAPI (blue). Scale bar = 10 µm. Quantification of the proportion of dopaminergic neurons (b) and GABAergic neurons (c) that were immunopositive for different classical cadherins (DAT+: n = 26, 39, 68, 23, 46 cells immunostained for N-cadherin, R-cadherin, cadherins -7, -8, and -11, respectively; GAD67+: n = 45, 23, 31, 26, 30 immunostained for N-cadherin, R-cadherin, cadherins -7, -8, and -11, respectively). Immunopositive cells were defined as cells in which the immunofluorescence signal for each cadherin was over 2.5 fold greater than average signal detected in secondary-only controls. (d-h) Quantification of somatic immunofluorescence signal levels for N-cadherin (n = 26 DAT+, 45 GAD67+ cells from 2 mice) (d), R-cadherin (n = 39 DAT+, 23 GAD67+ cells from 2 mice) (e), cadherin-7 (n = 68 DAT+, 31 GAD67+ cells from 2 mice) (f), cadherin-8 (n = 23 DAT+, 26 GAD67+ cells from 2 mice) (g) and cadherin 11 (n = 46 DAT+, 30 GAD67+ cells from 2 mice) (h) in DAT+, GAD67+ neurons and secondary-only controls (n = 23 DAT+, 18 GAD+ cells with anti-mouse secondary only; n = 75 DAT+, 50 GAD67+ cells with anti-rabbit secondary only) for each cell type. In all cell types (d-h), immunostaining levels for each of the cadherin isoforms were significantly greater than secondary-only controls (p<0.0001, 2-way ANOVA, significant main effect of immunostain condition, p<0.001, Bonferroni’s test post hoc) (i) Representative immunoblots identifying cadherins in the VTA. Sizes of cadherin isoforms detected were consistent with the predicted sizes of each protein (see Methods). Representative Data shown as mean ± SEM with individual cells (circles) overlaid.  62  2.3.2 Cadherins are Recruited to VTA Synapses During Cocaine CPP Drugs of abuse such as cocaine potentiate excitatory synapses onto dopaminergic neurons by enhancing the localization of Ca2+-permeable GluA1 homomers into synaptic membranes, which contributes to the formation and expression of drug-induced behaviours (Delva and Kowalczyk, 2009; Engblom et al., 2008; Mameli and Lüscher, 2011). After demonstrating that cadherin is required for LTP at synapses in the VTA (Figure 2.2b), we examined the role of cadherins in conditioned place preference (CPP), a behavioral assay that models the learned preference for previously neutral contextual cues driven by drugs of abuse (Tzschentke, 2007). In other brain regions, enhanced synaptic activity is associated with increased recruitment of cadherins to the synapse (Bozdagi et al., 2010; Mendez et al., 2010), as well as increased stability of cadherin at the synaptic membrane (Tai et al., 2007). Intact cadherin adhesion at synapses is also required for the acquisition of new memories (Schrick et al., 2007). We therefore hypothesized that the acquisition of cocaine-induced CPP is associated with increased insertion of cadherin to the synaptic membrane of VTA synapses. We used immunogold electron microscopy (EM) (validated in Figure 2.1) to examine nanometer-scale changes in the localization of cadherins and AMPAR subunits at synapses of the VTA following cocaine-induced CPP.  63   Figure 2.4 Cocaine-Induced CPP leads to Recruitment of Cadherin and GluA1 to Excitatory Synapses onto Dopaminergic Neurons in the VTA. (a) Experimental schedule for CPP and extinction experiments. Hab., habituation. (b) Cocaine administration produced robust CPP (#P < 0.001, significant interaction between treatment and test day, two-way repeated-measures ANOVA, F(6,60) = 4.422, **P < 0.01, Bonferroni’s test post hoc) that extinguished over 5 d. (c) EM of VTA synapse showing immunogold-labeled DAT, PSD-95 and cadherin (scale bar, 100 nm). (d) Cadherin shifted to the synaptic membrane of excitatory synapses following cocaine CPP (20 nm bins). (e) The relative percentage of cadherin at the synaptic membrane at excitatory synapses ([number of cadherin beads within 40 nm of the pre and postsynaptic membrane]/[ number of beads within 500 nm of the synaptic membrane], expressed as a percentage relative to saline controls) was significantly increased compared to saline controls following cocaine-induced CPP (P < 0.01, significant interaction, two-way ANOVA, F(4,41) = 4.999; **P = 0.0026, n.s.,P > 0.9999, Bonferroni’s test post hoc) but not in home cage controls, following extinction of CPP, following return to home cage for 6 d after CPP (CPP + HC), or following food CPP (NF: no food, PF: palatable food; see also Figure 2.7). CPP: n = 6 mice saline, 6 mice cocaine; HC: n = 4 mice saline, 3 mice cocaine; CPP + extinction: n = 5 mice saline, 6 mice cocaine; CPP + HC: n = 6 mice saline, 5 mice cocaine; food CPP: n = 4 mice NF, 6 mice PF. (f) The percentage of cadherin localized to the synaptic membrane at excitatory synapses was significantly correlated with time spent in conditioned chamber following cocaine CPP (linear regression, P = 0.0193, F(1,10) = 7.758), 6 mice per condition. (g) No change in cadherin localization was observed at inhibitory synapses following cocaine CPP. (h) The relative percentage of cadherin at the synaptic membrane was not changed at inhibitory synapses following cocaine CPP, home cage controls, extinction of CPP, or food CPP (no significant interaction, p=0.8399, two-way ANOVA, F(4,40) = 0.3537; n.s., P > 0.9999 Bonferroni’s test post hoc, CPP: n = 6 mice saline, 6 mice cocaine; HC: n = 4 mice saline, 3 mice cocaine; CPP + extinction: n = 4 mice saline, 5 mice cocaine; CPP + HC: n = 6 mice saline, 6 bd eg hRelative % cadherinlocalized to synaptic membranefiTime spent inconditioned chamber (%)Saline controlCocaine CPPDay0500 5000500 500Saline control Cocaine CPPExcitatory SynapsesTime in conditioned chamber (s)% cadherin localized to synaptic membrane% cadherin localized to synaptic membraneTime in conditioned chamber (s)cDistance from presynaptic (left) or postsynaptic (right) membrane (nm)Relative % cadherinlocalized to synaptic membrane0%10%20%30%40%50%0 500 1000 15000%10%20%30%40%50%0 500 1000 1500Inhibitory SynapsesSaline controlCocaine CPPr = -0.118 r = +0.6560500 500Saline control0500 500Cocaine CPP% of total cadherin% of total cadherin0%5%10%15%0%5%10%15%-5%0%+5%% change in cadherinfollowing cocaine CPP 0500 5000500 500Distance from presynaptic (left) or postsynaptic (right) membrane (nm)n.s.conditioning**Cocaine CPPj k lRelative % GluA1localized to PSDsynaptic membraneTime in conditioned chamber (s)% GluA1 localized to PSD synaptic membraneExcitatory SynapsesDistance from PSD synaptic membrane (nm)0 500 0 500 0 500**n.s.n.s. n.s.0%10%20%30%40%50%0 500 1000 15000%5%10%15%-5%0%5%0%5%10%15%20%0%5%10%15%20%-7.5%0.0%7.5%r = +0.661% of total GluA1% change in cadherinfollowing cocaine CPP % change in GluA1following cocaine CPP Saline control0%10%20%30%40%50%60%70%1 2 3 4 5 6 7 8 9 0 1 2 31 1 1 1# p = 0.0193p = 0.78p = 0.019Hab. Conditioning TestCocaine CPPExtinctionCPP tissuecollectedExtinction tissuecollectedCocaine (15 mg/kg) in conditioned chamberSaline in unconditioned chamberSaline in conditioned chambera1 2 3 4 5 6 7 8 9 10 11 12 13Saline controlFree access to both chambersDay:Saline controlCocaine CPPSaline controlCocaine CPPPRE POSTPRE POSTAt synaptic membrane(<40 nm from membrane)Cadherin (10 nm bead):Not at synaptic membranePSD-95 (15 nm bead)DAT (25 nm bead)0%5%10%15%Figure-2 (Bamji)0%50%100%150%200%350%CPP HomecageCPP +ExtinctionPFNFFoodCPPcoc.sal. coc.sal. coc.sal.CPP +HCcoc.sal.n.s.0%50%100%150%200%250%0%50%100%150%200%250%CPP HomecageCPP +ExtinctionPFNFFoodCPPcoc.sal. coc.sal. coc.sal.CPP +HCcoc.sal.CPP HomecageCPP +ExtinctionPFNFFoodCPPcoc.sal. coc.sal. coc.sal.CPP +HCcoc.sal.n.s.n.s. n.s. n.s.n.s.n.s. n.s. n.s.**250%300%64  mice cocaine; food CPP: n = 4 mice NF, 6 mice PF). (i) Cadherin localization to synaptic membrane at inhibitory synapses was not correlated with time spent in conditioned chamber following CPP. (j) GluA1 localization shifted toward the PSD membrane following cocaine CPP. (k) The relative percentage of GluA1 at the PSD membrane ([number of GluA1 beads within 30 nm of the PSD membrane]/[number of beads within 500 nm of the PSD membrane], expressed as a percent relative to saline controls) was significantly increased at excitatory synapses following cocaine-induced CPP (P < 0.01, significant interaction, two-way ANOVA, F(4,44) = 4.049, **P = 0.0041, n.s., P > 0.9999, Bonferroni’s test post hoc), but not in home cage controls, following extinction of CPP, following return to home cage for 6 d after CPP, or following food CPP. CPP: n = 6 mice saline, 6 mice cocaine; HC: n = 4 mice saline, 3 mice cocaine; CPP + extinction: n = 6 mice saline, 6 mice cocaine; CPP + HC: n = 6 mice saline, 6 mice cocaine; food CPP: n = 6 mice NF, 5 mice PF (l) Percentage of GluA1 localized to the PSD membrane was significantly correlated with time spent in conditioned chamber following cocaine-induced CPP (linear regression, P < 0.05, F(1,10) = 7.848), n = 6 mice per condition; >100 synapses were analyzed per group. Data shown as mean ± s.e.m. with data for individual mice (circles) overlaid.  CPP was induced in a three-chamber apparatus (Figure 2.4a), producing a robust increase in preference for the cocaine-paired, conditioned chamber (Figure 2.4b). Mice were then sacrificed and the VTA isolated by microdissection for immunogold EM (Figure 2.4c). We found that cocaine-induced CPP results in a striking redistribution of cadherin at excitatory synapses onto dopaminergic neurons in the VTA (Figure 2.4d). Indeed, the proportion of cadherin localized to the synaptic membrane increased by 86% after CPP (number of immunolabelled beads within 40 nm of the synaptic membrane divided by total beads at the pre and postsynaptic compartments; Figure 2.4e), though no changes in total levels of cadherin at synaptic compartments were detected (Figure 2.5). Moreover, analysis of individual mice demonstrated a strong positive correlation between the amount of cadherin at the synaptic membrane and time spent in the cocaine-paired conditioned chamber (r = +0.66, Figure 2.4f). The increased localization of cadherin to the synaptic membrane was observed at both pre- and post-synaptic compartments following cocaine CPP (Figure 2.6), which is consistent with increased trans-synaptic adhesion between cadherins.   65   Figure 2.5 Overall Levels of Cadherin and GluA1 at VTA Synapses are Unaffected by Acquisition and Extinction of CPP. For each behavioral condition, the total number of immunogold-labelled target proteins (cadherin or GluA1) at synaptic compartments (within 500 nm of the synaptic membrane) were counted in saline and cocaine groups, and expressed as a percentage of the saline group. Total immunogold-labelled cadherin at excitatory synapses (a) and inhibitory synapses (b) were unchanged following cocaine-induced CPP, home cage administration of cocaine, extinction of CPP, following return to home cage for 6 days after CPP (‘CPP + HC’), or Food CPP (NF: No food, PF: Palatable food) (c) Total immunogold-labelled GluA1 at excitatory synapses was also unchanged changed following cocaine-induced CPP, home cage administration of cocaine, extinction of CPP, return to home cage during extinction period after conditioning or Food CPP. Two-way ANOVA, Data shown as mean ± SEM with individual mice (circles) overlaid.   Figure 2.6 Time Spent in Cocaine-Conditioned Chamber is Correlated with Increased Cadherin localized to the Synaptic Membrane at both Pre- and Post-Synaptic Compartments of Excitatory Synapses onto Dopaminergic Neurons in the VTA. The percentage of cadherin localized to the synaptic membrane at excitatory synapses was significantly correlated with time spent in conditioned chamber following cocaine CPP in (a) presynaptic compartments (Linear regression, p<0.05, F(1,10) = 7.351), (b) postsynaptic compartments (Linear regression, p<0.05, F(1,10) = 5.143), and (c) both pre- and postsynaptic compartments analyzed together (Linear regression, p<0.01, F(1,22) = 13.44).   66  Notably, we saw no significant change in cadherin localization in control mice which received the same schedule of cocaine and saline administration in their home cages rather than in a novel environment (Figure 2.4e) or in mice where CPP was induced using palatable food rewards instead of cocaine (Figure 2.4e, Figure 2.7). This suggested that the effects observed after cocaine CPP were not due to general effects of cocaine or non-specific learning-induced plasticity, but were specifically attributable to the formation of drug-associated memories. Together, these findings indicate that insertion of cadherin into the synaptic membrane specifically occurs following the learned association between novel contextual cues and the effects of cocaine during behavioral conditioning. These changes in cadherin localization were also transient, and returned to baseline levels following active extinction of CPP or return of mice to home cages for an equivalent period of time without re-exposure to the CPP apparatus (Figure 2.4e). At inhibitory synapses, no changes in cadherin distribution were observed following cocaine CPP, extinction of cocaine CPP, food CPP or in home cage controls (Figure 2.4 g, h, i, Figure 2.7). We also observed no changes in cadherin localization at excitatory or inhibitory synapses onto glutamatergic or GABAergic neurons in the VTA following CPP (Figure 2.8), indicating the increase in cadherin localization to the synaptic membrane was specific to excitatory synapses onto dopaminergic neurons in the VTA. 67   Figure 2.7 Food CPP does not affect the Localization of Cadherin and GluA1 at VTA synapses. (a) Experimental schedule for induction of food CPP. Pairing of palatable food with the conditioned chamber produced robust CPP (p<0.05, significant interaction between genotype and test day, two-way RM ANOVA, F(1,10) = 8.325, *p< 0.05, Bonferroni’s test post hoc, n=6 mice control and palatable food). (b,c) No changes in the distribution of cadherin were observed at (b) excitatory synapses or (c) inhibitory synapses. (d) No redistribution of GluA1 was observed at excitatory synapses onto dopaminergic neurons in the VTA. See also Figure 2.4 e, h, k ‘Food CPP’ for analysis of relative % of cadherin and GluA1 localized to synaptic membrane. Data shown as mean ± SEM.   2.3.3 GluA1 is Recruited to VTA Synapses During Cocaine CPP We then used immunogold labelling to identify GluA1-containing AMPARs, and found that they exhibited the same pattern of insertion and removal from the synaptic membrane as cadherin in each of the behavioral conditions (Figure 2.4j). The proportion of GluA1-containing AMPARs localized to the post-synaptic density (PSD) membrane was significantly increased following CPP (121% increase, Fig. 2.4k), though total levels of GluA1 were unchanged at 68  synaptic compartments (Figure 2.5). As with cadherin, there was a strong positive correlation between the amount of GluA1 at the synaptic membrane and time spent in the cocaine-paired chamber for individual mice (r = +0.66, Fig. 2.4l), and GluA1-containing AMPAR localization was also unchanged compared to saline controls following extinction of CPP, in home cage controls, or following CPP using palatable food (Figure 2.4k, Figure 2.7). These data directly demonstrate that cocaine CPP drives the insertion of GluA1-containing AMPARs to the PSD membrane, which is thought to contribute to the cocaine-mediated increase in AMPA:NMDA receptor ratio at VTA synapses previously observed using electrophysiological techniques (Argilli et al., 2008; Borgland et al., 2004; Ungless et al., 2001). We also examined co-immunolabelling of GluA1 and cadherin together at individual VTA synapses following cocaine CPP and found that individual synapses with a greater proportion of cadherin localized to the synaptic membrane also had significantly more GluA1 localized to the membrane (Figure 2.9). This correlation between cadherin and GluA1 levels at individual synapses provided further support that cadherin acts to stabilize GluA1 homomers at potentiated synapses, consistent with our electrophysiological data demonstrating that cadherin stability was essential for STD LTP at VTA synapses (Figure 2.2 b). 