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The nigrostriatal dopamine system in the leucine-rich repeat kinase 2 G2019S knock-in mouse model of… Paschall, Sarah Afton 2016

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THE NIGROSTRIATAL DOPAMINE SYSTEM IN THE LEUCINE-RICH REPEAT KINASE 2 G2019S KNOCK-IN MOUSE MODEL OF PARKINSON’S DISEASE by  Sarah Afton Paschall  B.A., Western Washington University, 2010  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2016  © Sarah Afton Paschall, 2016 ii  Abstract Mutations on the leucine-rich repeat kinase 2 (LRRK2) gene are the most common variants responsible for idiopathic Parkinson’s disease (PD). Historically, PD is thought of as a late-stage neurodegenerative disease resulting from the loss of dopaminergic neurons in the substantia nigra pars compacta with the presence of Lewy pathology. Current treatments focus on the dopaminergic aspect of this disease, without addressing or successfully halting the underlying causes of this disease. The LRRK2 G2019S mutation is the single most common genetic risk factor for Parkinson’s disease and leads to increased kinase activity with subtle effects on the timing of nigrostriatal dopaminergic transmission. Here, using a genetically faithful G2019S knock-in (GKI) mouse model at multiple age points, although no differences were seen in monoamine, glutamate, or GABA release by in vivo microdialysis of the dorsolateral striatum, more temporally sensitive investigation revealed subtle release augmentation in mutants. Using fast-scan cyclic voltammetry to examine dopamine release and reuptake on a millisecond timescale in acute striatal slices at early (<3 months) and later age points (~12 months), alterations in dopamine release were observed with repeated stimulation and increased decay constant in mutants. In GKI mice dopamine release is augmented and responds differently across age points to pharmacological D2 receptor agonism and dopamine transporter (DAT) inhibition. LRRK2 is thought to be involved in synaptic vesicle storage and recycling and GKI mutation in mice leads to augmented dopamine release from an early age that is not attenuated by DAT inhibition, indicating other mechanisms (e.g. altered release / vesicle cycling) are affected and play a role in this alteration from early stages (<3 months, potentially earlier) in response to this mutation. Elucidation of the function of LRRK2 and the deleterious effects caused by the G2019S mutation will help target neuroprotective therapies to delay or halt disease progression. iii  Preface The following research presented in this thesis, was performed in the collaborative Centre for Applied Neurogenetics under the direction of Drs. Austen Milnerwood and Matthew Farrer. This thesis was written by me with feedback from Dr. Milnerwood. An introduction to slice physiology and initial setup of microdialysis paradigms came from Drs. Dayne Beccano-Kelly and Mattia Volta.  The work described in Section 2.3 was in part performed by myself (surgery, sampling, sample loading, chromatogram analysis, statistics, probe placement verification), with an increase in cohort size and HPLC maintenance by MV and Stefano Cataldi, and technical support from Sabrina Bergeron, Emma Mitchell, Silvia Turchetto, and Jesse Fox for sampling. JF was also responsible for histochemistry processing to verify probe placement and DAT / TH staining, imaging and analysis. Experiments performed in Section 2.4 were performed by myself with support from Igor Tatarnikov to increase sample sizes. I performed all statistical analysis and data presentation under the supervision of Dr. Milnerwood. All experiments described here were conducted in accordance with the Canadian Council of Animal Care and with the approval of the Animal Care Committee at the University of British Columbia. The animal protocol numbers, under which all experiments were conducted, were A11-0340 and A15-0038.  Work from Chapters 2.2 (methods) and 3.1 (results) have been previously published and was conducted in the Centre for Applied Neurogenetics at the University of British Columbia. I was responsible for the electrophysiology experiments included in this thesis. Beccano-Kelly, D.A., Volta, M., Munsie, L.N., Paschall, S.A., Tatarnikov, I., Co, K., Chou, P., Cao, L.-P., Bergeron, S., Mitchell, E., Han, H., Melrose, H.L., Tapia, L., Raymond, iv  L.A., Farrer, M.J., Milnerwood, A.J., 2015. LRRK2 overexpression alters glutamatergic presynaptic plasticity, striatal dopamine tone, postsynaptic signal transduction, motor activity and memory. Hum. Mol. Genet. 24, 1336–49. doi:10.1093/hmg/ddu543 v  Table of Contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iii Table of Contents ...........................................................................................................................v List of Figures ............................................................................................................................. viii List of Abbreviations ................................................................................................................... ix Acknowledgements ...................................................................................................................... xi Chapter 1: Introduction ................................................................................................................1 1.1 Parkinson’s disease ......................................................................................................... 1 1.1.1 Dopamine in Parkinson’s disease ............................................................................... 1 1.2 Striatal circuitry .............................................................................................................. 2 1.2.1 Direct and indirect pathways ...................................................................................... 3 1.2.2 D1 versus D2 receptor function .................................................................................. 4 1.2.3 Circuitry gone awry in Parkinson’s disease ................................................................ 6 1.3 Genetic Parkinson’s disease ............................................................................................ 7 1.3.1 Leucine-rich repeat kinase 2 ....................................................................................... 8 1.3.2 Dopamine & LRRK2 .................................................................................................. 9 1.3.3 LRRK2 G2019S ........................................................................................................ 10 1.4 Rationale for present experiments ................................................................................ 11 Chapter 2: Methods and materials .............................................................................................14 2.1 Subjects ......................................................................................................................... 14 2.2 Electrophysiology ......................................................................................................... 15 2.2.1 Slicing ....................................................................................................................... 15 vi  2.2.2 Equipment ................................................................................................................. 15 2.2.3 Sampling parameters ................................................................................................. 16 2.2.4 Data Analysis ............................................................................................................ 16 2.3 Basal dopamine tone, by in vivo microdialysis in the dorsolateral striatum ................. 17 2.3.1 Stereotaxic surgery.................................................................................................... 17 2.3.2 Sample collection and analysis by high-performance liquid chromatography ......... 19 2.3.3 Data Analysis ............................................................................................................ 20 2.4 Dopamine release and reuptake, by fast-scan cyclic voltammetry in acute striatal slices   ....................................................................................................................................... 21 2.4.1 Slicing ....................................................................................................................... 21 2.4.2 Equipment ................................................................................................................. 21 2.4.3 Sampling parameters ................................................................................................. 