69   Figure 2.8 No Changes in Localization of Cadherin at Synapses onto Non-Dopaminergic Neurons in the VTA Following Cocaine CPP. (a) Electron micrograph of VTA synapse showing immunogold-labelled GAD67, PSD-95 and cadherin (Scale bar = 100 nm). (b-c) No change in the localization of cadherin at excitatory (b) or inhibitory (c) synapses onto GAD67+ neurons was observed following cocaine CPP. The relative percentage of cadherin at the synaptic membrane at excitatory and inhibitory synapses ([#cadherin beads within 40 nm of the pre and postsynaptic membrane] / [ # beads within 500 nm of the synaptic membrane], expressed as a percent relative to saline controls) was also unchanged compared to saline controls following cocaine CPP (unpaired t-tests, n = 3 mice). (d) Electron micrograph of VTA synapse showing immunogold-labelled VGLUT2, PSD-95 and cadherin (Scale bar = 100 nm). (e-f) No change in the localization of cadherin at excitatory (e) or inhibitory (f) synapses onto VGLUT2+ neurons was observed following cocaine CPP. The relative percentage of cadherin at the synaptic membrane at excitatory and inhibitory synapses was also unchanged compared to saline controls following cocaine CPP (unpaired t-tests, n = 3 mice. > 100 synapses were analyzed per condition). Data shown as mean ± SEM with individual mice (circles) overlaid.  Figure 2.9 Increased Cadherin and GluA1 Localization to the Synaptic Membrane at Individual Synapses onto Dopaminergic Neurons in the VTA following Cocaine CPP. The proportion of GluA1 localized to the synaptic membrane was significantly higher in individual synapses with a greater proportion of cadherin localized to the synaptic membrane in (a) saline controls (p<0.0001 one-way ANOVA, F(2,8) = 71.57) and (b) following cocaine CPP (p<0.0001 one-way ANOVA, F(2,11) = 33.96) (c) Following cocaine CPP, a significantly smaller proportion of synapses in the VTA had ‘low’ cadherin localization to 70  the synaptic membrane, while a significantly greater proportion of synapses had a ‘high’ cadherin localization to the synaptic membrane (p<0.0001, significant interaction between treatment and cadherin groups, two-way ANOVA, F(2,15) = 18.40). **p< 0.01, **p< 0.01, ***p< 0.001 Bonferroni’s test post hoc, n=3 mice saline, n=4 mice cocaine CPP. Data shown as mean ± SEM with individual mice (circles) overlaid.  2.3.4 Cadherin Stabilization at VTA Synapses Reduces Cocaine CPP Given the strong correlation between CPP acquisition and cadherin localization at excitatory synaptic membranes in the VTA, we hypothesized that subcellular changes in cadherin localization may regulate cocaine-induced synaptic plasticity and behavioral conditioning. To test this, we increased cadherin at the synaptic membrane using a transgenic mouse line in which β-catenin levels are increased in dopaminergic neurons (Slc6a3:Cre/+;Ctnnb1lox(ex3)/lox(ex3) mice, termed here ‘DAT-Cre;β-catΔex3’ mice, Figure 2.10a;) (Bäckman et al., 2006; Harada et al., 1999). β-catenin is the major intracellular binding partner of all classical cadherins, and our lab has previously shown that elevating β-catenin levels using this approach significantly increases the stabilization of cadherin and AMPARs at the synaptic membrane in hippocampal neurons in vivo (Mills et al., 2014). DAT-Cre;β-catΔex3 mice exhibited a 48% reduction in cocaine-induced CPP compared to control mice (Figure 2.10b, Day 8). There was no significant difference between groups in the rate of extinction from day 8 to 9 (p = 0.5527, unpaired t-test, t(44) = 0.5983, n = 23 mice per condition). However, due to the decreased magnitude of CPP, DAT-Cre;β-catΔex3 mice returned to baseline levels of preference for the conditioned chamber after 1 day of extinction (day 9), compared to 3 days in controls (day 11). Behavioral changes in these mice were specific to cocaine-mediated CPP; DAT-Cre;β-catΔex3 mice appeared phenotypically normal, exhibited no changes in exploratory behaviour or basal locomotion (Figure 2.10c), and showed intact locomotor sensitization to repeated cocaine administration (Figure 2.10d). DAT-Cre;β-catΔex3 71  mice also showed no change in motor learning (Figure 2.10e), contextual fear conditioning (Figure 2.10f), food consumption (Figure 2.11), or CPP driven by food rewards (Figure 2.10g). The lack of change in tasks which require intact recognition of a novel context (contextual fear learning and food CPP) also indicated that impairments in spatial memory were not responsible for the reduction in cocaine CPP observed in DAT-Cre;β-catΔex3 mice. We also verified that, following β-catenin stabilization in DAT-Cre;β-catΔex3 mice, no subsequent changes in Wnt pathway targets were observed in dopaminergic neurons in the VTA, indicating that the observed effects on CPP were not due to alterations in Wnt signalling (Figure 2.13).  Figure 2.10 Stabilization of Cadherin by β-catenin at Synapses in the VTA Reduces Cocaine-Induced CPP. Scale bar, 10 mm. (a) b-catenin levels were significantly increased in DAT+ neurons (arrowheads) in the VTA of DAT-Cre;β-catΔex3 mice compared to adjacent DAT– cells (asterisks) and to DAT+ neurons in control mice (P < 0.001, significant interaction between genotype and cell type, two-way ANOVA, F(1,48) = 38.13, ***P < 0.001 Bonferroni’s test post hoc, wild type: n = 14 cells non-DAT, 10 cells DAT+, DAT-Cre;β-catΔex3: 16 cells DAT–, 12 cells DAT+). (b) Cocaine-induced CPP was significantly reduced in DAT-Cre;β-catΔex3 mice compared to controls (#P = 0.0440, significant interaction between genotype and test day, two-way repeated-measures ANOVA, F(6,264) = 2.194; *P = 0.0488, Bonferroni’s test post hoc, n = 23 mice per genotype). Preference for the cocaine-paired chamber returned to baseline after 3 d of extinction in control mice and 1 d of extinction in DAT-Cre;β-catΔex3 mice (+P < 0.01, ++P < 0.0001, significantly different from day 1, Dunnett’s test post hoc). (c,d) DAT-Cre;β-catΔex3  mice showed no differences in average speed (c) in the three-chamber CPP apparatus during habituation or after CPP (no significant interaction, two-way repeated measures ANOVA, F(6,154) = 0.2211; n.s., P = 0.9695, n = 10 01002003001 2 3 4 5 1 2 3 4 5cAverage speed (m/s)d eRotarod latency to fall (s)Day 1 Day 2ControlDAT-Cre;β-catΔex3% Time spent freezingDay 1 (foot shock)Day 2Pre Postn.s.0%10%20%30%40%50%60%n.s.050100150200250aRelative β-catenin immunofluorescenceDAT: DAT-Cre;β-catΔex3Control- + - +***DAT-Cre;β-catΔex3Controlβ-cateninDAT+DAPImerge *** *Day1 2 3Hab.ControlDAT-Cre;β-catΔex3Conditioning4 5 6Time in conditioned chamber (%)b*7 8 9 10 11 12 13# ++++++40%45%50%55%60%65%70%75%ControlDAT-Cre;β-catΔex3Figure-3 (Bamji)fCocaineDistance travelledper sesssion (m)DayCocaineControlDAT-Cre;β-catΔex3n.s.0102030405060701 2 3 4 5 6 7 17//gTime in conditioned chamber (%)n.s.ControlDAT-Cre;β-catΔex300.010.020.030.040.050.060.070.081 2 3 4 5 6 7 8 9 10 11 12 13ControlDAT-Cre;β-catΔex3n.s.Hab.ConditioningDayDayConditioning (food)0%25%50%75%1 2 3 4 5 6 7 8 9 10111230072  mice control, 14 mice DAT-Cre;β-catΔex3 ), and no differences in locomotor sensitization to cocaine (d) compared to littermate controls (no significant interaction, two-way repeated measures ANOVA, F(7,240) = 0.5123; n.s., P = 0.8249, n = 16 mice control, 16 mice DAT-Cre;β-catΔex3 ). (e) DAT-Cre;β-catΔex3 mice showed normal coordination and motor learning on an accelerating rotarod task (no significant interaction, two-way repeated measures ANOVA, F(9,260) = 0.3601, n.s., P = 0.9529, n = 13 mice control, 15 mice DAT-Cre;β-catΔex3 ). (f) DAT-Cre;β-catΔex3mice showed no change in the acquisition of contextual fear memory following a foot shock in a novel environment compared to littermate controls (no significant interaction, two-way ANOVA F(2,90) = 1.348; n.s., P = 0.2650, n = 16 mice control, 16 mice DAT-Cre;β-catΔex3 ) (g) DAT-Cre;β-catΔex3 mice showed no impairments in CPP driven by palatable food rewards (no significant interaction, two-way ANOVA F(1,27) = 0.1161, n.s., P = 0.7360, n = 17 mice control, 12 mice DAT-Cre;β-catΔex3 ). Data shown as mean ± s.e.m.   Figure 2.11 No Changes in Food Consumption or Body Weight in DAT-Cre;β-catΔex3 mice. No changes in consumption of low-fat food (a) or high-fat food (b) over a 24-hour period were observed in DAT-Cre;β-catΔex3 mice (unpaired t-tests, n=5 mice low-fat, 5 mice high-fat WT, n=6 mice low-fat, 5 mice high-fat DAT-Cre;β-catΔex3) indicating similar appetitive behavior. (c) At 4-6 weeks of age, no difference in average body weight was observed between age-matched DAT-Cre;β-catΔex3 mice and littermate controls (unpaired t-test, n=10 mice per group). Data shown as mean ± SEM with individual mice (circles) overlaid.   2.3.5 Cadherin Stabilization at VTA Synapses Blocks Synaptic Plasticity To determine why there was a marked attenuation of cocaine CPP in DAT-Cre;β-catΔex3 mice, we used immunogold EM to examine the distribution of cadherin, GluA1 and GluA2 at excitatory synapses onto VTA dopaminergic neurons after CPP. We found that the proportion of cadherin localized to the synaptic membrane was significantly increased in DAT-Cre;β-catΔex3 mice under basal conditions (~77% increase) (Figure 2.12 a, b). However, unlike control mice, DAT-Cre;β-catΔex3 mice did not exhibit additional recruitment of cadherin to the synaptic membrane during cocaine-mediated CPP. Additionally, both the removal of GluA2-containing AMPARs and the insertion of GluA1-containing AMPARs driven by cocaine CPP were blocked 73  in DAT-Cre;β-catΔex3 mice (Figure 2.12 c-f). There was also no significant change in total levels of cadherin (p = 0.3567), GluA1 (p = 0.8557) or GluA2 (p = 0.5683) in DAT-Cre;β-catΔex3 mice (Figure 2.13 c, d, e).   Figure 2.12 Stabilization of Cadherin at Synapses in the VTA Prevents the Removal of GluA2-Containing AMPARs and Blocks the Insertion of GluA1-Containing AMPARs. Immunogold EM was used to identify differences in cadherin, GluA2, and GluA1 localization after cocaine CPP in wild type and DAT-Cre;β-catΔex3 . (a,b) Cadherin localization to the synaptic membrane was increased under basal conditions in DAT-Cre;β-catΔex3 mice, and recruitment of additional cadherin to the synaptic membrane during CPP was blocked (P = 0.0453, significant interaction between treatment and genotype, two-way ANOVA, F(1,8) = 5.613; *P = 0.0191, n.s., P > 0.9999, Bonferroni’s test post hoc, n = 3 mice per condition; >100 synapses were analyzed per group). (c,d) The removal of GluA2 from the PSD membrane at excitatory synapses following CPP was blocked in DAT-Cre;β-catΔex3 mice (P =0.0015, significant interaction between treatment and genotype, two-way ANOVA, F(1,8) = 22.07; **P < 0.01, n.s., P > 0.9999, Bonferroni’s test post hoc, n = 3 mice per condition). (e,f) The insertion of GluA1 in the PSD membrane at excitatory synapses onto dopaminergic neurons following CPP was blocked in DAT-Cre;β-catΔex3 mice (P = 0.0073, significant interaction between treatment and genotype, two-way ANOVA, F(1,11) = 10.75; *P = 0.0117, **P = 0.0080, n.s., P > 0.9999, Bonferroni’s test post hoc, n = 3 mice control saline, n = 3 mice control cocaine; n = 5 DAT-Cre;β-catΔex3 mice saline, 4 DAT-Cre;β-catΔex3 mice cocaine). Data shown as mean ± s.e.m. with data for individual mice (circles) overlaid.  Relative GluA1 atPSD synaptic membranee fd% of total GluA2Controlc% of total GluA1 Saline control Cocaine CPP Saline control Cocaine CPPControl n.s.* *DAT-Cre;β-catΔex3Relative GluA2 atPSD synaptic membraneDAT-Cre;β-catΔex3Controln.s.** **DAT-Cre;β-catΔex3ba% of total cadherinRelative cadherinat synaptic membraneControln.s.*DAT-Cre;β-catΔex3Saline control Cocaine CPPSaline control Cocaine CPP Saline control Cocaine CPPDistance from presynaptic (left) or postsynaptic (right) membrane (nm)0500 500n.s.0%100%200%300%0%50%100%150%0%50%100%150%200%250%Distance from PSD synaptic membrane (nm)0 500 0 500 0 500 0 500Distance from PSD synaptic membrane (nm)0 500 0 500 0 500 0 500Control DAT-Cre;β-catΔex3Control DAT-Cre;β-catΔex3Saline control Cocaine CPP0%5%10%15%20%25%0%5%10%15%20%25%0%5%10%15%20%25%0%5%10%15%20%25%0%5%10%15%20%25%0%5%10%15%20%25%0%5%10%15%20%25%0%5%10%15%20%25%0%5%10%15%0%5%10%15%0%5%10%15%0%5%10%15%0500 5000500 5000500 500% of total GluA2% of total GluA1% of total cadherincoc.sal. coc.sal.coc.sal. coc.sal.coc.sal. coc.sal.PRE POSTFigure-4 (Bamji)74  In addition to interacting with GluA1 (Nuriya and Huganir, 2006), cadherins can also interact directly and indirectly with the GluA2 subunit of AMPARs (Saglietti et al., 2007; Silverman et al., 2007). Consequently, these data indicate that aberrantly increasing cadherin localization to the synaptic membrane prior to CPP stabilizes GluA1/2 heteromers present at VTA synapses under basal conditions, which blocks cocaine-induced insertion of GluA1 homomers and results in significantly reduced cocaine CPP.  Figure 2.13 No Changes in DAT-Cre;β-catΔex3 Mice of Overall Expression of Wnt Targets in VTA Dopamine Neurons or Levels of Cadherin, GluA2 or GluA1 at VTA Synapses. (a) Representative images of immunostaining for Wnt targets in VTA dopaminergic neurons (DAT; dopamine transporter). No significant differences in Wnt target expression were observed dopaminergic neurons in of DAT-Cre;β-catΔex3 and control mice (Scale bar = 5 µm), indicating that the increase in β-catenin (a component of the Wnt signalling pathway) in DAT-Cre;β-catΔex3 mice did not result in alterations in Wnt signalling. This finding is consistent with our previous work demonstrating that stabilization of β-catenin in adult hippocampal neurons using this transgenic line did not result in increases in Wnt target mRNA or protein levels (Mills et al., 2014) and evidence that differentiated neurons are less sensitive to fluctuations in cytoplasmic β-catenin levels (Kratz et al., 2002), which may be mediated by greater control of nuclear transport of β-catenin in neurons (Schmeisser et al., 2009). (b) Quantification of immunostaining for Wnt targets, no differences between groups were observed (two-way ANOVA, n=8, 12 (Axin2), n=9, 12 (Lef1), n=18, 13 (c-Jun), n=13, 9 (c-Myc) dopaminergic neurons from WT, DAT-Cre;β-catΔex3, respectively). No changes in total levels of immunogold-labelled cadherin (c), GluA2 (d) or GluA1 (e) were observed in DAT-Cre;β-catΔex3 mice and littermate controls under basal conditions or following cocaine CPP (n=3 mice, >100 synapses per group). Two-way ANOVA, data shown as mean ± SEM with individual cells or mice (circles) overlaid.  75   To functionally verify these changes in AMPAR trafficking, we also examined spike-timing-dependent LTP at synapses in the VTA and found that this form of LTP was abolished in DAT-Cre;β-catΔex3 mice (Figure 2.14 a). Additionally, treatment with NASPM (1-naphthyl acetyl spermine trihydrochloride), a selective antagonist of GluA2-lacking AMPARs, reduced excitatory postsynaptic potential (EPSP) amplitude back to basal levels in control mice, indicating that the enhanced EPSP amplitude observed following LTP induction was the result of the insertion of GluA2-lacking AMPARs. In contrast, NASPM treatment had no effect on EPSP amplitude in DAT-Cre;β-catΔex3 mice, demonstrating that GluA2-lacking AMPARs were not recruited to these synapses following the spike-timing-dependent LTP protocol (Figure 2.14 a). These data confirmed that increasing cadherin at the synaptic membrane in DAT-Cre;β-catΔex3 mice results in the retention of GluA2-containing AMPARs at the membrane and prevents the insertion of GluA2-lacking AMPARs which are crucial for the strengthening of these synapses. 76   Figure 2.14 Stabilization of Cadherin at Synapses in the VTA Blocks LTP by Retaining GluA2-Containing AMPARs and Preventing the Insertion of GluA2-lacking AMPARs. (a) STD LTP in the VTA was abolished in naive DAT-Cre;β-catΔex3 mice (#P < 0.001, significant interaction between genotype and time, two-way repeated-measures ANOVA, F(52, 468) = 2.644, *P < 0.05, Bonferroni’s test post hoc, n = 5 cells in 5 mice wild type, 6 cells in 6 mice DAT-Cre;β-catΔex3 ). Treatment with NASPM reversed LTP in control mice, but had no effect on EPSP amplitude in DAT-Cre;β-catΔex3 mice. (b) Increased AMPAR:NMDAR ratio 24 h after cocaine administration observed in control mice was abolished in DAT-Cre;β-catΔex3 mice (P = 0.0290, two-way ANOVA, significant interaction between genotype and drug treatment, F(1,39) = 5.143, *P = 0.0321, n.s., P > 0.9999, Bonferroni’s test post hoc, n = 10 cells in 4 mice control saline, 11 cells in 5 mice control cocaine, 12 cells in 6 mice DAT-Cre;β-catΔex3 saline, 10 cells in 5 mice DAT-Cre;β-catΔex3  cocaine). (c) Increased rectification index of AMPAR EPSCs 24 h after cocaine administration observed in control mice was absent in DAT-Cre;β-catΔex3 mice (P < 0.01, two-way ANOVA, significant interaction between genotype and drug treatment, F(1,31) = 7.641, **P = 0.0032, n.s., P > 0.9999, Bonferroni’s test post hoc, n = 9 cells in 3 mice control saline, 8 cells in 3 mice control cocaine, 9 cells in 3 mice DAT-Cre;β-catΔex3 saline, 9 cells in 3 mice DAT-Cre;β-catΔex3 cocaine). (d,e) The frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) (d) and miniature inhibitory postsynaptic currents (mIPSCs) (e) onto dopaminergic neurons in the VTA were unchanged in DAT-Cre;β-catΔex3 mice compared to controls, indicating that basal excitatory and inhibitory synaptic transmission at these synapses was unaltered in the VTA of DAT-Cre;β-catΔex3 mice (n = 9 cells in 3 mice control, 8 cells in 3 mice DAT-Cre;β-catΔex3 , unpaired t-tests, P > 0.05). Data shown as mean ± s.e.m. with individual data for cells (circles) overlaid. Figure-5 (Bamji)00.51.01.5010203001230102030Control:Cocaine0.00.51.01.52.02.50%50%100%150%200%0 10 20 30 40 50Normalized EPSP amplitude# Spike-timing LTP protocol*NASPMTime (min)ControlDAT-Cre;β-catΔex3b1 2 31 2 31 2 3cadControl DAT-Cre;β-catΔex3coc.sal. coc.sal.AMPA/NMDA Ratio0.00.30.60.91.2eRectification Index10/4 11/5 11/6  10/5Control DAT-Cre;β-catΔex3coc.sal. coc.sal.Control:DAT-Cre;β-catΔex3:coc.sal.coc.sal.Control:DAT-Cre;β-catΔex3:*Control:DAT-Cre;β-catΔex3:SalineDAT-Cre;  β-catΔex3: 0.5-1.0-0.59/3 8/3 9/3 9/3n.s. ** n.s.CocaineSalinemEPSC Frequency (Hz)mEPSC Amplitude (pA)mIPSC Frequency (Hz)mIPSC Amplitude (pA)Control:DAT-Cre;β-catΔex3:Control:DAT-Cre;β-catΔex3:n.s.Control DAT-Cre;β-catΔex3n.s. n.s. n.s.-75 -50 -25 25 500.5-1.0-0.5-75 -50 -25 25 50Control DAT-Cre;β-catΔex3 ControlDAT-Cre;β-catΔex3ControlDAT-Cre;β-catΔex3coc.sal.coc.sal.9/3 8/3 9/3 8/3 9/3 9/3 8/38/377   We then examined changes in AMPAR/NMDAR ratio and inward rectification of AMPA EPSCs 24 hours after cocaine administration in control and DAT:Cre;βcatΔex3 mice. In control mice we found that, consistent with previous studies, cocaine administration caused a significant increase in AMPAR/NMDAR ratio (Figure 2.14 b) and inward rectification of AMPA EPSCs (Figure 2.14 c) indicating increased insertion of GluA2-lacking AMPARs at synapses onto VTA DA neurons (Wolf and Tseng, 2012). However, these increases were completely absent in DAT:Cre;βcatΔex3 mice (Figure 2. 14 b, c), consistent with our immuno EM data showing that GluA1 at the synaptic membrane is increased in control mice but not DAT:Cre;βcatΔex3 mice following cocaine CPP. To confirm that these changes were not due to earlier, developmental disruptions, we also examined the morphology, density and function and VTA synapses in DAT-Cre;β-catΔex3 mice. EM analysis demonstrated that the size and density of excitatory and inhibitory synapses in the VTA was unchanged in DAT-Cre;β-catΔex3 mice compared to controls (Figure 2.15). We also found no differences in the frequency or amplitude of miniature excitatory postsynaptic currents or miniature inhibitory postsynaptic currents onto VTA dopaminergic neurons in DAT:Cre;βcatΔex3 mice compared to control mice (Figure 2.14 d, e). Together, these data demonstrate that increasing cadherin at the synaptic membrane prior to behavioural training results in a significant reduction in cocaine CPP through the aberrant retention of GluA1/2 AMPARs, and the prevention of GluA1 homomer membrane insertion to VTA synapses (Figure 2.16). 78   Figure 2.15 No Changes in DAT-Cre;β-catΔex3 Mice to Morphology or Density of VTA Synapses. (a) Representative images of VTA synapses in DAT-Cre;β-catΔex3 mice and littermate controls. No significant differences in length (b) or synapse density (c) of excitatory or inhibitory synapses were observed between groups (two-way ANOVA, n= 3 mice per group). Data shown as mean ± SEM with individual mice (circles) overlaid.  2.4 Discussion Here we demonstrate that cadherin plays a critical role in synaptic plasticity in the VTA and behavioral conditioning driven by cocaine. We show that cadherins are widely expressed in the VTA and are essential for the potentiation of excitatory synapses onto dopaminergic neurons. Using immunogold EM, we observed a strong correlation between cocaine-induced CPP in wild-type mice and the insertion of cadherin and GluA1-containing AMPARs into the synaptic membrane of these synapses. These changes in cadherin and AMPAR localization were specific to cocaine-induced CPP, and were not observed when mice were given the same schedule of 79  cocaine and saline administration in their home cage, nor following CPP induced by palatable food rewards. In DAT-Cre;β-catΔex3 mice, we found that stabilization of cadherin at VTA synapses was sufficient to reduce the magnitude of cocaine-induced CPP. This behavioral effect was associated with disruptions in the plasticity of excitatory synapses formed onto dopaminergic neurons; stabilization of cadherin led to the abolishment of LTP induced by both cocaine administration and STD stimulation in these mice. Indeed, our immunogold EM data demonstrated that cocaine-induced internalization of GluA2-containing AMPARs and the subsequent insertion of GluA2-lacking AMPARs into the membrane was blocked in DAT-Cre;β-catΔex3 mice. Our findings suggest that, in wild-type mice, increased cadherin at the synaptic membrane acts to stabilize newly inserted GluA1 homomers during cocaine CPP. This is supported by evidence that cadherin interacts with the GluA1 subunit and stabilizes GluA1-containing AMPARs (Nuriya and Huganir, 2006), as well as by our data showing that intact cadherin adhesion is required for STD LTP at VTA synapses, which is also mediated by insertion of GluA1-homomers, as is potentiation of these synapses by cocaine. Cadherin-mediated stabilization of GluA1 homomers may therefore contribute to increased strength of excitatory synapses onto dopaminergic neurons, as well as prolonged postsynaptic Ca2+ influx through these AMPARs. Potentiation of these synapses is also likely to enhance the activity of dopaminergic neurons and increase dopamine release onto target structures of the mesocorticolimbic system, leading to further downstream changes in synaptic plasticity and circuit activity that contribute to addiction. 80   Figure 2.16 Model of Changes in Cadherin and AMPAR Subunit Localization in Control and DAT-Cre;β-catΔex3 Mice during CPP. (a,b) Wild-type mice. (a) Under basal conditions, the population of AMPARs at excitatory inputs to dopaminergic neurons is composed of GluA1-GluA2 heteromers (Argilli et al., 2008). Cadherins regulate the dynamic localization of AMPARs through direct and indirect interactions with GluA1 and GluA2 (Nuriya and Huganir, 2006; Saglietti et al., 2007; Silverman et al., 2007; Gorski et al., 2005). (b) During cocaine-mediated CPP, activity is enhanced at excitatory inputs to dopaminergic neurons, driving the removal of GluA1-GluA2 heteromers and the insertion of Ca2+-permeable GluA1 homomers into AMPA receptor ‘slots’ in the PSD (Bellone and Lüscher, 2006; Mameli et al., 2007). Enhanced synaptic activity also leads to increased levels of cadherin at the synaptic membrane. Cadherins are then situated to associate with and stabilize GluA1 homomers at the synaptic membrane, contributing to the potentiation of these synapses underlying behavioral changes in CPP. (c,d) DAT-Cre;β-catΔex3 mice. (c) Under basal conditions, elevated levels of β-catenin promote the stability of cadherin, resulting in an increase in cadherin localized to the synaptic membrane. Cadherins associate with GluA1-GluA2 heteromers (Nuriya and Huganir, 2006; Saglietti et al., 2007; Silverman et al., 2007), enhancing their stability at the synaptic membrane. (d) During cocaine-mediated CPP, the removal of GluA1-GluA2 heteromers is prevented owing to their stabilization by increased synaptic cadherin in DAT-Cre;β-catΔex3  mice. GluA1-GluA2 heteromers are retained in available AMPAR slots, preventing the insertion of GluA1-containing AMPARs and the potentiation of these synapses. Thus, stabilizing synaptic cadherin in DAT-Cre;β-catΔex3 mice disrupts the cocaine-induced switch in AMPAR composition and reduces CPP.  Excitatory synapses onto dopaminergic neurons have previously been implicated in contextual conditioning and reward learning (Stuber et al., 2008; Harris and Aston-Jones, 2003), Glutamatergicpresynaptic inputsDopaminergicneuronNa+ Ca2+ Na+Cocaine-inducedchange in AMPARcompositionBasal conditions Cocaine CPP   Basal Conditions Cocaine CPP   DAT-Cre;β-catΔex3 mice Wildtype mice - Cocaine-induced plasticity at glutamatergic inputs onto dopaminergic neurons in the VTAGluA1/2heteromersretained atsynapseGluA1homomerinsertion blockedNo change inAMPAR composition;reduced CPPNa+GluA1/2heteromersremovedGluA1homomersinsertedEnhancedActivityNa+CadherininsertedIncreasedcadherinat synapseStabilization of GluA1/2 AMPARsCadherin stabilizesGluA1 homomersat synapsesCadherinAMPAR - GluA1/2 heteromerAMPAR - GluA1 homomerSynaptic VesiclePostsynaptic Density (PSD)AMPAR slotsβ-cateninLegendEnhancedActivitya bc dCadherinGluA1/2heteromerIncreased levelsof β-cateninβ-cateninFigure-6 (Bamji)81  and our data demonstrate a strong relationship between increased cadherin at these synapses and drug-induced behavioral changes in wild-type mice. Increased cadherin at the synaptic membrane was correlated with the magnitude of CPP in individual mice, which suggests that increased cadherin adhesion may be a mechanism that contributes to drug-induced increases in the stability and potentiation of VTA synapses, promoting the ‘hard-wiring’ of synaptic traces of drug-associated memories and behaviors. We speculate that increased strength and stability of VTA synapses mediated by cadherin adhesion may also contribute to sensitization to drug-associated cues, which can trigger relapse both in humans and animal models of addiction even after extended periods of abstinence from drug-taking (Steketee and Kalivas, 2011). In DAT-Cre;β-catΔex3 mice, stabilization of cadherin at VTA synapses before cocaine administration prevented the removal of GluA2-containing AMPARs and blocked the insertion of GluA1-containing AMPARs. This disruption of cocaine-induced changes in AMPAR localization was associated with attenuated CPP and the abolishment of STD LTP. These impairments contrast with observations in the hippocampus, where stabilization of cadherin leads to impairments in LTD and behavioral flexibility but has no effect on LTP or acquisition of spatial memory (Mills et al., 2014). The key difference in plasticity between these regions appears to be the requirement for removal of GluA2-containing AMPARs for LTP at VTA synapses. It has been proposed that the number of AMPARs at synapses onto dopaminergic neurons is limited by the number of ‘slots’ where receptors can associate with scaffold proteins in the PSD (Bellone and Lüscher, 2006; Kessels and Malinow, 2009). Indeed, inhibiting the internalization of GluA2-containing AMPARs by disrupting GluA2–PICK1 (protein interacting with C kinase-1) interactions abolishes cocaine-induced increases in AMPAR:NMDAR ratio that are typically mediated by the insertion of GluA2-lacking AMPARs (Bellone and Lüscher, 2006). 