22 2.4.4 Pharmacology ........................................................................................................... 23 2.4.5 Data Analysis ............................................................................................................ 23 2.5 Statistics ........................................................................................................................ 23 Chapter 3: Results........................................................................................................................24 3.1 Field electrophysiology: ex vivo analysis of extracellular striatal glutamate plasticity demonstrates intact LTD in mice overexpressing hLRRK2 ..................................................... 24 3.2 Microdialysis: in vivo analysis of striatal extracellular basal neurotransmitter tone and effects of D2R agonism are unaltered in LRRK2 G2019S knock-in mice ............................... 26 3.2.1 Dopamine and its metabolites: DA, DOPAC, and HVA .......................................... 27 3.2.2 Serotonin and its metabolite: 5-HT and 5-HIAA ...................................................... 28 3.2.3 Glutamate and GABA ............................................................................................... 28 vii  3.3 Fast-scan cyclic voltammetry: ex vivo analysis of striatal extracellular dopamine release and reuptake in acute brain slices from LRRK2 G2019S knock-in mice ..................... 29 3.3.1 Evoked release of dopamine is altered with repeated stimulations in GKI mice under basal conditions ..................................................................................................................... 29 3.3.2 Quinpirole: D2 agonism alters repeated release in young GKI mice ....................... 31 3.3.3 GBR-12909: DAT inhibition alters DA release differently in young and old GKI mice ................................................................................................................................... 31 Chapter 4: Discussion ..................................................................................................................42 4.1 LRRK2 and its role in PD, clues from hLRRK2 OE .................................................... 42 4.2 LRRK2 G2019S-induced alterations to the DA system ............................................... 44 4.3 Limitations and future directions .................................................................................. 50 4.3.1 DAT/D2 quantification, functionality ....................................................................... 51 4.3.2 Individual stimulation of striatal inputs .................................................................... 51 4.4 Conclusions ................................................................................................................... 52 Bibliography .................................................................................................................................54  viii  List of Figures Figure 1. Local electrical stimulation in acute striatal slices revealed subtle synaptic alterations, but normal long-term depression in LRRK2 hWT OE mice. ....................................................... 33 Figure 2. Microdialysis in the dorsolateral striatum of young LRRK2 GKI mice revealed no genotypic alterations in basal levels or turnover of monoamines and metabolites....................... 35 Figure 3. Microdialysis in the dorsolateral striatum of old LRRK2 GKI mice revealed no genotypic alterations in basal levels or turnover of monoamines and metabolites....................... 36 Figure 4. Microdialysis in the dorsolateral striatum of young and old LRRK2 GKI mice revealed no genotypic alterations in glutamate and GABA tone. ............................................................... 37 Figure 5. Fast-scan cyclic voltammetry in slices from the dorsolateral striatum of young LRRK2 GKI mice revealed alterations to repeated stimulation and decay under basal conditions, increased sensitivity to D2 agonism, and decreased sensitivity to DAT inhibition relative to WT littermates. ..................................................................................................................................... 38 Figure 6. Fast-scan cyclic voltammetry in slices from the dorsolateral striatum of old LRRK2 GKI mice revealed no alterations in electrically evoked dopamine release, but with alterations to decay under basal conditions, while the inhibition of DAT revealed alterations to evoked DA release relative to WT littermates. ................................................................................................ 40  ix  List of Abbreviations 5-HT = 5-hydroxytryptamine / serotonin 6-OHDA = 6=hydroxydopamine AC = adenylyl cyclase ACh = acetylcholine aCSF = artificial cerebral spinal fluid ANOVA = analysis of variance AP = anterior/posterior BAC = bacterial artificial chromosome BL = baseline cAMP = cyclic adenosine monophosphate DA= dopamine DARPP-32 = DA and cAMP-regulated phosphoprotein 32 DAT = dopamine transporter DOPAC = 3,4-dihydroxyphenylacetic acid DV = dorsal/ventral EtOH = ethanol FSCV = fast-scan cyclic voltammetry GABA = γ-aminobutyric acid GBR-12909 =1-[2-[Bis-(4-fluorophenyl)methoxy]-ethyl]-4-[3-phenylpropyl]piperazine dihydrochloride GKI = LRRK2 G2019S knock-in Glu = glutamate x  GPe = globus pallidus pars externa HFS = high-frequency stimulation HPLC = high performance liquid chromatography HVA = homovanillic acid KI = knock-in KO = knock-out L-dopa = Levodopa LRRK2 = leucine-rich repeat kinase 2 ML = medial/lateral MPTP = 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ms = millisecond nM = nanomolar NT = non-transgenic OE = over-expressing PD = Parkinson’s disease PKA = protein kinase A RM ANOVA = repeated measures analysis of variance SEM = standard error of the mean SNpc = substantia nigra pars compacta SPN = spiny projection neuron TH = tyrosine hydroxylase V = Volts WT = wild-type xi  Acknowledgements I want to thank the entirety of the Centre for Applied Neurogenetics at UBC for supporting me and making science fun, but especially my supervisor Dr. Austen Milnerwood and peers Chelsie Kadgien and Naila Kuhlmann who kept me going with questions, edits, treats, and jokes throughout my time here.  To my new friends in Vancouver and my continuing friends from before, thank you for being so lovely, encouraging, and full of life through this process and always. Special thanks are owed to my family for their constant support and love throughout my life; I wouldn’t be here without you. 1  Chapter 1: Introduction  1.1 Parkinson’s disease Parkinson’s disease, first described by James Parkinson in 1817, is the second most prevalent neurodegenerative disease. Typically characterized as a movement disorder due to resting tremor, rigidity, postural instability, and bradykinesia, it is often the visible motor symptoms that lead patients to the clinic for diagnosis. However, these motor symptoms are preceded by non-motor, peripheral and cognitive symptoms (decreased olfaction, impotence, fatigue, sleep disturbances, depression, anxiety, psychosis, and dementia), which are often more debilitating and are not ameliorated by current treatment options (Goldman and Postuma, 2014). Mild cognitive impairment is seen in 20-50% of new patients, while dementia is exhibited in 10% of diagnoses and increasing to 80% within 20 years, demonstrating the severity of PD’s non-motor symptoms (Goldman and Postuma, 2014; Hely et al., 2008; Weintraub and Stern, 2005). Pathologically, PD is confirmed post-mortem by visible degeneration of the dopaminergic neurons of the substantia nigra pars compacta (SNpc) and the presence of Lewy pathology, proteinaceous aggregates containing high levels of alpha-synuclein (aSyn) along with other proteins (Spillantini and Goedert, 2006; Volta et al., 2015). Dopamine (DA) replacement therapy, often in the form of the DA precursor L-dopa, is at present the most effective treatment for motor symptoms; however, it does nothing to halt progression of the disease and often comes with debilitating side effects (e.g. dyskinesia) (Chesselet and Richter, 2011). 