82  Since cadherin can stabilize AMPARs at synapses through interaction with both the GluA1 and GluA2 subunits (Nuriya and Huganir, 2006; Saglietti et al., 2007; Silverman et al., 2007), this suggests that in DAT-Cre;β-catΔex3 mice increased cadherin at the synaptic membrane stabilized GluA2-containing AMPARs that occupied available slots in the PSD and prevented the insertion of GluA1 homomers. Our electrophysiological data confirmed the functional identity of AMPARs at VTA synapses in DAT-Cre;β-catΔex3 mice, demonstrating that GluA2-containing AMPARs were indeed retained at these synapses and insertion of GluA2-lacking AMPARs was blocked, leading to abolishment of LTP. Our findings also show that the timing of cadherin stabilization at the synaptic membrane had a critical effect on AMPAR trafficking and plasticity at VTA synapses. In wild-type mice, increased cadherin localization to the synaptic membrane during CPP resulted in the stabilization of GluA1 homomers that were also inserted during CPP. However, in DAT-Cre;β-catΔex3 mice, increased cadherin localization to the synaptic membrane occurred before CPP, which resulted in the stabilization of GluA1-GluA2 heteromers present under basal conditions. In both cases, the effect of cadherin was consistent: increased cadherin at the synaptic membrane was found to stabilize the AMPARs that were present at that time. However, the differences in timing of when cadherin was stabilized and the type of AMPAR subunits present at each time led to different effects in LTP and CPP observed between DAT-Cre;β-catΔex3 mice and controls. Notably, while CPP was reduced in DAT-Cre;β-catΔex3 mice, we observed no changes in cocaine-induced locomotor sensitization in these mice compared with controls. This finding is consistent with studies showing that CPP and sensitization are not necessarily directly correlated and may be mediated by distinct circuits and neural adaptations in the brain driven by cocaine. 83  Disruption of the dopaminergic projections from the substantia nigra, but not VTA, has been shown to abolish locomotor sensitization to cocaine while leaving CPP intact (Beeler et al., 2009). Additionally, an analysis of CPP and locomotor sensitization in different inbred mouse lines found no correlation between the two behaviors, with some strains exhibiting robust CPP but no locomotor sensitization, or vice versa (Eisener-Dorman et al., 2011). Finally, no correlation was found to exist between CPP and sensitization within individual mice in a behavioral task designed to test both parameters simultaneously (Seymour and Wagner, 2008). Our findings also demonstrate interesting differences in CPP driven by cocaine and food rewards. Cocaine CPP, but not food CPP, increased both cadherin and GluA1 localization to the synaptic membrane at excitatory synapses onto dopaminergic neurons in the VTA. Consistent with this, stabilization of cadherin (and GluA1-GluA2 heteromers) at the synaptic membrane of these synapses in DAT-Cre;β-catΔex3 mice markedly decreased the magnitude of cocaine CPP, but had no effect on food CPP. These findings are consistent with a number of studies indicating that changes in dopaminergic neuron activity and behavior driven by food rewards may be mediated by mechanisms other than increased LTP at VTA synapses. Food-related peptide hormones have been shown to play a major role in mediating behavioral changes driven by food (Meye and Adan, 2014), in some cases by acting directly on VTA dopamine neurons to regulate their activity (Abizaid et al., 2006). Additionally, pharmacological disruption of NMDARs and metabotropic glutamate receptors have both been shown to have opposite effects on CPP driven by food rewards and CPP driven by drugs of abuse (Herzig et al., 2005; Yonghui et al., 2006), which further suggests that major differences exist in the mechanisms underlying these different forms of CPP. 84  What are the molecular mechanisms responsible for the changes in cadherin localization observed following CPP? In hippocampal neurons, cadherins have been shown to undergo constitutive turnover at the synaptic membrane under basal conditions (Tai et al., 2007). We have previously shown that synaptic activity leads to post-translational changes in the cadherin-binding protein δ-catenin, increasing its association with cadherin and augmenting cadherin’s retention at the synaptic membrane (Brigidi et al., 2014). This results in increased trans-synaptic adhesion and the stabilization of postsynaptic AMPARs by cadherin, causing long-lasting increases in synapse strength, size and stability over time. An important direction for future research will be to determine whether these same mechanisms drive changes in cadherin localization during activity- and drug-induced plasticity at synapses in the VTA. The finding that cadherins regulate AMPAR trafficking at synapses in the VTA supports an emerging view that structural and scaffolding proteins may be of central importance in synaptic plasticity due to their role in mediating the recruitment and removal of postsynaptic receptors (Granger and Nicoll, 2014; Sheng et al., 2013). Since the finding that LTP can be induced at synapses as long as extrasynaptic glutamate receptors are present (Granger and Nicoll, 2014), there has been a shift of focus to identify the proteins that control the insertion, removal and stabilization of AMPARs during different forms of synaptic plasticity. The present study provides further evidence that cadherins are key molecules that control AMPAR trafficking during plasticity underlying different forms of learning and memory. Additionally, our findings demonstrate that cadherins play a critical role in synaptic plasticity outside of the hippocampus, where they have typically been studied, and likely have specialized functions in mediating different forms of synaptic plasticity throughout the CNS. Another important goal for future studies will be determining the specific cadherin subtypes responsible for mediating different 85  forms of synaptic plasticity throughout the brain. In the VTA, N-cadherin likely plays a major role in activity and cocaine-induced plasticity examined in the present study, as the HAV peptide used to block STD LTP at VTA synapses has been shown to specifically disrupt N-cadherin trans interactions (Tang et al., 1998), and as N-cadherin is critical for plasticity at excitatory synapses in other brain regions such as the hippocampus (Benson and Tanaka, 1998; Bozdagi et al., 2010; Mendez et al., 2010; Tang et al., 1998). However, cadherins have highly similar structure and functional redundancy, and other cadherin subtypes expressed in dopaminergic neurons may therefore contribute as well. Our study provides mechanistic insight into how mutations in cadherin adhesion complex proteins could contribute to susceptibility or resilience to addiction. Clinically, substance dependence is defined by several notable symptoms (2000). The physiological symptoms include tolerance to the drug, meaning the individual needs to consume markedly increased amounts in order to achieve the desired level of intoxication, and withdrawal, meaning the individual experiences negative physiological or psychological effects when they stop drug-taking. Furthermore, there are often behavioural symptoms and social consequences that characterize addiction. Specifically, individuals spend a great deal of time in activities that are necessary to obtain the drug, and give up important social or occupational activities due to their use of the drug (2000). Genome-wide association studies in substance abusers have identified increased prevalence of clustered single nucleotide polymorphisms in δ-catenin, which stabilizes cadherin at synapses, and α-catenin, which tethers the cadherin adhesion complex to the actin cytoskeleton (Liu et al., 2006). While the functional consequences of these polymorphisms are unknown, we speculate that changes in the function or expression levels of δ-catenin or α-86  catenin could affect the stabilization of cadherin and AMPARs at the synaptic membrane of VTA synapses, which our findings show is sufficient to alter drug-induced synaptic plasticity and behavior. Cadherin adhesion-complex proteins may therefore be targets of interest for future studies investigating genetic risk factors for addiction.  87  Chapter 3: zDHHC5-Mediated Palmitoylation Promotes Excitatory Synapse Formation Palmitoylation is a form of lipid modification that has important effects on the localization and function of synaptic proteins. The zDHHC family of palmitoyl acyl-transferases (PATs) mediate this modification of substrates. In neurons, palmitoylation of synaptic proteins can play a role in synapse formation and synaptic plasticity. Previous work examining zDHHC5 has indicated that this PAT is localized at the plasma membrane of dendritic spines, and can change its localization in response to synaptic activity in order to palmitoylate substrates localized in the dendritic shaft. Here we report that zDHHC5 contributes to the formation of excitatory synapses in part by promoting the stability of dendritic spines. We find that the palmitoylation function of zDHHC5 and its localization at the plasma membrane of dendritic spines are important for excitatory synapse formation.    3.1 Introduction Protein palmitoylation is the reversible addition of the fatty acid, palmitate, to cysteine residues of a substrate protein. This post-translational modification is likely important for normal synaptic function, as over 40% of all known synaptic proteins are substrates of palmitoylation (Sanders et al., 2015). Protein palmitoylation is mediated by a family of multi-pass transmembrane proteins containing a conserved aspartate-histidine-histidine-cysteine motif that located within a zinc finger-like domain (zDHHC). The zDHHC motif is required for the enzyme’s palmitoyl-acyltransferase (PAT) function.  88  zDHHC5 is a PAT that is ubiquitously expressed in the brain, with high expression in the cortex, hippocampus, cerebellum and thalamus (Lein et al., 2007). Within neurons, zDHHC5 is highly localized to dendrites. Where Thomas and colleagues (2012) found that the closely related PAT zDHHC8 but not zDHHC5 is highly localized to synapses, we previously found that zDHHC5 is particularly localized to most excitatory synapses and approximately half of inhibitory synapses (Brigidi et al., 2015). Given that zDHHC5 exhibits changes in localization in response to synaptic activity, this discrepancy may be due to differences in culture conditions and the overall activity of neurons in the culture.  In neurons, zDHHC5 has been shown to palmitoylate the synaptic scaffolding protein, GRIP1b (Thomas et al., 2012) and the cadherin-binding protein, δ-catenin (Brigidi et al., 2014). Although zDHHC5 palmitoylates GRIP1b in a constitutive manner (Thomas et al., 2012), this PAT is critical for activity-mediated palmitoylation of δ-catenin (Brigidi et al., 2014). δ-catenin palmitoylation is mediated by the trafficking of zDHHC5 from the synaptic membrane of dendritic spines to the dendritic shaft upon NMDAR activation, where it can act on δ-catenin (Brigidi et al., 2015). zDHHC5’s localization at the postsynaptic membrane is mediated through its binding to PSD-95, which occurs through a PDZ binding motif localized at the zDHHC5 C-terminal tail. Interestingly, a de novo zDHHC5 single nucleotide variant (SNV) that would impair zDHHC5 interaction with PSD-95 has been identified in a patient with schizophrenia (Fromer et al., 2014). This SNV introduces a premature stop codon at residue 648, which results in the loss of the last 68 amino acids of zDHHC5, including the PDZ binding motif. This suggests that this mutant form of zDHHC5 might interfere with its proper localization and function.  89  However, little is known about zDHHC5’s role in the development of the nervous system. Given the role that zDHHC5 plays in the palmitoylation of synaptic proteins; we sought to determine whether zDHHC5 also is necessary for dendrite outgrowth and synapse formation. In the current study, we find that zDHHC5 palmitoylation function and proper localization to postsynaptic membranes is critical for the stability of dendritic spines, and contributes to the formation of excitatory synapses.   3.2 Materials and Methods 3.2.1 cDNA Constructs Control shRNA, shRNA against zDHHC5, and HA-zDHHC5 were kind gifts from R. Huganir (Johns Hopkins University). HA-zDHHC5 that was resistant to RNA interference (denoted by #) was generated as previously described (Brigidi et al., 2014). The GFP-zDHHC5 construct was generated by PCR of zDHHC5 from HA-zDHHC5 (accession number: NM_00139388) using an EcoRI-tagged forward primer and a BamHI-tagged reverse primer and pasted into pEGFP-C1 backbone using EcoRI and BamHI sites as previously described (Brigidi et al., 2015). The E648* point mutation was generated by site-directed mutagenesis using a QuikChange kit (Agilent Technologies, 200523) with the GFP-zDHHC5 and HA-zDHHC5# constructs as templates. The Y533E point mutation was generated by site-directed mutagenesis using a QuikChange kit (Agilent Technologies, 200523) as previously described (Brigidi et al., 2015).  3.2.2 Cell Cultures Hippocampi were isolated from embryonic day (E) 18 Sprague-Dawley rats as previously described (Xie et al., 2000) and plated at a density of 130 cells per mm2. Neurons were 90  transfected with Lipofectamine 2000 (ThermoFisher, 11668019) at 10 days in vitro (DIV) following the manufacturer’s recommendations and used for experiments at 13 DIV. HEK293T cells were transfected using Lipofectamine 2000 (ThermoFisher, 11668019) according to the manufacturer’s recommendations. HEK293T cells were transfected at ~70-80% confluency and incubated for 48 h before harvesting for biochemistry.  