1.1.1 Dopamine in Parkinson’s disease The death of nigrostriatal midbrain dopaminergic neurons in the SNpc is a defining characteristic of PD. Nigral cell death, and preceding degeneration of nigrostriatal terminals, results in a loss of 2  dopaminergic input into the striatum (STR) and causes an imbalance in striatal processing and output that is responsible for action selection and movement (Calabresi et al., 2014). Much has been learned from SNpc toxic-lesion models that replicate the end-stage PD cell loss (6-OHDA, MPTP, paraquat, rotenone, etc.) and they have proved valuable in designing therapies for alleviating PD motor symptoms, e.g., L-dopa, dopamine agonists, and deep-brain stimulation (Chesselet et al., 2008). Approximations vary widely on the amount of nigral DA neurons degenerated at the time of motor symptom onset (from 0 to 30%, some variation explained due to staining for requisite DA synthesizing enzyme TH which would disappear prior to DA cell loss), but the most rapid phase of dopaminergic neuron depletion is in the first 4-5 years after clinical diagnosis based on motor symptoms. After this initial ~5y period, the level of cell death stabilizes indicating a need for the development of rapid neuroprotective therapy and the requisite disease biomarkers, prior to symptomatic DA neuronal loss (Kordower et al., 2013). Despite their strengths, animal models mimicking late-stage lesions fail to translate to neuroprotective therapy, given that they cannot inform upon the causes, onset, or progression of PD (Chesselet and Richter, 2011).    1.2 Striatal circuitry Axons of nigral cells in the SNpc project to the striatum (STR); a large subcortical nucleus involved in motor and cognitive action selection. The STR is often referred to as the gateway to the basal ganglia, a group of interconnected subcortical nuclei that regulate a wide range of brain functions (Hikosaka et al., 2000; Mink, 2003; Yin and Knowlton, 2006). As the entry point to the basal ganglia, the STR functions as a hugely integrative structure receiving input from the cortex, thalamus, and midbrain carrying information for action selection, salience, and locomotion 3  (Gerfen and Surmeier, 2011; Kreitzer, 2009; Matsumoto et al., 2001).The STR is comprised mainly of GABAergic spiny-projection neurons (SPNs; 95%), but also aspiny GABAergic and cholinergic interneurons (Calabresi et al., 2014; Dubé et al., 1988). The dorsal STR specifically is implicated in Parkinson’s disease, as the target of DA from the SNpc, and victim of nigral deterioration in late-stage disease. SNpc neurons are spontaneously active at low frequencies, providing a basal DA tone to the STR which can be bi-directionally altered by either phasic bursts or transient pauses of activity (Tritsch and Sabatini, 2012). This two-way function of DA neurons becomes even more dynamic with diverse target cells and various DA receptor subtypes.  1.2.1 Direct and indirect pathways The SPNs integrate incoming glutamatergic information from the cortex and thalamus with dopaminergic input from the SNpc, and form two output pathways, classically the direct and indirect pathways. The SPN projections of the direct pathway lead to the internal segment of the globus pallidus and the substantia nigra pars reticulate (SNpr). Direct pathway GABAergic inhibition of the SNpr, which is also GABAergic, leads to a disinhibition of excitatory glutamatergic thalamic neurons that project to the cortex, resulting in locomotor activation (Calabresi et al., 2014). Indirect pathway SPNs project indirectly to the SNpr via the globus pallidus pars externa (GPe) and subthalamic nucleus. Inhibition of the GABAergic neurons of the GPe, disinhibit the glutamatergic neurons of the subthalamic nucleus. This disinhibition of excitatory subthalamic neurons activates the GABAergic SNpr neurons projecting to the thalamus, ultimately reducing locomotion.  In addition to these divergent pathways, complex recurrent striatal microcircuits are formed by interneurons and collaterals modulating SPN activity (Gerfen and Surmeier, 2011). Tracing studies in rats have also shown overlap in these traditionally distinct pathways; 60% of 4  labeled neurons were found projecting along the direct pathway while having collateral terminal fields in the GPe possibly providing a way for the direct pathway to modulate the indirect pathway (Calabresi et al., 2014; Cazorla et al., 2014). Although these pathways can be teased apart, they typically work in concert with firing increases in both direct and indirect SPNs when animals initiate action (Cui et al., 2013). Thus, while the direct and indirect pathways were previously thought of as separate, parallel systems, recent research has forced reappraisal. It is becoming increasingly accepted that these pathways are in fact, 1) intertwined structurally and functionally, 2) driven by both the intensity of glutamatergic activation, and 3) the amount and precise timing of DA release (Calabresi et al., 2014, 2007).  1.2.2 D1 versus D2 receptor function Classically, direct and indirect SPNs are distinguished by the expression of either D1 or D2 dopamine receptors, respectively. Although enriched on the cell bodies and dendrites of SPNs, D1 and D2 DA receptors are also found on a wide variety of striatal interneurons, as well as presynaptically on nerve terminals and axonal varicosities of both glutamatergic and dopaminergic striatal inputs (Tritsch and Sabatini, 2012). These two distinct DA receptor families are coupled to separate intracellular G-proteins with opposing effects on adenylyl cyclase (AC) signaling pathways, leading to contrasting downstream signals. The activation of AC by D1 stimulation leads to the production of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) activation, while D2 stimulation inhibits AC activation and ensuing effects (Tritsch and Sabatini, 2012). The activation of PKA leads to increased excitability potentially promoting long-term potentiation at synapses due to the phosphorylation and regulation of many cellular substrates including voltage-gated K+, Na+ and Ca2+ channels, ionotropic glutamate and GABA receptors and transcription factors (Tritsch and Sabatini, 2012). 5  PKA activation also targets DA and cAMP-regulated phosphoprotein 32 (DARPP-32) which integrates signals from multiple neurotransmitters to bidirectionally modulate PKA activity, either blocking an inhibitor of PKA after D1 activation or conversely upon D2 stimulation becoming a potent inhibitor of the PKA pathway (Tritsch and Sabatini, 2012). Conversely, activation of D2 receptors decreases excitability and promotes long-term depression at excitatory synapses by inhibiting AC and therefore limiting PKA activation while activating inward-rectifier K+ channels and decreasing Ca2+ currents by the activation of phospholipase C (Tritsch and Sabatini, 2012).  Both D1 and D2 receptors can also alter membrane trafficking of Ca2+ channels, NMDA receptors, and GABAA receptors, leading to further modification of the membrane surface.  D2 receptors are of particular interest in PD due to their high expression in the STR where they are found on the indirect pathway SPNs, as autoreceptors on DA neurons, and at corticostriatal and nigrostriatal terminals negatively regulating synthesis and release. The affinity of D2 receptors for DA is reported to be 10- to 100-fold stronger than D1 receptors, indicating that low levels of DA would favor D2 activation, further decreasing DA output on the aforementioned locations (Tritsch and Sabatini, 2012). Using in vivo optogenetics, bidirectional effects on locomotion have been seen with excitation of either D1 or D2 expressing SPNs with the alleviation of freezing, bradykinesia, and locomotor initiation deficits in a mouse model of PD upon stimulation of the direct pathway SPNs (Kravitz et al., 2010). The majority of DA receptors are on non-dopamine neurons, however DA receptors in the form of autoreceptors are present on DA neurons themselves to provide feedback inhibition that controls cell firing and the synthesis, release, and uptake of DA (Ford, 2014). These D2-subtype autoreceptors are located on the soma and dendrites of SNpc neurons as well as on their axon terminals in projection areas 6  (Beaulieu and Gainetdinov, 2011; Ford, 2014). Autoreceptors are inherent feedback regulators, both directly though the activation of K+ conductance and indirectly by controlling downstream expression of tyrosine hydroxylase (TH, enzyme in DA metabolism) and the dopamine transporter (DAT, main synaptic reuptake mechanism for DA), ultimately leading to a decrease in excitability of DA neurons and the synthesis/release of DA (Ford, 2014). The role of D2 autoreceptors in regulating the dopamine system has been examined in mice with a conditional knock-out of D2 receptors only from DA neurons (autoreceptor-null) (Anzalone et al., 2012; Bello et al., 2011; Ford, 2014). These mice have normal levels of D2 receptors on other neurons (heteroreceptors) but lack D2-autoreceptor mediated inhibition of DA release from DA terminals and hyperpolarizations of the cell body (Anzalone et al., 2012; Bello et al., 2011). Presynaptic D2 receptor agonism leads to locomotor inhibition, while studies antagonizing presynaptic D2 autoreceptors can remove this feedback leading to an increase of synthesis and release of DA (Shi et al., 2000). The overlapping combination of the direct and indirect pathways with the dichotomous relationship of D1 and D2 receptors and associated G-protein signaling pathways creates a highly dynamic and fine-tuned system. 1.2.3 Circuitry gone awry in Parkinson’s disease  Estimates put the length of human nigral DA cell axons at 4m long, forming around 2.5 million striatal synapses. Conversely, very similar DA cells in the ventral tegmental area, which are spared in PD, are estimated to form only around 30,000 synapses (Bolam and Pissadaki, 2012). The highly arborized nature of the nigral axons may lead to increased vulnerability due to a massive metabolic demand (Bolam and Pissadaki, 2012); furthermore, in this light it is clear that the removal of an individual nigrostriatal DA neuron projection will affect vast numbers of target neurons (Tritsch and Sabatini, 2012). When DA input to the STR is reduced in PD and these 7  nigrostriatal neurons degenerate, the GABAergic outputs to the nigra or globus pallidus are affected differently and consequently the originally balanced gateway is disturbed, leading to an imbalance and hyperexcitability of the pathways controlling movement (Calabresi et al., 2014). This can be ameliorated temporarily by adding dopamine precursor L-DOPA; however, as this does nothing to halt the disease progression, ultimately there is nothing left with which to interact.   1.3 Genetic Parkinson’s disease Recent discoveries indicate that gene mutations linked to familial parkinsonism currently account for 2-5% of all PD and up to 41% in certain populations (Lesage et al., 2005); furthermore, mutations linked to late onset PD are increasingly being reported (Vilariño-Güell et al., 2014, 2011) and the proteins in which they reside are functionally related (Volta et al., 2015). The holistic understanding that is beginning to emerge from protein pathways implicated in several familial forms of PD may also be relevant to idiopathic PD. Early structural and neurochemical alterations have been reported in asymptomatic PD mutation carriers as well as mouse models allowing for the assessment of whether these changes contribute to the subsequent loss of terminals and neurons seen in the late disease stage (Beccano-Kelly et al., 2014; Helmich et al., 2015; Nandhagopal et al., 2008; Sossi et al., 2010). Characteristic biochemical and pathological features of PD (loss of striatal dopamine and nigrostriatal neurons) have been replicated at late stages in some genetic mouse models allowing for the study of precursory disease mechanisms at earlier time points (Chesselet and Richter, 2011). Longitudinal investigation is necessary to provide biomarkers for disease progression and neuroprotective treatment strategies. Many knock-out (KO) and over-expression (OE) animal 8  models have been developed to aid in this query. KO models, entirely rid of the gene in question, can help to answer some basic questions about loss of function. OE animal models with increased expression of the gene in question are helpful for interpreting gain of function, but are limited by non-physiological transgene expression patterns and levels with varying levels of enhanced expression. Alternatively, subtle knock-in (KI) mice expressing the disease causing mutation are genetically faithful and physiologically relevant, and arguably best suited for investigating pathophysiological processes and designing interventions to delay or prevent disease onset and progression. 1.3.1 Leucine-rich repeat kinase 2 Mutations of the large multidomain enzyme leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of PD and produce a form of parkinsonism that is clinically indistinguishable from idiopathic PD (Paisán-Ruı́z et al., 2004; Zimprich et al., 2004). LRRK2 disease is characterized by an asymmetrical tremor presentation with a good response to L-dopa and a slow progression with a lower risk of cognitive decline than idiopathic PD though there are currently few complete longitudinal studies (Alcalay et al., 2013; Paisán-Ruı́z et al., 2013). Postmortem analysis of mutation carriers revealed a fairly heterogeneous LRRK2-associated neuropathology, mainly characterized by loss of DA neurons and the presence of Lewy pathology, but with other variations also found, e.g. taupathology (Paisán-Ruı́z et al., 2013).  Though the exact function of LRRK2 has yet to be elucidated, evidence points towards its role in neuronal homeostasis, vesicular dynamics, and neurotransmitter release. LRRK2 has been shown to bind multiple synaptic vesicle cycle regulatory proteins and be involved in synaptic vesicle recycling and distribution (Piccoli et al., 2011). Recently, a subset of Rab GTPases have been shown to be key LRRK2 substrates that upon overactive kinase activity seen 9  in some mutations leads to increased phosphorylation of Rab proteins which are involved in transporting molecules and cargoes around cells, potentially deregulating intracellular trafficking between cytosol and target membrane compartments by  increasing Rab accumulation in the membrane (Steger et al., 2016). Previous work in our lab using neuronal cultures from LRRK2 mice showed normal synaptic release and synapse number with only discrete reductions in glutamatergic activity and synaptic protein levels in KO lines, contrasted with an increase in synapse density but not event frequency in OE cultures (Beccano-Kelly et al., 2014). Phenotypically KO mice appear relatively normal when assessed for motor function, exploratory behavior, motivation, and anxiety levels, as well as being cognitively comparable to NT littermates on spatial, recognition, and both short- and long-term memory tasks; together the data indicate that PD-causing LRRK2 mutations are unlikely to produce disease due to a loss of function (Beccano-Kelly et al., 2015). Contrastingly, LRRK2 OE mice have altered dopamine levels and short-term synaptic plasticity, behavioral hypoactivity, and impaired recognition memory (Beccano-Kelly et al., 2015, 2014). LRRK2 has also been implicated in the regulation of synaptic vesicle release along with aSyn where they appear to interact and possibly overlap in function (Carballo-Carbajal et al., 2010; Lin et al., 2009). It is apparent that LRRK2 is a complex protein with several PD-causing mutations that increase kinase activity (Jaleel et al., 2007; West et al., 2005) and is linked to multiple other proteins in the endo- and exocytotic pathways required for synaptic vesicle and receptor recycling (Trinh and Farrer, 2013; Volta et al., 2015). 1.3.2 Dopamine & LRRK2 The loss of SNpc DA neurons and STR DA terminals is a feature of both LRRK2 and idiopathic PD (Lin and Farrer, 2014; Paisán-Ruı́z et al., 2013). Asymptomatic human LRRK2 mutation carriers show early dopaminergic alterations that progress over time; starting as increases in DA 10  turnover (prior to decreases as the disease progresses). This indicates dysfunction in presymptomatic PD unrelated to simplistic loss of DA (Nandhagopal et al., 2008; Sossi et al., 2010). Interestingly, this biphasic alteration to DA is also observed in an aSyn PD mouse model (Chesselet and Richter, 2011; Lam et al., 2011). In a LRRK2 R1441C mutant knock-in mouse model the mutation resulted in behavioral hypoactivity and insensitivity of nigral cell firing patterns to D2 receptor activation via quinpirole, suggesting that this LRRK2 mutation impairs D2 receptor function (Tong et al., 2009). Previous work in our lab using LRRK2 BAC mice over-expressing either the human wild-type (WT) or G2019S mutation has also shown increased D2 receptor activity at young time points with D2-dependent alterations to striatal short-term plasticity, along with behavioral hypoactivity and recognition memory impairments associated with decreased striatal DA tone and abnormal signal integration (Beccano-Kelly et al., 2014; Melrose et al., 2010). It seems LRRK2 acts at the nexus of dopamine and glutamate signaling in the striatum, affecting many things, from DA levels and signal transduction to presynaptic glutamate release via D2R-dependent synaptic plasticity. 