3.2.3 Immunocytochemistry Immunocytochemistry experiments were performed as previously reported (Sun and Bamji, 2011). Briefly, cultured neurons were fixed in 4% paraformaldehyde/4% sucrose, permeabilized with 0.1% Triton-X, and blocked in 10% goat serum for 1 h at room temperature. Primary antibodies were diluted in 1% goat serum and applied to neurons overnight at 4˚C. Secondary antibodies were also diluted in 1% goat serum, and applied to neurons for 1 h at room temperature. Coverslips were mounted with Prolong Gold (ThermoFisher, P36930). Primary antibodies were as follows: PSD-95 (mouse monoclonal, IgG2a isotype, 1:500, abcam, ab2723), gephyrin (mouse monoclonal, IgG1 isotype, 1:500, Synaptic Systems, 147 011), VGLUT1 (guinea pig, 1:500, EMD Millipore, AB5905). Secondary antibodies were as follows: goat-anti-mouse IgG2a AlexaFluor 568 (Life Technologies, A21134), goat-anti-mouse IgG1 AlexaFluor 647 (Life Technologies, A21240), goat-anti-guinea pig AlexaFluor 633 (Life Technologies, A21105), goat anti-mouse AlexaFluor 568 (Life Technologies, A11019), goat anti-mouse AlexaFluor 633 (Life Technologies, A21050).  91  3.2.4 Immunogold EM  Samples from mouse CA1 hippocampus were processed as described previously (Mills et al., 2017). Briefly, brains were sliced into 250 µm-thick sections by vibratome and pieces of CA1 hippocampus (<1 mm in all dimensions) were dissected from slices and cryoprotected in 30% glycerol overnight at 4˚C. Samples were plunge-frozen in liquid ethane at -170˚C in an EM cryopreparation chamber (Leica) and transferred to a 1.5% uranyl acetate solution in 100% methanol, kept at -90˚C in a Leica EM AFS for 30 h. The temperature was gradually increased and samples were infiltrated with HM-20 acrylic resin (Electron microscopy sciences, Hatfield, PA). Samples were set up in capsules containing pure resin and polymerized under UV light for 24h. Tissue sections were cut at 85 nm using a Diatome diamond knife and a Leica ultramicrotome. Sections were collected on 300-mesh, formvar coated nickel grids (Electron Microscopy Sciences).   Post-embedding immunostaining was performed on the EM grids as described previously (Mills et al., 2017). Briefly, grids were rinsed with distilled water and immersed in a bead of TTBS with 0.1% Triton-X, 0.1% sodium borohydride and 50 mM glycine. Nonspecific binding was blocked wth 2% BSA in TTBS with 0.1% Triton-X. Primary antibodies against zDHHC5 (rabbit, Sigma Prestige, HPA014670) or PSD-95 (rabbit, Frontier Institute, Af628) were diluted in 2% BSA in TTBS with 0.1% Triton-X. Grids were immersed in 15 µl beads of diluted primary antibody overnight at room temperature in a humidified chamber. The next day, grids were rinsed in TTBS with 0.1% Triton-X. Secondary antibodies were diluted in 2% BSA in TTBS with 0.1% Triton-X and 0.05% polyethylene glycol (PEG). Girds were immersed in 15µl beads of secondary antibody (Electron microscopy sciences, goat-anti-rabbit 15 nm, cat. no. 25112; goat-anti-rabbit 10 nm, cat. no. 25108) for 1.5 h. Grids were rinsed in TTBS with 0.1% Triton-X, 92  in Milli-Q H2O and dried. Grids were then lightly counterstained with 2% uranyl acetate and Reynold’s lead citrate. Images were collected at 98,000x magnification on a Tecnai G2 Spirit transmission electron microscope (FEI Company, Eindhoven, the Netherlands).  To analyze zDHHC5 immunogold labelling, the distance of all immunogold-labelled zDHHC5 to the pre- or postsynaptic membrane was measured. To quantify excitatory and inhibitory synapse density in the CA1 hippocampus, the number of PSD-95 positive and negative synapses was quantified within a 2500 µm2 region of the hippocampus. All images were acquired and analyzed blind to the genotype of each mouse.   3.2.5 Fluorescence Recovery After Photobleaching Dendritic spines within 100 µm of the cell body were imaged every 10 s for 4 min after photobleaching. Spines were identified using dsRed. A 1-µm-diameter circular region of interest (ROI) was photobleached within a spine head using the tornado function in Fluoview software (Olympus). The fluorescence of GFP-zDHHC5 in the photobleached ROI was quantified over time using Fluoview software. The recovery of fluorescence intensity (R) was determined by normalizing the intensity at a specific time (Ft) using the formula: R = (Ft – F0)/(Fi – F0), where F0 is the fluorescence at the time of photobleaching, and Fi is the fluorescence before photobleaching. To account for passive bleaching, a 1 µm ROI in an adjacent spine was tracked over time and quantified. The fluorescence intensity within the photobleached ROI was normalized to this control, non-photobleached ROI for each scan. Normalized fluorescence recovery data was collected in Prism software (GraphPad), analyzed and fit to a single exponential model, which generates plateau values for the mean R-value among cells in each condition. Plateau values ± s.e.m. were statistically compared in Prism software. 93   3.2.6 Spine Turnover Analysis Dendrites within 200 µm of the cell body were imaged every 30s for 15 min. Spines were visually identified and categorized as stable if they were present throughout all images, and as unstable if they retracted or transiently appeared and retracted during the 15 minute imaging period. The presence or absence of GFP-zDHHC5 in stable and unstable spines was manually determined.  3.2.7 Immunoblot Assay Western blotting was performed as previously described (Brigidi et al., 2014; Sun and Bamji, 2011) . HEK293T cells were homogenized in an ice-cold lysis buffer containing 1% IGEPAL CA-630 (Sigma), 50 mM Tris-HCl, pH7.5, 150 mM NaCl and 10% glycerol, supplemented with phenylmethanesulfonyl fluoride solution and a protease inhibitor cocktail with ethylenediaminetetraacetic acid (Roche). Brain tissue was homogenized in ice-cold RIPA buffer (ThermoFisher, CAT# 89900). Proteins were cleared by centrifugation at 14,000g for 30 min at 4˚C. Proteins were separated by SDS-PAGE and probed with antibodies against zDHHC5 (1:1,000; Sigma Prestige, HPA014670), HA (1:1,000; Sigma, H9658), and GAPDH (1:1,000; abcam, ab9484). Bands were visualized using enhanced chemiluminescence (Pierce Biotechnology) on a C-DiGit Chemiluminescence Western Blot Scanner (LI-COR).   3.2.8 Image Analysis and Quantification Confocal images for synapse density analyses were subjectively thresholded using ImageJ software while the observer was blind to condition. Puncta were identified as a thresholded 94  fluorescence cluster with an area between 0.05 and 3 µm2. Puncta area was determined using ImageJ. An ImageJ colocalization plugin was used to assess colocalization between VGLUT1 and PSD-95 puncta (http://rsb.info.nih.gov/ij/plugins/colocalization.html). Points of colocalization were defined as regions of >4 pixels in size with a > 50 intensity ratio between the two channels.   3.2.9 Statistical Analysis All data values are expressed as the mean ± s.e.m. For all imaging experiments, the n numbers shown refer to the number of cells used per condition over 3 separate cultures, with the exception of Figure 3.8C-E, where n refers to the number of spines and is specified within figure legends. Statistical significance was determined by Student’s t test, one-way or two-way ANOVA with Bonferroni’s test post hoc using Prism where indicated. Statistical significance was assumed when p < 0.05. All figures were generated using Illustrator CS6 software (Adobe Systems, Inc.)  3.3 Results 3.3.1 zDHHC5 is Localized to Pre- and Postsynaptic Compartments of Hippocampal Synapses We first examined the localization of zDHHC5 at synaptic compartments in the stratum radiatum of the CA1 region of the hippocampus. Previous research has identified many presynaptic proteins as substrates for zDHHC5 (Li et al., 2010), however in neurons, zDHHC5 has only been examined in postsynaptic compartments (Brigidi et al., 2015; Thomas et al., 2012). We used zDHHC5 gene-trapped (zDHHC5-gt) mice and wildtype littermate controls to examine zDHHC5 protein localization in the CA1 hippocampus. The zDHHC5-gt mice have previously 95  been shown to exhibit no more than 7% expression of a non-functional zDHHC5 protein fragment in brain tissue compared to wildtype samples (Li et al., 2010), and therefore these mice serve as suitable control. Adult zDHHC5-gt mice and littermate controls were sacrificed and CA1 hippocampus was isolated by microdissection for immunogold EM. We found that zDHHC5 was localized mainly to the synaptic membrane of postsynaptic terminals, with a large proportion of immunostaining also localized to the synaptic membranes of presynaptic terminals (Figure 3.1). Immunostaining was negligible in zDHHC5-gt control samples.  Figure 3.1 zDHHC5 is Localized to Pre- and Postsynaptic Compartments of Hippocampal Synapses. (a) EM of hippocampal synapse showing immunogold-labeled zDHHC5. Scale bar, 100 nm. (b) zDHHC5 immunogold quantification in wildtype and zDHHC5-gt hippocampus (20 nm bins). Pre, presynaptic (yellow); Post; postsynaptic (red).  3.3.2 Membrane-Associated zDHHC5 Promotes Excitatory Synapse Formation via its PAT Function  Previous work has shown that zDHHC5 is localized to ~80% of excitatory and ~47% of inhibitory synapses in hippocampal neurons (Brigidi et al., 2015). We therefore sought to determine whether zDHHC5 is involved in promoting the formation of excitatory and inhibitory synapses. We transiently transfected primary rat hippocampal neurons at 10 DIV with GFP plus zDHHC5 shRNA. At 13 DIV, we immunostained for the excitatory synapse marker PSD-95, and the inhibitory synapse marker, gephyrin. To quantify the density of excitatory and inhibitory 96  synapses forming onto transfected cells, we used the GFP cell fill to mask the immunostained images (see Figure 3.2) so we could measure the density of synapses forming specifically onto transfected cells.   Figure 3.2 Masking of Immunostaining Images using GFP Cell Fill from Transfected Neurons. Right column: unmasked images of GFP cell fill, PSD-95 immunostaining and gephyrin immunostaining. Left column: the same images after masking to present immunostaining only within the transfected dendrite.   We observed a significant reduction in the density of excitatory but not inhibitory synapses in cells expressing zDHHC5 shRNA, a phenotype that was rescued in cells expressing zDHHC5 shRNA plus wildtype shRNA-resistant zDHHC5 (Figure 3.3). Excitatory synapse density was also significantly reduced in cells expressing zDHHC5 shRNA and a palmitoylation deficient DHHC5 mutant construct (zDHHS5), indicating that the palmitoylation function of zDHHC5 is necessary to promote excitatory synapse formation (Figure 3.3).  97   Figure 3.3 zDHHC5 Expression and Membrane Localization Affects Excitatory but not Inhibitory Synapse Density. (A) Confocal images of hippocampal neurons transfected with the indicated constructs and immunostained for the excitatory synapse marker, PSD-95 (magenta) and the inhibitory synapse marker, gephyrin (cyan). Scale bars, 5 µm. (B) shRNA knockdown of zDHHC5 results in a significant reduction in excitatory synapse density, which can be rescued by shRNA-resistant wildtype zDHHC5 and phosphomimetic Y533E mutant, but not by palmitoylation deficient zDHHS5 or the truncated E648* mutant. Inhibitory synapse density was not altered in any of these conditions (two-way ANOVA, significant interaction between condition and synapse type, F(5, 472) = 5.223, p = 0.0001, **p < 0.01, ***p < 0.001, Bonferroni’s test post hoc, Excitatory synapses: scramble shRNA n = 52 cells, 98  shRNA n = 49 cells, shRNA + WT# = 41 cells, shRNA + zDHHS5# = 34 cells, shRNA + E648*# = 29 cells, shRNA + Y533E# = 37 cells. Inhibitory synapses: scramble shRNA n = 53 cells, shRNA n = 49 cells, shRNA + WT# = 43 cells, shRNA + zDHHS5# = 33 cells, shRNA + E648*# = 28 cells, shRNA + Y533E# = 36 cells). Data shown as mean ± s.e.m.  We were also interested in the role of a de novo zDHHC5 mutation that was identified in a patient with schizophrenia (Fromer et al., 2014). This point mutation introduces a premature stop codon at residue 648, resulting in a premature truncation and the loss of the last 68 amino acids of zDHHC5 including the PDZ binding motif, which has been shown to bind to PSD-95 (Brigidi et al., 2015). We first expressed this E648* mutant in HEK cells and performed Western blots to ensure that it produced a stably expressed protein (Figure 3.4) When zDHHC5 shRNA and an shRNA-resistant form of the E648* mutant zDHHC5 were expressed in hippocampal neurons, we saw a significant reduction in excitatory synapse density compared to the scrambled shRNA control (Figure 3.3). This suggests that the E648* mutant affects zDHHC5’s synapse-promoting function.   Figure 3.4 Validation of E648* Mutant Expression and zDHHC5 Antibody Specificity. (A) Expression of WT and E648* mutant zDHHC5-HA constructs in HEK cells. The E648* mutation results in a stably expressed protein that can be detected by HA antibody and zDHHC5 antibody. (B) Western blot for zDHHC5 in cortical and hippocampal lysates from wildtype (WT) and zDHHC5-gt (gt) brains show that zDHHC5 immunoblotting is specific to zDHHC5 protein.  99   We were also interested to determine whether the localization of zDHHC5 at the plasma membrane was important for the formation of excitatory synapses. To examine this, we used a phosphomimetic form of zDHHC5 where AP2µ-mediated endocytosis of zDHHC5 is disrupted (Brigidi et al., 2015). As a result, this form of zDHHC5 is more strongly localized to synaptic membranes. To determine whether membrane localization of zDHHC5 is involved in synapse formation, we expressed zDHHC5 shRNA and the Y533E mutant form of zDHHC5 in hippocampal neurons, and saw that the excitatory synapse phenotype was no different from control neurons. This suggests that zDHHC5’s proper localization at the postsynaptic membrane is necessary for excitatory synapse formation (Figure 3.3). We did not see any differences in inhibitory synapse density across all conditions (Figure 3.3). Typically, we define synapses as being points of colocalization between pre- and postsynaptic markers. To confirm the knockdown of zDHHC5 leads to a reduction in the density of excitatory synapses, we performed the same experiment and immunostained for excitatory pre- and postsynaptic markers to confirm the results we observed when using PSD-95 alone as an excitatory synapse marker. We immunostained for VGLUT1 and PSD-95, and examined the density of colocalized puncta in each condition, observing the same changes in excitatory synapse density as when PSD-95 density alone was measured (Figure 3.5). This indicates that the PSD-95 marker is a suitable indicator of excitatory synapses in these conditions. 