1.3.3 LRRK2 G2019S The most common LRRK2 mutation leading to PD is the G2019S point mutation, contributing to 2-6% of familial late-onset autosomal dominant PD with lifetime risk as high as 80% in the most vulnerable populations (Brice, 2005; Lin and Farrer, 2014). Non-manifesting carriers of the G2019S LRRK2 mutation demonstrated cognitive alterations with impaired executive function (Thaler et al., 2012) but intact working memory (Thaler et al., 2015) as would be predicted from striatal dysfunction. The glycine residue altered in this mutation is conserved throughout all eukaryotic LRRK2 homologs indicating its potential importance (Nichols et al., 2005). This single amino acid substitution in the kinase domain of LRRK2 leads to a two- to threefold 11  increase in kinase activity, recently shown to target the Rab GTPase family which is involved in transporting various cargoes around the cell (Paisán-Ruı́z et al., 2013; Steger et al., 2016). In mouse cortical neuronal cultures, one copy of mutant G2019S has a more pronounced effect than that of an OE model harboring an almost 3-fold increase in LRRK2 protein, observed as elevated synaptic release (by way of increased glutamate release without a change to synapse density) indicating the stark influence of the increase in kinase activity caused by this mutation (Beccano-Kelly et al., 2014). With the knowledge of genetic causes of Parkinson’s disease, and the tools at hand, we are able to investigate the role of this specific LRRK2 G2019S mutation by examining genetically faithful, subtle knock-in mouse models.   1.4 Rationale for present experiments Parkinson’s disease is the most prevalent neurodegenerative disorder after Alzheimer’s disease, with limited therapies that do not halt disease progression, leading to debilitating motor and cognitive decline. With recent genetic advances we now have the opportunity to study DA in the brains of human mutation carriers prior to symptom onset and diagnosis of PD. We are also able to mimic physiologically relevant genetic mutations in mouse models that we can examine longitudinally.  Previously mentioned BAC OE mouse models have fundamental caveats caused by random (BAC) gene insertion, exogenous control of expression patterns and parallel endogenous murine homologue expression. Therefore, examination of DA transmission in subtle, physiologically relevant G2019S knock-in mouse models with endogenous regulation of gene expression patterns / levels will help lead to a better understanding of LRRK2 physiology and the pathophysiology of LRRK2 mutations.  12  To this point, partial characterization of these LRRK2 G2019S KI (GKI) mice has previously been conducted. Work in primary mouse cortical cultures comparing LRRK2 OE, KO, and GKI, revealed LRRK2 regulates glutamatergic activity, evidenced by increased synaptic release in GKI cultures, in the absence of any change to synapse density (Beccano-Kelly et al., 2014). A longitudinal motor study found hyperkinetic behavior that was resistant to typical age-related decline in GKI mice, which was reversed with kinase inhibitors and altogether absent in kinase-dead LRRK2 mutation D1994S, pointing to the increased kinase activity with G2019S mutation as a cause (Longo et al., 2014). Unpublished work from our lab has also shown that GKI mice are hyperactive at a young age, but this normalizes to similar levels as their WT littermates with age. In GKI mice at 12 months, a decrease in striatal basal and drug-evoked extracellular DA are seen in addition to mitochondrial abnormalities at advanced ages (Yue et al., 2015). Overall, basal DA levels have been assessed by microdialysis at 6 and 12 months where they were normal and reduced, respectively. Analysis of motor behavior, assessed by 2 different laboratories, yielded inconsistent results concerning locomotion. However, parallel comparison of basal DA levels and rigorous behavioral testing over time has not been conducted. Furthermore, microdialysis assays the extracellular content of dopamine over a 15-minute period, and many alterations to rapid physiological DA release and reuptake kinetics would not be detected in this way. Herein, my objective was to confirm previous results and extend our understanding of the function of the nigrostriatal dopamine system in GKI mice. This was conducted longitudinally to determine the effects of this mutation upon the DA system with respect to behavioral performance and dysfunction over time. Basal DA tone (and its metabolites DOPAC and HVA, along with 5-HT, 5-HIAA, glutamate, and GABA) as well as rapid release and reuptake kinetics, 13  were assessed in the dorsolateral striatum by microdialysis and fast-scan cyclic voltammetry, respectively. Pharmacological manipulations were also employed to tease out subtle alterations and how they might relate to behavioral changes. Understanding the effect this mutation has on the physiology of dopaminergic neurons and whole animal behavior will lead to a better understanding of PD pathophysiology and mechanistically targeted therapeutic approaches. 14  Chapter 2: Methods and materials  2.1 Subjects All mice were bred in house at the UBC Centre for Disease Modeling. Mice initially created during Dr. Matthew Farrer’s time at Mayo Clinic Jacksonville were rederived in house onto the same C57BL/6 background from Jackson Laboratories (Maine, USA). The overexpressing (OE) mice overexpress the human WT LRRK2 gene about 3-20 fold using vector pBACe3.6.  GKI mice (used for the majority of this thesis) were made with a targeted vector for mLRRK2 exon 41 G2019S on a C57BL6/J background, and express LRRK2 with species appropriate region, regulation, and anatomical distribution, eliminating potential confounds from non-specific effects of protein overexpression that may present in other models (such as artificial transgenic insertion, overexpression, and the potentially compensatory contribution of endogenous murine LRRK2 function). Animals were kept in one of two temperature controlled (19-22° C) units on The University of British Columbia’s campus on a twelve-hour regular light/dark schedule (7am-7pm light) in clear cages with ad libitum access to chow and water. At weening, animals were group housed with same-sex littermate groups of up to 5, with nesting material, a nesting dome, and a tube for enrichment. After surgery, animals were housed individually in larger cages so as to not cause interference with the microdialysis probe from cage-mates or low-hanging wire rack. Mice were classified as either young (2-3 months) or old (12+ months) in the following experiments and only males were used.  15  2.2 Electrophysiology Previous work from our lab indicated short-term plasticity changes in OE mice, seen as reduced facilitation in response to a paired-pulse stimulation (Beccano-Kelly et al., 2015). Here, basal transmission, short- and long-term striatal plasticity were evaluated by field excitatory postsynaptic potential recordings in acute slices from young adult LRRK2 OE mice. 2.2.1 Slicing  Following rapid decapitation, the brain was isolated and submerged in cold modified aCSF (cutting solution: aCSF with MgCl2 brought to 5mM) for one minute to cool, then bisected and glued to the cutting plate against an agar block. 300μm coronal sections were taken on a vibratome (Leica VT1200 S, Concord, Ontario; Feather 2-edge razor blades, Ted Pella, Redding, CA, USA) and sections were transferred to a holding chamber in aCSF (in mM: 130 NaCl, 10 glucose, 26 NaHCO3, 3 KCl, 1 MgCl2H2O, 1.25 NaH2PO4 (monobasic monohydrate), 2 CaCl2; pH 7.2-7.4, mOsm 290-310, in MilliQ water) perfused with carbogen (95% oxygen, 5% carbon dioxide, Praxair) at room temperature for at least one hour prior to experiments. A post-mortem ear notch was also collected for verification of genotype. 2.2.2 Equipment After a minimum of one hour for slices to recover, a single section was transferred to electrophysiological set up consisting of a Kinetic Systems Vibraplane Anti-Vibration Table with Faraday Cage (MA, USA) containing a microscope (Olympus BX51WI Research Fixed Stage Microscope) with a temperature controlled (23°C-25°C, Warner Instrument Corporation, CT, USA) continuous flow bath chamber with a pump (1-2mL/min, Argos Evac, IL, USA). A stimulating bipolar electrode (made in house, nickel80/chromium20 wire, Advent Research Materials Ltd, Oxford, England) was controlled by ClampEx software (pClamp, Molecular 16  Devices, CA, USA) through an optical stimulus isolator (A365, World Precision Instruments, FL, USA) and placed (MX130R, Siskiyou, OR, USA) into the dorsal lateral striatum. The field recording glass microelectrode (Ag/AgCl, Molecular Devices, in aCSF; in borosilicate thin wall pipette, OD = 1.50mm, ID = 1.17mm, Warner Instruments; pulled on Model P-1000, Sutter Instruments, CA, USA: tip 2-3µm, taper 4-5mm, R 1-4Meg) was placed (MP-285 Micromanipulator with Controller, Sutter) along the corticostriatal axonal tracts 200-300µm ventral to the stimulating electrode. Stimulation parameters were executed through ClampEx software, recordings were digitized and acquired at 10kHz (Digidata 1440A and Multiclamp 700B amplifier, Molecular Devices). 2.2.3 Sampling parameters Field excitatory postsynaptic potentials (fEPSPs) were first evoked with increasing stimulus intensity from 100μA to 700μA to generate an Input/Output plot using a rectangular stimulus of 100μs at 0.033Hz (30sec inter-pulse interval (IPI)). For the rest of the experiments, stimulation intensity was set at 60-80% maximum response, for paired-pulse and LTD paradigms. Paired-pulse events were measured by two stimulus events with an IPI of 20ms Δ 40ms. To assay plasticity a standard LTD paradigm was used: a minimum 10 min stable baseline with 0.033Hz stimulation, followed by high-frequency stimulation (4x 100 pulses at 100Hz, with an intertrain interval of 10s at time zero), followed by one hour of 0.033Hz stimulation.  2.2.4 Data Analysis Data was analyzed in Clampfit 10.4 (pClamp, Molecular Devices). The last five minutes of the post-induction period were compared to the last 5 minutes of the ten minute baseline to investigate LTD. N2 (dendritic / population spike peak amplitude) in relation to N1 (volley amplitude) was analyzed to assess plasticity and ensure no increase or loss of axon recruitment 17  occurred over the duration of the experiment.  Data are presented as mean ± SEM where n is the number of slices with the total animal count in parenthesis.  2.3 Basal dopamine tone, by in vivo microdialysis in the dorsolateral striatum Microdialysis involves the use of a probe with a semi-permeable, size-selective membrane perfused with aCSF to collect by osmosis and concentration gradient, neurotransmitters and metabolites in the extracellular space of the area of interest. Here, the probe was placed into the dorsolateral striatum to examine the nigrostriatal function of our GKI mice by investigation of DA and its metabolites (DOPAC, HVA), 5-HT, 5-HIAA. This allows for the quantification of tonic neurotransmitter levels at various age points in awake, freely moving mice. This technique also permits pharmacological manipulation by reverse microdialysis to compare basal tone to availability. Samples collected via this method are analyzed with high-performance liquid chromatography (HPLC) and compared to standards of known concentration to extrapolate the amount of neurotransmitter present in the area of interest.  Due to the emerging hypothesis that overactive glutamatergic input may eventually lead to toxic hyperexcitation and its known role in striatal plasticity (Calabresi et al., 2014; Morales et al., 2013), GABA and glutamate levels were also analyzed. As D2 autoreceptors modulate corticostriatal terminals, and previous work implicates D2-autoreceptor alterations in OE mice, the D2 agonist quinpirole was applied (Akopian and Walsh, 2007; Bamford et al., 2004; Beccano-Kelly et al., 2015; Calabresi et al., 2014). 2.3.1 Stereotaxic surgery Mice were weighed (Salter, Kent, UK) prior to anesthetization by 5% vaporized isoflurane mixed with oxygen (Pharmaceutical Partners of Canada, ON, Canada; medical grade O2, Praxair) 18  in an induction chamber. Once surgical plane was attained, the mouse was transferred to a heating pad (Life brand, available at pharmacy) on the stereotax and affixed with ear bars (David Kopf instruments, Tujunga, CA, USA) for the duration of the surgery mice were held under 1.5-2.5% isoflurane to maintain an appropriate level of anesthesia. Ophthalmic ointment was applied to the eyes (Refresh Lacri-Lube, ON, Canada), the surgical site was shaved (Wahl Clipper Corp, IL, USA) and sterilized (70% EtOH, iodine), injection of Mepivacaine (2%, Hospira, Inc., QC, Canada) was made along the midline extending slightly past bregma to lambda prior to incision (all surgical tools from Fine Science Tools, BC, Canada). After identification of bregma, a hole was drilled (51449, Stoelting, IL, USA) through the skull (StereoDrive software, Neurostar, Tübingen, Germany; +0.5mm AP, -2.1mm ML from bregma, Paxinos and Franklin, 2004) for probe insertion and another contralaterally and anterior to bregma was started for the anchoring screw (CMA 743 1021, CMA Microdialysis AB, Harvard Apparatus, MA, USA) which was then inserted. A probe (1mm dialyzing membrane, 240µM outer diameter, 6000 Dalton molecular weight cutoff; CMA 7, Harvard Apparatus, MA, USA), flushed with Ringer’s solution (in mM: 147 NaCl, 3 KCl, 1.2 MgCl26H2O, 1.2 CaCl2) at a rate of 1.5µl/min for the duration of the surgery by using a gas-tight syringe (500µL, Hamilton Company, NV, USA) and a syringe pump (4004, CMA, Harvard Apparatus) was slowly lowered (-2.8mm DV from dura) into the dorsal lateral striatum and affixed with a layer of Krazy glue (Elmer’s Products Canada, Corp., ON, Canada) followed by a head cap of dental cement (glass ionomer cement, GC FujiCEM 2, GC America Inc, IL, USA). Saline (50µL, 0.9% Sodium Chloride, Hospira, Inc.) and Metacam (1mL/kg, 0.5%, Boehringer Ingelheim, ON, Canada) injections were given and the animal was allowed to recover on a heating pad before solo placement into a large home cage until sampling the following day.  19  2.3.2 Sample collection and analysis by high-performance liquid chromatography The day following surgery, animals were weighed and checked for overall wellness prior to sampling. Mice were placed into the clear sampling bowls (CMA 120, CMA, Harvard Apparatus, MA, USA) with free access to food and water and attached to a constant perfusion of a modified Ringer’s solution (identical pump and Ringer’s solution as surgery) at 1.5 µL/min. Samples were collected every 15 minutes into a collection tube (250µL, Fisher) with 2µL [10 mM] acetic acid (HPLC grade, Sigma) starting 2 hours after the onset of perfusion.  Due to the length of the lines, thirty minutes before the start of collection, the solution was switched to one of 50µM quinpirole (Tocris) to be collected after four baseline samples. Ringer’s solution was put back on after the first baseline sample was collected to facilitate four samples post-drug infusion. Samples were then frozen at -80ºC until analysis by high-performance liquid chromatography with electrochemical detection. We utilized the ALEXYS LC-ECD Neurotransmitter base system in combination with the SCC FLEX I, UHPLC and Monoamines SSC kit, and GABA/Glu SSC kit with LC Step-gradient upgrade kit. The system consisted of a pump, an autosampler kept at 4°C, a LINK noise reducer, and detector with electrochemical flowcell controlled by Clarity (pump: LC 110, autosampler: AS 110, detector: Decade II, flowcell: VT-03, Clarity: software version 6.1; Antec, Leiden, The Netherlands). DOPAC, 5-HIAA, DA, HVA, and 5-HT (RT: 2.57 ± 0.2 min, 3.45 ± 0.2 min,  3.91 ± 0.2 min, 5.17 ± 0.2 min, 7.90 ± 0.2 min, respectively) were measured with a column at 40°C (1mm ID, 10cm length, Acuity UPLC® BEH C18, 1.7 µm, Waters, Ireland), cell potential set at 0.8V vs. salt bridge (mobile phase, in mM: 100 phosphoric acid, 100 citric acid, 0.1 EDTA, pH 3.0, 600mg/L OSA, 8% v/v acetonitrile) was pumped at a rate of 50µl/min. Calibration standards were in concentrations of 1, 10, 25, 50, 100 nM for each metabolite as well 20  as mixtures of all analytes at each concentration. Volume of samples and standards run was 5µL in all cases. Glutamate and GABA (RT: 3.40 ± 0.4 min, 8.30 ± 0.4 min, respectively) were measured at 200µl/min, (mobile phase in mM: 50 phosphoric acid, 50 citric acid, 0.1 EDTA, pH 3.50, 2% v/v acetonitrile), with a column at 40°C (1mm ID, 2cm length, Acquity UPLC® HSS T3, 1.8µm, Waters, Ireland), cell potential set at 0.85V vs salt bridge, and a post-separation step gradient applied between 10 and 12 min to elute GABA (same mobile phase composition except 50% v/v acetonitrile). 5µL of sample was subjected to in-needle derivatization with 1µL OPA reagent (0.025g OPA, 250µL MeOH, 250µL 1M sulphite solution (0.126 sodium sulphite, 1mL water), 4.5mL 0.1M Borate buffer (0.618g boric acid, pH 10.4 with 50% NaOH, 100mL total)). Calibration standards were in concentrations of (GABA nM: glutamate µM) 50:0.5, 100:1, 150:1.5, 200:2, 250:2.5. To make standards, respective powder from Tocris was dissolved in MilliQ water to make 10mM standard, which was then diluted to 1mM stock solution with percholoric acid (0.1M; HPLC grade, Sigma), then diluted down in acidified Ringer’s solution to keep consistent with acid having been added to our samples to avoid DA degradation.  