100   Figure 3.5 Excitatory Synapse Density measured by the Colocalization of Presynaptic Marker VGLUT1 and Postsynaptic Marker PSD-95. (A) The density of excitatory synapses shows the same pattern of results when quantified by the colocalization of VGLUT and PSD-95 puncta as when quantified by PSD-95 puncta alone (see Figure 3.3D). shRNA knockdown of zDHHC5 results in a significant reduction in excitatory synapse density, which can be rescued when zDHHC5 shRNA and an shRNA-resistant wildtype zDHHC5 or phosphomimetic Y533E mutant are also expressed, but not when zDHHC5 shRNA and a palmitoylation deficient zDHHS5 or the truncated E648* mutant are expressed (one-way ANOVA, F(5, 226) = 9.981, p < 0.0001, *p < 0.05, **p < 0.01, ***p < 0.0001, Bonferroni’s test post hoc, scramble shRNA n = 64 cells, shRNA n = 32 cells, shRNA + WT# = 37 cells, shRNA + zDHHS5# = 26 cells, shRNA + E648*# = 29 cells, shRNA + Y533E# = 44 cells). (B) Quantification of PSD-95 puncta density from this dataset shows the same pattern of results as the previous experiment where cells were immunostained for PSD-95 with gephyrin, instead of PSD-95 with VGLUT1 (one-way ANOVA, F(5, 229) = 11.04, **p < 0.01, ***p < 0.0001, Bonferroni’s test post hoc, scramble shRNA n = 66 cells, shRNA n = 32 cells, shRNA + WT# = 37 cells, shRNA + zDHHS5# = 26 cells, shRNA + E648*# = 30 cells, shRNA + Y533E# = 44 cells. (C) Confocal images of hippocampal neurons transfected with the indicated constructs and immunostained for the postsynaptic excitatory synapse marker, PSD-95 (magenta) and the presynaptic excitatory synapse marker, VGLUT1 (cyan). Scale bars, 5 µm.  101  3.3.3 zDHHC5 does not Regulate Dendrite Length or Complexity Previous work has shown that zDHHC5-mediated palmitoylation of the lipid raft protein, flotillin-2, is critical for flotillin-2 oligomerization neuronal stem cell cultures (Li et al., 2012b), and that flotillin-2 overexpression promotes filopodia extension in cultured cell lines (Neumann-Giesen et al., 2004). We therefore examined the effects of zDHHC5 on dendrite outgrowth. Using primary hippocampal cultures, we transiently transfected neurons at 10 days in vitro (DIV) with GFP cell fill, zDHHC5 shRNA (previously validated in Brigidi et al., 2014) and shRNA-resistant rescue constructs described in the previous section (Chapter 3.3.2) (# = shRNA-resistant). Cells expressing shRNA or shRNA with zDHHC5 shRNA-resistant constructs had similar overall dendritic length and complexity compared to control cells expressing a scrambled shRNA (Figure 3.6)  Figure 3.6 zDHHC5 does not Affect Dendrite Outgrowth or Complexity. The knockdown of zDHHC5 or knockdown and rescue with shRNA-resistant constructs (# = shRNA-resistant) does not affect summed dendritic length (A) or Sholl profile (B) of primary hippocampal neurons (scramble shRNA n = 45 cells, shRNA n = 25 cells, shRNA + WT# = 28 cells, shRNA + zDHHS5# = 26 cells, shRNA + E648*# = 28 cells, shRNA + Y533E# = 41 cells.  102  3.3.4 zDHHC5 Promotes Excitatory Synapse Formation in vivo To further understand the role of zDHHC5 in vivo, we examined the density of excitatory and inhibitory synapses in the CA1 hippocampus by electron microscopy. Excitatory and inhibitory synapses can be differentiated based on their morphology, where excitatory synapses are asymmetric, and inhibitory synapses are symmetric. These synapses can also be distinguished based on immunostaining for synaptic markers such as PSD-95. In order to more conclusively differentiate between excitatory and inhibitory synapses, EM samples from homozygous zDHHC5 gene-trapped mice and wildtype controls were immunostained for PSD-95 to identify excitatory synapses (antibody validated previously; see Liu et al., 2016; Mills et al., 2017), and the density of excitatory and inhibitory synapses was examined (Figure 3.7). The number of PSD-95 positive excitatory synapses, but not inhibitory synapses was significantly decreased in zDHHC5-gt mice compared to wildtype controls (two-way ANOVA, significant interaction between genotype and synapse type, F(1, 8) = 8.753, p = 0.0182). Although excitatory synapses were significantly larger than inhibitory synapses, there was no significant interaction between genotype and synapse size.   Figure 3.7 zDHHC5 Expression Affects Excitatory but not Inhibitory Synapse Formation in vivo.  (A) Electron micrograph from CA1 hippocampus with immunolabelling for PSD-95 to differentiate excitatory synapses (black arrows) and inhibitory synapses (white arrow). Scale bar = 500 nm. (B) A significant reduction in the density of excitatory but not inhibitory synapses in the hippocampus of DHHC5-gt mice (two-way ANOVA, 103  significant interaction between genotype and synapse type, F(1, 8) = 8.753, p = 0.0182, **p<0.01 Bonferroni’s test post hoc, n = 3 mice per genotype). (C) Although inhibitory synapses were significantly smaller than excitatory synapses (two-way ANOVA, main effect of synapse type, F(1, 8) = 15.96, p = 0.0040, n = 3 mice per genotype), no differences in synapse size were observed across genotypes. Data shown as mean ± s.e.m.  3.3.5 zDHHC5 E648* Mutation Affects Protein Localization and Mobility PSD-95 binding to zDHHC5 through PDZ interactions has been shown to be important to stabilize zDHHC5 at the synaptic membrane. Indeed, co-expression of PSD-95 and zDHHC5 results in significantly more zDHHC5 at the synapse surface compared to cells expressing zDHHC5 alone (Brigidi et al., 2015). We therefore hypothesized that the E648* mutant would be improperly localized outside dendritic spines, and would traffic in and out of spines more readily than wildtype zDHHC5. To examine this, we first transfected cells at 10 DIV with a dsRed cell fill and either wildtype or E648* mutant forms of GFP-tagged zDHHC5. At 13 DIV we fixed and immunostained cells for PSD-95. We masked the immunostaining images using the cell fill image, and examined the colocalization of GFP-zDHHC5 with PSD-95 immunostaining. We observed significantly less colocalization of PSD-95 with E648* compared to wildtype zDHHC5 (Figure 3.8A, B). 104   Figure 3.8 A zDHHC5 Truncation Mutant found in a Patient with Schizophrenia Results in Improper Localization and Increased Mobility of the Protein.  (A) PSD-95 is significantly less colocalized with E648* mutant GFP-zDHHC5 compared to WT GFP-DHHC5 (unpaired t-test, t(62) = 3.644, p = 0.0006, n = 31 wildtype cells, 33 E648* cells).(B) Confocal images of hippocampal neurons transfected with the indicated constructs and immunostained for the excitatory synapse marker, PSD-95 (grey) Scale bars, 5 µm. (C) The mobile fraction of GFP-zDHHC5 (fluoresecence within the ROI at the 4-minute timepoint normalized for photobleaching; mean ± s.e.m (unpaired t-test, t(61) = 2.739 ,p = 0.0081, n = 37 wildtype spines, 26 E648* spines). (D) Normalized fluorescence recovery of GFP-zDHHC5 wildtype, and E648* mutant. Points with error bars represent the mean ± s.e.m., and solid lines represent a single exponential fit. n = 37 GFP-zDHHC5-WT spines and 26 GFP-zDHHC5-E648* spines. (E) Fluorescence within a photobleached ROI (white circle) was initially photobleached at 0s within a 1- µm ROI. Scale bars, 1 µm. Data shown as mean ± s.e.m. 105  We next examined the role of the E648* mutation in regulating the trafficking and mobility of zDHHC5 in postsynaptic spine heads using fluorescence recovery after photobleaching (FRAP; Figure 3.8C-E). Primary hippocampal neurons were transfected with either wildtype or E648* mutant DHHC5 tagged with GFP, together with dsRed to outline transfected neurons. GFP-DHHC5 puncta localized to dendritic spines were identified and a region of interest (ROI) was photobleached using a 405 nm laser. The fluorescence recovery of wildtype or E648* GFP-DHHC5 within the photobleached ROI was measured over 4 minutes of time-lapse imaging. In control neurons expressing the wildtype zDHHC5, the recovery of GFP-zDHHC5 plateaued at 29.6 ± 4.0% (mean ± s.e.m.) at 4 minutes after photobleaching. In contrast, the E648* form of GFP-zDHHC5 was significantly more mobile, with a recovery of 47 ± 4.9% (unpaired t-test, t(61) = 2.739 , p = 0.0081). Together, these results indicate that the de novo E648* mutation found in a schizophrenia patient results in significantly reduced localization of zDHHC5 at postsynaptic sites, and increased zDHHC5 mobility (Figure 3.8).  3.3.6 zDHHC5 Localization at Dendritic Spines Promotes Spine Stability  Given that the loss of zDHHC5 results in reduced excitatory synapse density in hippocampal neurons both in vitro (Figure 3.3, 3.5) and in vivo (Figure 3.7B), we hypothesized that zDHHC5 localization in postsynaptic spines might either promote the formation of spines or inhibit the elimination of spines and as a result, stabilize nascent excitatory synapses, which could account for the increase in excitatory synapse density. To examine this, we transfected cultured hippocampal neurons with GFP-zDHHC5 and dsRed cell fill at 10 DIV.   At 12-13 DIV, we imaged live transfected neurons for a 15-minute period to observe spine turnover. We saw that the majority of spines were stable over the 15-minute imaging 106  period (88% ± 1.9%). Of all the spines that remained stable over the imaging period, 94% were associated with GFP-DHHC5 puncta, while only 6% of stable spines were not associated with GFP-DHHC5 puncta. Moreover, only 10% of unstable spines were associated with GFP-DHHC5 puncta while 90% of unstable spines lacked GFP-DHHC5 puncta (Figure 3.9). This highly suggests that the presence of zDHHC5 at postsynaptic spines increases their stability. Together, these data support the finding that zDHHC5 promotes the formation of excitatory synapses by promoting the stability of dendritic spines, and zDHHC5 localization at the synaptic membrane is necessary for the formation or stability of excitatory synapses.   Figure 3.9 GFP-zDHHC5 Localization in Spines Promotes Spine Stability. (A) Live imaging of spine turnover in GFP-zDHHC5 transfected neurons indicates that a significantly higher percentage of stable spines contain GFP-zDHHC5, while a significantly higher percentage of unstable spines do not contain GFP-zDHHC5 (two-way ANOVA, significant interaction between GFP-zDHHC5 and spine stability, F(1, 92) = 520.5, p < 0.001, Stable spines: n = 28 cells, Unstable spines: n = 20 cells. ***p < 0.001 Bonferroni’s test post hoc). (B) Representative images of live neurons transfected with GFP-zDHHC5 and dsRed cell fill. White arrows indicate spines that are stable throughout 15-minute imaging period, yellow arrows indicate spine that retracts during the imaging period. Data shown as mean ± s.e.m.  3.4 Discussion The results from this study indicate that zDHHC5 expression stabilizes dendritic spines, and promotes the formation or stability of excitatory synapses. When endogenous zDHHC5 expression was knocked down using shRNA, excitatory synapse density was significantly reduced (Figure 3.4D). Although this phenotype could be rescued by the expression of the 107  zDHHC5 shRNA with an shRNA-resistant form of wildtype zDHHC5, a palmitoylation deficient zDHHS5 was unable to rescue the phenotype, indicating the importance of zDHHC5’s palmitoylation function in promoting excitatory synapse formation. Additionally, the expression of a de novo E648* truncation mutant could not rescue the phenotype. The E648* mutant was identified in a screen of patients with schizophrenia (Fromer et al., 2014). As this mutant lacks the PDZ binding motif necessary for zDHHC5 to interact with postsynaptic scaffolding protein PSD-95, we saw that the E648* mutant was significantly more mobile than wildtype zDHHC5 (Figure 3.7C-E), and was not strongly localized to postsynaptic spines (Figure 3.7A, B). We hypothesize that the mislocalization of zDHHC5 impairs its ability to promote excitatory synapse formation. Indeed, the expression of zDHHC5 shRNA with phosphomimetic mutant Y533E, a mutation that disrupts AP2µ-mediated endocytosis of zDHHC5 (Brigidi et al., 2015), showed normal excitatory synapse formation (Figure 3.4D). The Y533E mutant is more strongly localized to the plasma membrane in neurons (Brigidi et al., 2015), and we saw that the expression of this mutant rescued the phenotype, resulting in an excitatory synapse density that was similar to control neurons. Furthermore, when the same experiment was performed but neurons were instead immunostained for the presynaptic excitatory synapse marker VGLUT1 and the excitatory postsynaptic synapse marker PSD-95, we saw the same pattern of results (Figure 3.5), suggesting that immunostaining for PSD-95 alone was a suitable marker for excitatory synapses. These findings were further supported by a live imaging experiment where we overexpressed GFP-zDHHC5 and monitored spine turnover. Significantly more stable spines contained GFP-zDHHC5, where significantly fewer transient spines contained GFP-zDHHC5 (Figure 3.9). As the majority of excitatory synapses form onto dendritic spines, this further supports the finding that zDHHC5 contributes to excitatory synapse formation, perhaps by 108  promoting spine stability. Together, these data suggest that zDHHC5’s PAT function and localization at the synaptic membrane are critical for the formation of excitatory synapses.  Using immunogold EM, we detected differences in the density of excitatory synapses within the stratum radiatum of the CA1 hippocampus in zDHHC5 gene-trapped mice compared to wildtype controls. This further supports the data from our in vitro studies, which indicate that zDHHC5 expression is necessary for excitatory synapse formation.  