2.3.3 Data Analysis Chromatographs were analyzed with Clarity software (Clarity 6.1, Antec, Netherlands) to find the concentration of DOPAC, 5-HIAA, DA HVA, 5-HT, GABA & Glu in nM against a calibration made from the appropriate standards, as well as the relative variations in percent from baseline samples. One-way analysis of variance (1-way ANOVA) was used to analyze neurotransmitter levels and ratios, while 2-way ANOVA was used to look at drug effect over time. Uncorrected Fisher’s LSD was used for multiple comparisons and t-tests for direct pairwise comparisons. Probe placement was verified in the majority of animals by sectioning (30µm) and 21  staining for Iba1 (activated microglial marker) with GFAP (astroglial stain) and DAPI (denotes nuclei), while a subset of these were stained for TH (dopaminergic neuron marker as tyrosine hydroxylase/TH is an enzyme needed to make DA), DAT (main DA transporter), and DAPI (nucleus) to quantify TH and DAT levels.  2.4 Dopamine release and reuptake, by fast-scan cyclic voltammetry in acute striatal slices Fast-scan cyclic voltammetry (FSCV) allows for rapid, millisecond timescale analysis of dopamine release and reuptake in response to electrical stimulation. Due to the fact that DA can be oxidized at low voltage, selective electrochemical detection can be used based on a voltage-dependent oxidation and reduction processes (John and Jones, 2007). A carbon-fiber microelectrode was placed in the dorsolateral striatum the entry point for dopaminergic innervation from the SNpc. The examination of DA release and reuptake in acute slices also allows for the application of pharmacological compounds to isolate and probe differences in release patterns and reuptake kinetics.  2.4.1 Slicing Slice preparation for voltammetry was identical to that of electrophysiology. 2.4.2 Equipment The same electrophysiology rig described in Chapter 2.2.2 was used for the following experiments. Flow rate was from 1-2mL/min. The recording of voltammetric responses was done with the Invilog In Vivo Voltammetry Full Setup (Invilog Research Ltd., Kuopio, Finland) containing the potentiostat, stimulator with isolation unit and head stage, USB X Series Multifunction DAQ (National Instruments, Austin, TX, USA), and included acquisition and 22  analysis software components. The voltammetry carbon fiber electrodes (diameter: 32µm, length: 30µm, sensitivity: 21-40nA/µM) were prefabricated by Invilog and controlled on a motorized micromanipulator (PCS-6000, EXFO, Mississauga, ON, Canada) and used against the included Ag/AgCl recording electrode. 2.4.3 Sampling parameters The stimulation paradigm delivered through Clampex consisted of an Input/Output, 5 single baseline pulses, a paired-pulse, and a train, followed by single pulses during drug wash-in and a repeat of the baseline, paired-pulse, and train in drug. All stimulations were using a 150µs rectangular stimulation with a two-minute IPI. The Input/Output paradigm consisted of increasingly intense single pulse stimulation events to find ~70% of the maximum response, starting at 50µA and increasing until plateau (100µA, 200µA, 300µA, etc) which was then set as stimulation intensity for the rest of the experiment. Five single pulses were then given to achieve a baseline, followed by a paired-pulse (4sec IPI). During drug wash in, single pulse stimulations were continued every two minutes for ten minutes or drug effect was seen. A repeat of the 5 single simulations, paired-pulse, and train were then recorded in the drug condition. For FSCV recording, a triangular waveform from -400mV to 1200mV to -400mV was passed through the carbon-fiber microelectrode over 10ms in a 100ms period (10Hz); markers were set at 3.2 and 3.5ms to catch the oxidation of DA between 700 and 800mV.  At the end of each sampling day, a three point calibration of the carbon fiber electrode was done (final concentrations 0.µM, 0.5µM, 1.0µM DA in aCSF). Field recordings were taken as well, secondary to FSCV recordings, with recording electrode placed further from stimulation than carbon-fiber microelectrode for FSCV recording. 23  2.4.4 Pharmacology Quinpirole ((-)-Quinpirole hydrochloride, Tocris Biosciences, Bristol, UK), a presynaptic D2R agonist, was used in some experiments in a 50nM working solution in aCSF. GBR-12909 (1-[2-[Bis-(4-fluorophenyl)methoxy]-ethyl]-4-[3-phenylpropyl]piperazine dihydrochloride; Tocris Biosciences), a DAT inhibitor preventing DA reuptake into the pre-synapse (also may simultaneously inhibit the release of DA), was used where noted in a 1µM working solution in aCSF. 2.4.5 Data Analysis Extracellular dopamine concentration versus time is determined from DA oxidation current in background subtracted voltammetric current collected from Invilog Analysis program. This was then processed in Clampfit (10.4, pClamp, Molecular Devices) to find peak amplitude and decay tau. Basal fatigue amplitudes (Fig 5C & 6C) were normalized to P1, while quinpirole fatigue amplitudes (Fig 5F & 6F) were normalized to 1st and 2nd space-trial peaks (prior to drug wash in) to account for any genotypic effects of previous baseline and paired-pulse stimulation. Data are presented as mean ± SEM where n is the number of slices with the total animal count in parenthesis.  2.5 Statistics Once data was analyzed with the appropriate software, numbers were imported and statistics, ANOVA and Uncorrected Fisher’s LSD or appropriate post-hoc tests as noted were run in GraphPad Prism, with alpha set at p=0.05 (version 6.01 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com). 24  Chapter 3: Results  3.1 Field electrophysiology: ex vivo analysis of extracellular striatal glutamate plasticity demonstrates intact LTD in mice overexpressing hLRRK2 Long-term depression (LTD) is arguably the best characterized form of plasticity at striatal SPN glutamatergic synapses (Gerfen and Surmeier, 2011). Using high-frequency stimulation of striatal inputs in acute slices, glutamatergic synaptic transmission can be depressed (Lovinger et al., 1993). By stimulating (and recording) in the dorsolateral STR, the milieu consisting of corticostriatal and thalamostriatal glutamatergic afferents, SPNs, interneurons, and dopaminergic afferents can be examined for effects on glutamatergic synaptic plasticity. Dopamine has been known to play a modulatory role in this form of plasticity, as DA receptors are found on corticostriatal terminals where D2 receptors decrease excitatory post synaptic currents and paired-pulse plasticity via the retrograde endocannabinoid CB1 receptor to decrease corticostriatal transmission (Akopian and Walsh, 2007; Yin and Lovinger, 2006). Spike-timing-dependent plasticity experiments have uncovered that release of DA and the specific timing of that release can have varying effects on synaptic plasticity (Yagishita et al., 2014). Various animal models of PD have shown altered striatal synaptic connectivity and plasticity including in dopamine-depletion models (Dauer and Przedborski, 2003; Ellens and Leventhal, 2013) as well as prior to dopaminergic loss in alpha-synuclein over-expressers (Wu et al., 2010) and mutants (Kurz et al., 2010). Previous, work from our lab indicated a role of D2 receptors on short-term plasticity changes in OE mice, seen as reduced facilitation in response to a paired-pulse stimulation by whole cell-patch clamp that was reversed by presynaptic D2 receptor antagonism (Beccano-Kelly et al., 2015). Here, synaptic function was evaluated using field excitatory 25  postsynaptic potential recordings in acute slices from both young and old LRRK2 OE mice to assess the effect of increased LRRK2 expression on corticostriatal plasticity.  The relationship between the stimulus input and the response output can inform about synaptic connectivity, release efficacy, and postsynaptic responsiveness (Akopian and Walsh, 2007; Platt et al., 2012; Wu et al., 2010). Locally applied electrical stimulation and recording in the dorsolateral striatum revealed no differences between NT and OE mice in fEPSP compound dendritic/population spike (N2) peak amplitude when normalized to fiber volley (N1) amplitude, with increasing stimulation (Input/Output paradigm, Fig 1A: 2-way RM ANOVA, Interaction p=0.6413), suggesting that basic release and responsiveness of striatal glutamate transmission is grossly normal. Paired-pulse responses were conducted as the ratio of two temporally close stimuli (P2/P1) can inform on presynaptic release probability. The increased (or decreased) amplitude of the second pulse (P2) relative to the first (P1) can indicate facilitation (or depression) of presynaptic plasticity due to genetic mutation. Synapses with increased facilitation have an initially low probability of release (Pr), as leftover Ca2+ in the terminal from a first pulse (that was subthreshold for a release event) will increase the likelihood of a release event at the second pulse. There were no significant differences between OE or NT littermates for PPR (20ms-300ms IPI, ∆40) indicating no significant alterations in Pr due to LRRK2 overexpression though there were some suggested subtle trends towards