We did not see any changes in inhibitory synapse formation in any condition in vitro (Figure 3.2D) or in vivo (Figure 3.6). Previous work shows approximately 47% of inhibitory synapses contain zDHHC5 (Brigidi et al., 2015), however our results show that the expression of zDHHC5 does not affect inhibitory synapse formation. The substrates palmitoylated by zDHHC5 at inhibitory synapses are not known, but it is possible that these substrates do not affect inhibitory synaptogenesis. We also found that zDHHC5 expression did not affect dendrite outgrowth or complexity. This was interesting given that zDHHC5 palmitoylates flotillin-2 (Li et al., 2012b), which has been shown to facilitate the formation of filopodia-like structures when expressed in cell lines (Neumann-Giesen et al., 2004). Indeed, palmitoylation has been shown to influence dendritogenesis and spinogenesis through other substrates such as neural cell adhesion molecule (NCAM), Ca2+-calmodulin-dependent protein kinase type 1G (CAMK1G), and LIM kinase 1 (George et al., 2015; Ponimaskin et al., 2008; Takemoto-Kimura et al., 2007).  In this work, we examine the effects of a clinical zDHHC5 variant identified in a patient with schizophrenia. Schizophrenia is a chronic mental disorder that is characterized by positive symptoms and negative symptoms (Crow, 1981; Thompson et al., 2004). The positive symptoms include hallucinations, delusions, and unusual or dysfunctional ways of thinking, and the 109  negative symptoms include flat affect, reduced feelings of pleasure in everyday activities and difficulty beginning new activities. Furthermore, individuals with schizophrenia often exhibit cognitive symptoms including poor executive functioning, trouble focusing and working memory impairments (Elvevag and Goldberg, 2000). Indeed, these cognitive symptoms are often identified years before the more obvious positive symptoms associated with schizophrenia. Our research identifies a functional effect of the clinical zDHHC5 variant when expressed in neurons. We find that the E648* variant is more mobile and improperly localized compared to wildtype zDHHC5. This variant contributes to a reduced number of excitatory synapses in neurons, and may have a causal role in schizophrenia.  When examining the pre- and post-synaptic localization of zDHHC5 at hippocampal synapses using immunogold EM, we detected a significant proportion of zDHHC5 localized to the presynaptic plasma membrane (Figure 3.1). Although the current study was focused on the importance of postsynaptically localized zDHHC5 on excitatory synapse formation, it is possible that the zDHHC5-mediated palmitoylation of presynaptic proteins also affects synapse formation and plasticity. Indeed, a SILAC study of wildtype and DHHC5 gene-trapped neural stem cell cultures identified synaptic vesicle proteins such as synaptobrevins 2 and 3, Rab3a, Rab8a, and the secretory carrier membrane protein scamp5 as potential targets of zDHHC5 palmitoylation (Li et al., 2012b). Future studies should follow up on the role of zDHHC5 at the presynaptic terminal. Here we have shown that zDHHC5 PAT function and localization at the plasma membrane are necessary for excitatory synapse formation. Although beyond the scope of this study, it is tempting to speculate which substrate zDHHC5 palmitoylates in order to promote synapse formation. The few confirmed substrates of zDHHC5 palmitoylation are GRIP1b, δ-110  catenin, the G-protein coupled somatostatin receptor 5, and the STREX variant of voltage-activated potassium channels (Thomas et al., 2012; Brigidi et al., 2014; Kokkola et al., 2011; Tian et al., 2010). Palmitoylated δ-catenin has been shown to increase synapse size and synaptic strength in response to activity (Brigidi et al., 2014), however δ-catenin is mainly localized in dendritic shafts when it is not palmitoylated (Brigidi et al., 2015). Given that zDHHC5 undergoes subcellular changes in localization from spines to the dendritic shaft in response to stimulation (Brigidi et al., 2015), the palmitoylation of δ-catenin seems to contribute more to activity mediated changes rather than synaptogenesis. GRIP1b is also a potential candidate. Indeed, lipid attachment targets GRIP1b to Transferrin-positive endosomes in dendrites, where it is positioned to influence AMPAR recycling, and the overexpression of zDHHC5 accelerates the recycling of AMPARs (Thomas et al., 2012). It is possible that palmitoylated GRIP1b might increase the turnover of AMPARs in dendrites, which could result in increased AMPAR delivery to nascent synapses. Studies thus far suggest that GRIP1b palmitoylation has a greater effect on AMPAR cycling (Setou, 2002, Mao, 2010, Thomas, 2012), but more work is needed to determine whether the palmitoylation of GRIP1b is necessary for synapse formation. Of course, unidentified targets of zDHHC5 palmitoylation likely contribute to synaptogenesis. Future studies should further examine the targets of zDHHC5 palmitoylation in neurons.  111  Chapter 4: Conclusion The work presented in this thesis provides insight into the role of cadherin adhesion complexes in synaptic plasticity in the mesolimbic dopamine circuitry, and identifies a new role for zDHHC5 in excitatory synapse formation. We show that cadherins play an important role in synaptic plasticity and behavioural conditioning with drugs of abuse such as cocaine. Additionally, we find that the palmitoyl aclytransferase zDHHC5 is not only a critical regulator of cadherin adhesion complexes and synaptic plasticity (Brigidi et al., 2014) but is also important for the formation of excitatory synapses. Given the role that zDHHC5 plays in the palmitoylation and changes in synaptic localization of δ-catenin, future studies should examine whether zDHHC5 can influence the cadherin adhesion complex at synapses onto dopamine neurons in response to drugs of abuse.  4.1 The Role of Cadherins in Plasticity in the Ventral Tegmental Area The majority of previous research on the role of cadherins at the synapse has examined cadherin function in hippocampal neurons. Although some preliminary studies of mRNA expression of classical cadherins identified them in the midbrain (Hertel et al., 2008), their role in synaptic plasticity had not been studied in this brain region. Our findings show that transsynaptic cadherin-cadherin interactions contribute to synaptic strengthening at excitatory inputs formed onto VTA dopamine cells, and that cadherins serve to stabilize the AMPA receptors that are present at these excitatory synapses. In the hippocampus, increased synaptic activity is associated with increased cadherin recruitment to synapses (Bozdagi et al., 2010; Mendez et al., 2010) and increased cadherin stability at the synaptic membrane (Tai et al., 2007).  112  We used a β-catenin stabilized transgenic mouse model (DAT-Cre;βcat∆ex3) to increase cadherin’s stability at the synaptic membrane of dopamine cells, finding that this increased cadherin stability resulted in increased AMPA receptor stability at synapses. This increase in stability caused impairments in STD LTP and conditioned place preference (CPP). These findings were initially counterintuitive, as previous work in the hippocampus has shown that using the same approach to stabilize cadherins at hippocampal synapses results in normal LTP, but impaired LTD resulting in behavioural inflexibility (Mills et al., 2014). We discovered that the increase in cadherin blocked the AMPAR redistribution that normally occurs in response to cocaine, where Ca2+ impermeable GluA1/2 heteromers are swapped out for Ca2+ permeable GluA1 homomers. Previous work from Bellone et al. (2006) supports our findings. They found that the cocaine-mediated redistribution of AMPARs at excitatory synapses onto VTA dopamine neurons relies on the interaction between protein interacting with C kinase-1 (PICK1) and GluA2 subunits (Bellone and Lüscher, 2006). Most importantly, Bellone et al’s results show that when this interaction between GluA2 and PICK1 is blocked using a peptide, the insertion of GluA1 homomers in response to cocaine was impaired. Bellone et al. speculate that the number of ‘slots’ for AMPA receptors may be limited at postsynaptic sites, such that GluA1/2 heteromers need to be removed from synapses to make room for the insertion of Ca2+-permeable, GluA1 homomers, a hypothesis which is further supported by our findings. We show that increased cadherin stability at VTA synapses results in impaired AMPA receptor redistribution after cocaine exposure. N-cadherin has been shown to interact with GluA1 and GluA2 (Nuriya and Huganir, 2006; Saglietti et al., 2007; Silverman et al., 2007), so we suspect that this increase in cadherin at synaptic sites stabilizes the GluA1/2 heteromers that are present under basal 113  conditions. As a result, there are no free “slots” for the insertion of GluA1 homomers, and synaptic plasticity is impaired. Our findings emphasize the importance of studying the function of synaptic proteins in multiple brain regions and cell types. The majority of studies examining the molecular mechanisms of synaptic proteins have been limited to hippocampal neurons, both in vitro and in vivo. Although the well-understood circuit of the hippocampus is an asset for initial studies of synaptic proteins, we show that the same “molecular rules” do not necessarily apply in all cell types. Further studies could examine whether other synaptic proteins that have been well studied in the hippocampus have different roles in other brain regions such as the mesocorticolimbic circuit.  4.2 β-catenin and Wnt Signaling in Dopaminergic Cells Although Wnt signaling was not the main focus of this thesis, the findings in Chapter 2 provide useful insight into the relationship between β-catenin expression levels and Wnt signaling in fully differentiated dopaminergic neurons. We saw that increases in β-catenin in differentiated dopamine neurons did not result in any changes in Wnt signaling detected by immunohistochemistry. Furthermore, we saw no changes in overall morphology of VTA synapses, and no differences in electrophysiological measures of basal synapse function in the VTA.   Wnt signaling regulates dopamine cell differentiation in the ventral midbrain during development. Several Wnt signaling pathway members has been shown to regulate the proliferation and specification of dopamine progenitor cells in the midbrain (Prakash et al., 2006; Castelo-Branco et al., 2010; Sousa et al., 2010). The loss of β-catenin in midbrain progenitor 114  cells results in reduced dopamine cell neurogenesis (Tang et al., 2009). Furthermore, when a stabilized form of β-catenin is expressed in midbrain progenitor cells, there is a significant expansion in the progenitor cell population (Tang et al., 2010). Interestingly, the cell cycle is perturbed in progenitor cells expressing the stabilized β-catenin, and as a result they fail to differentiate into dopamine neurons (Tang et al., 2010). Clearly, Wnt signaling plays a critical and complex role in the differentiation of midbrain dopamine neurons.  Although Wnt signaling has significant effects in the developing midbrain, the adult brain seems to be minimally affected by by β-catenin levels (Misztal et al., 2017). When mice were chronically treated with widely used anti-psychotic lithium, which is known to inhibit GSK3, β-catenin levels were increased throughout the brain. However, β-catenin levels were not elevated in cellular nuclei within the cortex or hippocampus, only in the thalamus. Misztal et al. (2017) found that this was because the thalamus has higher expression of TCF7L2, and this LEF1/TCF family protein was mediating the activation and nuclear transport of β-catenin (Misztal et al., 2017). It is not clear what effect Wnt signaling has in differentiated neurons, although some studies suggest that it might affect activity dependent gene regulation (Abe and Takeichi, 2007) and anxiety-like behaviour in a zebrafish model (Misztal et al., 2017). These findings show that β-catenin’s effect on Wnt signaling may be much more influential during development than in terminally differentiated neurons.   4.3 zDHHC5 in Hippocampal Synaptogenesis In Chapter 3, we identify an important role for zDHHC5 in excitatory synapse formation. We observe that the loss of zDHHC5 expression in vivo results in a significant reduction in excitatory synapse formation in the hippocampus. Furthermore, our experiments in hippocampal 115  culture demonstrate that zDHHC5 must be expressed, must be able to palmitoylate it’s substrates, and must be properly localized to postsynaptic spines in order to promote the formation of excitatory synapses. Based on these results, we can speculate that zDHHC5 likely palmitoylates a substrate or a group of substrates localized in the postsynaptic spine, and the palmitoylation of these substrates helps to stabilize dendritic spines and therefore promotes either the formation of excitatory synapses, or the stabilization of nascent excitatory synapses.  zDHHC5 has been shown to palmitoylate GRIP1b, flotillin-2, the spliced STREX variant of large conductance calcium- and voltage- activated potassium (BK) channel, somatostatin receptor 5, and δ-catenin (Brigidi et al., 2014; Kokkola et al., 2011; Li et al., 2012b; Thomas et al., 2012; Tian et al., 2010). Given that the palmitoylation of δ-catenin results in increased interaction with N-cadherin, and N-cadherin can contribute to synapse formation, it’s possible that the zDHHC5-mediated palmitoylation of δ-catenin could contribute to excitatory synapse formation. However, previous work suggests that δ-catenin is palmitoylated in an activity dependent manner and contributes to synaptic plasticity rather than synapse formation.  The zDHHC5-mediated palmitoylation of GRIP1b targets this scaffolding protein to recycling endosomes, where it can influence AMPAR cycling. It’s possible that palmitoylated GRIP1b might increase AMPAR delivery to nascent synapses, however this has not been directly examined. Recent work has shown that the actin regulator LIM kinase-1 (LIMK1) is palmitoylated, and that the palmitoylation of LIMK1 is necessary for dendritic spine and synapse formation (George et al., 2015). Although the PAT necessary for LIM kinase-1 palmitoylation has not been identified, it’s also possible that zDHHC5 contributes to LIMK1 palmitoylation, and in this way can contribute to synaptogenesis, however this has not been examined. Further 116  work must be done to determine which proteins palmitoylated by zDHHC5 contribute to synaptogenesis.  4.4 Methodological Strengths and Limitations In Chapter 2 and 3, quantitative analysis of immunogold EM was used to determine the localization of cadherin, GluA1, GluA2, and zDHHC5 at synapses. Immunogold EM was a critical method for our studies as it allows for incredibly high-resolution imaging of protein localization at synaptic terminals, and allowed us to observe changes in the proportion of GluA1, GluA2 and cadherin localized to synaptic membranes. This level of detail was crucial as the presence of these proteins at the synaptic membrane indicated that they were positioned to participate in transsynaptic adhesion between pre- and postsynaptic compartments (in the case of cadherin), or produce postsynaptic reseonses to neurotransmitter release (in the case of GluA1 and GluA2, components of AMPARs). Conventional approaches such as confocal microscopy would not have provided high enough resolution to differentiate between proteins localized to the synaptic membrane and proteins localized within the synaptic compartment in recycling endosomes. Although changes in AMPARs at synapses onto VTA dopamine neurons were also detected using electrophysiological methods (see Figure 2.14), there is no electrophysiological output that could be used to detect changes in cadherin localization. The work in this thesis demonstrates that immunogold EM can be analyzed quantitatively and can provide useful insight into changes in protein localization in response to synaptic activity and drugs of abuse. Surely this will be a powerful approach for future studies of cadherins, zDHHCs, and other proteins at the synapse. 117  Although this technique is powerful, there are a few limitations to quantitative immunogold EM. One limitation is that we cannot determine the interactions or binding partners of the individual proteins detected. For example, we cannot be certain that cadherins localized to the synaptic membrane are actively interacting transsynaptically with cadherins from the opposing synaptic membrane. In comparing the relative proportion of cadherin at the synaptic membrane between mice that underwent cocaine mediated CPP and saline controls, we can infer that a significant increase in cadherin adhesion is likely. However, it is possible that association with different binding partners or post-translational modifications might modulate cadherin trans-synaptic interactions. Therefore, the actual amount of cadherin adhesion at VTA synapses is not directly testable using immunogold EM. Similarly, though we can observe changes in the relative proportion of GluA1 monomers localized to synapses following cocaine-mediated CPP, we cannot determine the identities of the other glutamate receptor subunits that the GluA1 monomers might be associated with in tetramer form. In examining GluA1 and GluA2 localization (see Figure 2.12), we can infer that a “subunit switch” is occurring, where GluA1/2 heteromers are removed from VTA synapses, and GluA1 homomers are inserted at VTA synapses. However, the occurrence of this change in AMPAR composition (and its impairment in β-catenin stabilized mice) was also measured using electrophysiological techniques (see Figure 2.14). The data from our studies show that immunogold EM is complementary to electrophysiological techniques, and these two techniques can be used together to provide a greater understanding of changes in synaptic protein composition.  In Chapter 2, we used a transgenic approach to manipulate the membrane stability of cadherin in dopaminergic cells. Given that in the DAT-Cre;βcat∆ex3 mice, β-catenin is stabilized in all dopamine cells, it is possible that the effects we observe are due to differences in synapse 118  function in other dopaminergic neurons, (i.e. nigrostriatal dopamine neurons). However, dopaminergic release by dopamine cells in the nigrostriatal circuit is critical for the production of movement rather than drug preference. We examined general locomotor activity using the open field test and motor learning using the accelerating rota-rod and did not see any differences in the DAT-Cre;βcat∆ex3 mice compared to littermate controls, suggesting that the increased β-catenin levels in dopamine cells has a more robust affect on drug responses than locomotor activity. However, another concern with this transgenic approach is the potential effects of increased β-catenin levels at dopaminergic presynaptic terminals, which project to the NAc and prefrontal cortex. We did not examine whether presynaptic increases in β-catenin and cadherin stability have any effect on dopaminergic output, however previous work has shown that stabilizing β-catenin at presynaptic terminals in the hippocampus had subtle effects on synaptic vesicle release. Specifically, the increased β-catenin resulted in impaired responses to repeated stimulation, suggesting that the mobilization or release of synaptic vesicles might be impaired due to the increased β-catenin (Mills et al., 2014). This suggests that perhaps the DAT-Cre;βcat∆ex3mice would have impairments in dopamine release in the NAc. This could also contribute to their reduced preference for cocaine, in addition to the impaired plasticity seen at the excitatory inputs formed onto these dopamine neurons.   4.5 Future Directions The findings presented in this thesis help to elucidate the role of cadherin adhesion complexes in the mesolimbic reward circuitry, and determine the role of a cadherin adhesion complex regulator, zDHHC5 in synapse formation and dendrite outgrowth. There are several future directions that could continue from this research, which include a further examination of 119  the role of cadherin adhesion complexes in drug-mediated plasticity in the mesocorticolimbic circuit, determining the presynaptic targets of zDHHC5-mediated palmitoylation, and determining whether zDHHC5 can contribute to drug-mediated plasticity in the mesocorticolimbic reward circuit.  4.5.1 Role of Cadherin Adhesion Complexes in Drug-Mediated Plasticity in Mesolimbic Circuitry The findings from Chapter 2 have important implications for the mechanisms underlying the initial stages of drug addiction. The immunogold EM studies examining protein localization in wildtype mice demonstrate that cocaine mediated CPP causes a redistribution of cadherin and GluA1 to the synaptic membrane at excitatory synapses onto dopamine neurons in the VTA. Furthermore, we show that interfering with synaptic plasticity at excitatory inputs to VTA dopamine neurons has a protective effect against the acquisition of drug preference.  An important future direction for this work will be to tease apart any circuit differences in cadherin localization and function. It would be of interest to determine whether inputs to specific dopamine cell populations exhibit changes in cadherin localization in response to cocaine. Indeed, the VTA dopamine cells are a heterogeneous group, with different response characteristics depending on the brain region to which they project (Lammel et al., 2011). Furthermore, glutamatergic input from specific brain regions has been shown to drive preference and aversive behaviour (Lammel et al., 2012). Given the limitations of immunogold EM, it was not possible for us to examine differences in cadherin localization at synapses onto specific dopamine cell subpopulations, or at synapses projecting from specific brain regions. However, 120  using a viral vector strategy, a study of the role of cadherins in specific dopamine cell subpopulations would still be challenging, but could be attempted.  We also find that although the potentiation of excitatory synapses onto VTA dopamine neurons is important for the acquisition of CPP, changes in the strength of these synapses does not seem to be integral for the extinction of CPP. We found that after mice extinguished their preference for a drug-paired chamber by repeated exposure to the behaviour box with no further exposure to cocaine, the amount of cadherin and GluA1 localized to excitatory synapses onto VTA dopamine neurons was no different from saline control mice. However, we saw the same result when mice were conditioned with cocaine, and then housed in their home cage for 6 additional days with no further exposure to the behaviour box or cocaine (see Figure 2.4 e,k ‘CPP + Extinction’, ‘CPP + HC’) . This suggests that the increase in membrane-associated cadherin and GluA1 at excitatory synapses onto VTA dopamine neurons is a transient occurrence that likely drives increased dopamine neuron activity during and immediately following cocaine exposure, and leads to downstream synaptic changes in other regions such as the NAc. Others have shown that cocaine-mediated increases in the strength of synapses onto VTA dopamine neurons drives synaptic plasticity in the NAc, where cocaine-induced reduction in the AMPAR/NMDAR ratio is observed (Mameli et al., 2009). However, the potentiation of VTA synapses themselves does not necessarily encode drug preference. Indeed, extinction of conditioned responses is thought to be a separate form of learning rather than the erasure of the initially formed memory (Botreau et al., 2006; Maren and Quirk, 2004; Quirk, 2002). This will be important to consider if our work is ever adapted for a therapeutic context: it is unlikely that treatments for addiction will arise from targeting cadherin stability at excitatory inputs formed onto dopamine neurons, but rather from targeting the downstream plasticity that is initially 121  driven by the transient, drug-mediated increase in synaptic strength at VTA inputs. Together, these results suggest that studying downstream regions of the mesocorticolimbic circuit is important to understanding the addicted brain. A potential future direction for this research would be to examine changes in cadherin localization at synapses forming on the NAc medium spiny neurons, to determine whether cadherin localization might play a role in the synaptic plasticity observed in the NAc after exposure to drugs of abuse such as cocaine. Indeed, there are increases in the number of Ca2+ permeable GluA1 homomer AMPARs at synapses onto medium spiny neurons (MSNs) in the NAc immediately after cocaine self-administration, and it’s thought that this increase in AMPARs might contribute to the incubation of craving observed in this model of addiction (Conrad et al., 2008; Mameli et al., 2009; McCutcheon et al., 2011a, 2011b). Given that cadherins have been shown to interact with AMPARs and stabilize their localization at the postsynaptic membrane, it’s possible that changes in cadherin membrane localization play a role in this type of plasticity in the NAc.   4.5.2 Presynaptic Targets of zDHHC5 Palmitoylation In Chapter 3, we examined the localization of zDHHC5 at pre- and postsynaptic terminals by immunogold EM and identified a significant proportion of zDHHC5 localized to the presynaptic terminal. There is much to be learned about the presynaptic role of zDHHC5. First of all, given that zDHHC5 has a PDZ binding domain at the C-terminal tail, it would be interesting to determine if zDHHC5 can interact with presynaptically localized PDZ domain proteins. Indeed, a number of PDZ domain-containing scaffolding proteins including CASK (Lin-2 in C. elegans), Velis (Lin-7 in C. elegans), and MINT1 (Lin-10 in C. elegans) are localized to presynaptic sites (Kim and Sheng, 2004) and could potentially interact with zDHHC5. Indeed, 122  CASK, Velis and MINT1 can form a complex, and it is suggested that they might link the core active zone proteins with scaffolding proteins and cell adhesion molecules (Südhof, 2012). The PDZ domain containing protein PICK1 is present at both synaptic and non-synaptic sites, and has also been shown to interact with presynaptic proteins (Kim and Sheng, 2004). It is possible that zDHHC5 interacts with one or several of these PDZ domain proteins at presynaptic sites, which would position zDHHC5 to palmitoylate putative presynaptic substrates. Second, the presynaptic substrates of zDHHC5 palmitoylation must be further examined. Preliminary SILAC screens on neural stem cell cultures have identified several synaptic vesicle proteins such as synaptobrevins 2 and 3, Rab3a, Rab8a, and the secretory carrier membrane protein scamp5 as potential targets of zDHHC5 palmitoylation (Li et al., 2012b), but these potential substrates would need to be validated in neurons. Previous research has shown that proteins that contribute to synaptic vesicle fusion including SNAP25, cysteine string protein, synaptobrevin 2, and synaptotagmin 1 are substrates of palmitoylation (Prescott et al., 2009). It will be important to determine if zDHHC5-mediated palmitoylation of presynaptic proteins regulates synaptic vesicle release. Indeed, the presynaptically localized PAT zDHHC17 has been shown to regulate synaptic vesicle exocytosis in drosophila (Ohyama et al., 2007; Stowers and Isacoff, 2007), so perhaps zDHHC5 plays a complementary role. Third, as zDHHC5 localization at the postsynaptic terminal is altered in response to activity, it would be of interest to determine if similar activity-dependent mechanisms drive changes in zDHHC5 localization presynaptically.  4.5.3 Role of zDHHC5 in Drug-Mediated Plasticity in the Ventral Tegmental Area The role of zDHHC5 in synaptogenesis was examined in Chapter 3, however previous work has shown that zDHHC5 palmitoylates the cadherin adhesion complex protein, δ-catenin, 123  in an activity-dependent manner (Brigidi et al., 2014, 2015). It would be of interest to determine whether zDHHC5 functions in an activity dependent manner in VTA dopamine neurons, as much of the work on zDHHC5 in neurons has focused on hippocampal neurons. In situ hybridization studies indicate that zDHHC5 is expressed in the midbrain (Lein et al., 2007), however it is unclear whether this PAT is expressed in dopamine neurons of the VTA. First, the expression of zDHHC5 in the midbrain, and the cell types expressing zDHHC5 in this region must be confirmed. Cocaine-mediated CPP results in an increase in cadherin localized to the synaptic membrane of VTA synapses, and the activity-mediated palmitoylation of δ-catenin results in increased δ-catenin at dendritic spines and increased association with N-cadherin. Therefore, it would be interesting to determine whether zDHHC5 mediated palmitoylation of synaptic substrates such as δ-catenin could contribute to cocaine-mediated synaptic plasticity, and how zDHHC5’s activity might work to alter synapse strength in this region of the brain. Furthermore, if more substrates of zDHHC5 palmitoylation are identified, it would be of interest to determine how their palmitoylation might affect synapse plasticity in the mesocorticolimbic reward circuitry.  124  Bibliography Abe, K., and Takeichi, M. (2007). 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