an increase in OE (Fig 1B: 2-way RM ANOVA, Interaction p=0.8895). Although basal transmission was unaltered, during high frequency stimulation (HFS) the augmentation of fEPSPs (evaluated as the percent change in secondary, tertiary, and quaternary peak amplitude when normalized to P1), was reduced in OE (Fig 1C: 2-way RM-ANOVA, Genotype F1,29=4.466, p=0.0433) potentially indicative of less efficient rapid-rerelease. This fatigue during HFS did not seem to have an effect on LTD under 26  the current paradigms; as there were no differences between genotypes in the degree of LTD despite a trend to increased depression in OE animals, exemplified by a sustained 5% reduction in OE after one hour (Fig 1D: 2-way RM ANOVA Genotype p=0.37). Comparison of the last five minutes of LTD recording revealed no significant difference in the magnitude of synaptic depression between genotypes (Fig 1E: t-test p=0.2191). As nigral dopamine neurons intermittently fire in phasic burst patterns, increased fatigue in OE animals during HFS could be indicative of nigrostriatal malfunction in LRRK2 OE mice. The timeline for DA signal transduction effects upon autoreceptors and subsequent downstream signaling pathways occurs over a range from several hundred milliseconds up to several seconds (Ford, 2014; Schmitz et al., 2002) which would be the correct range for seeing alterations during HFS but not PPR.   3.2 Microdialysis: in vivo analysis of striatal extracellular basal neurotransmitter tone and effects of D2R agonism are unaltered in LRRK2 G2019S knock-in mice Microdialysis of the dorsolateral striatum can reveal alterations in basal tone of neurotransmitters and their metabolites in awake, freely moving mice and is conducive to pharmacological manipulation by reverse-microdialysis. Basal tone of dopamine and other neurotransmitters (its metabolites DOPAC & HVA, 5-HT and its metabolite 5-HIAA) was assessed due to the known loss of dopaminergic neurons in PD including in LRRK2 mutation carriers and genetic mouse models (Chesselet and Richter, 2011; Kordower et al., 2013; Sossi et al., 2010; Volta et al., 2015). We have previously reported reduced dopamine tone in young and old LRRK2 OE mice (Beccano-Kelly et al., 2015; Melrose et al., 2010) as well as in 12- (but not 6-) month old GKI mice (Yue et al., 2015). Due to the emerging hypothesis that overactive glutamatergic input may eventually lead to toxic hyperexcitation and its known role in striatal plasticity (Calabresi et al., 27  2014; Morales et al., 2013), GABA and glutamate levels were also analyzed. As D2 receptors modulate corticostriatal terminals, and previous work implicates D2-autoreceptor alterations in OE mice, the D2 agonist quinpirole was applied (Akopian and Walsh, 2007; Bamford et al., 2004; Beccano-Kelly et al., 2015; Calabresi et al., 2014). 3.2.1 Dopamine and its metabolites: DA, DOPAC, and HVA In both young and old GKI mice, basal DA tone was similar across all genotypes though there was a strong trend towards a decrease in tone in older animals by 1-way ANOVA (young, Fig 2A: p=0.8065; old, Fig 3A: p=0.1962). Discrepancy between this finding and our previous research that showed a significant decrease in DA tone in old GKI mice (Yue et al., 2015) could potentially be attributed to the fact that previously female mice were used in addition to males, and here males were environmentally enriched, potentially delaying the transition to hypodopaminergia. Ratios of extracellular metabolites relative to DA can indicate alterations to degradation versus reuptake. No difference was seen at either age point in basal tone of the DA metabolites DOPAC and HVA, nor ratios of DA to either metabolite individually or combined (1-way ANOVA: young, Fig 2B-F: DOPAC p=0.4450; HVA p=0.4415; DOPAC/DA p=0.5749; HVA/DA p=0.5515; DOPAC+HVA/DA p=0.5132; old, Fig 3B-F: DOPAC p=0.4837; HVA p=0.5597; DOPAC/DA p=0.8225; HVA/DA p=0.4447; DOPAC+HVA/DA p=0.5435) indicating no impairment of DA turnover in LRRK2 GKI mice. As D2 autoreceptor activation results in feedback inhibition, agonism of these receptors would be expected to decrease subsequent DA release. The D2 agonist quinpirole lead to an unexpected increase in DA release in young WT animals (not the expected decrease in DA tone) and produced no genotype specific alteration in DA release from baseline at either age point 28  (young, Fig 2G: p=0.3400; old, Fig 3G: p=0.4870). At this level (spatially and temporally) D2 receptor activation did not affect DA release indicating it is unsullied by G2019S knock-in. 3.2.2 Serotonin and its metabolite: 5-HT and 5-HIAA Serotonin (5-HT) has been implicated in sleep disturbance and other non-motor symptoms of Parkinson’s disease (Goldman and Postuma, 2014; Weintraub and Stern, 2005). No difference was seen at either age point in basal levels of 5-HT or its metabolite 5-HIAA by 1-way ANOVA (young, Fig 2H-I: 5-HT p=0.0701; 5-HIAA p=0.2525; old, Fig 3H-I: 5-HT p=0.2863; 5-HIAA p=0.6826), nor the ratio of 5-HT to 5-HIAA at either age (1-way ANOVA: young, Fig 2J: p=0.9092; old, Fig 3J: p=0.9585). It seems 5-HT tone and synthesis is unaltered in response to G2019S knock in at both young and old ages in GKI mice. 3.2.3 Glutamate and GABA In both young and old GKI mice, there was no difference in basal glutamate levels, though there was a strong trend towards a decrease in young animals (1-way ANOVA: young, Fig 4A: p=0.0640; old, Fig 4C: p=0.6149). Similarly, the data acquired for GABA revealed no changes between genotypes, however the number of animals was too low to draw a firm conclusion (1-way ANOVA: young, Figure 4B: n too small to run statistics; old, Fig 4D: p=0.9227). Even though some samples were re-run to try and increase data points, various factors (small number of animals, variable freeze/thaws, poor standards) have rendered this information inconclusive as it stands.  29  3.3 Fast-scan cyclic voltammetry: ex vivo analysis of striatal extracellular dopamine release and reuptake in acute brain slices from LRRK2 G2019S knock-in mice Microdialysis enables the investigation of transmitter tone over 15 minute periods; however it does not give any information about the rapid release and reuptake kinetics of the dopamine system. Although no alterations were seen in basal tone, subtle alterations might not be expected to be isolated with such poor temporal resolution. Fast scan cyclic voltammetry (FSCV) allows for investigation of DA in the extracellular space of acute striatal slices on a millisecond timescale.  A voltage that is sufficient to oxidize DA is applied through the carbon fiber microelectrode causing a current flow at the electrode surface indicative of the DA concentration in the vicinity of the tip when compared to standards of known concentration (John and Jones, 2007). This current is measured rapidly (100ms) to provide release and reuptake kinetics under basal conditions and pharmacological manipulation. Basic DA release and responsiveness is examined by assessing increasing stimulus intensity (Input/Output) and maximum release. Basal release was assessed over five stimulations with a 2-minute IPI. Paired-pulse ratios (4-second IPI) were examined to assess synaptic facilitation. The pharmacological manipulation of D2 autoreceptors with quinpirole or dopamine transporter (DAT) function with GBR-12909 is also possible with this method allowing for the investigation of subtle alterations in the system beyond basic tone. 3.3.1 Evoked release of dopamine is altered with repeated stimulations in GKI mice under basal conditions In young and old GKI mice, single pulse electrically evoked dopamine peak release is unaltered under basal conditions in the dorsolateral striatum. There were no significant effects of genotype 30  (or interaction between genotype and stimulus intensity) upon dopamine peak responses by 2-way RM-ANOVA (young, Fig 5A: Genotype p=0.99674; Interaction p=0.9828; old, Fig 6A: Genotype p=0.7542; Interaction p=0.9782), nor in the maximum concentration of dopamine released (1-way ANOVA: young, Fig 5B: p=0.7900; old, Fig 6B: p=0.7332). Over a baseline of five single peaks in young animals, the peak amplitude of P3, P4, and P5 normalized to P1 was significantly different by genotype (and interaction between genotype and pulse) with WT animals decreasing while GKI animals increased in amplitude; indicating a resistance to fatigue in young GKI mice (2-way RM ANOVA, Fig 5C: Genotype F2,84=4.080, p=0.0204; Interaction F8,336=2.306, p=0.0203; multiple comparisons by Uncorrected Fisher’s LSD: P3 WT v Het p=0.0095, WT v Homo p=0.0009; P4 WT v Het p=0.0052, WT v Homo p=0.0557; P5 WT v Het p=0.0027, WT v Homo p=0.0056). This difference in repeated release was not seen in old animals (Fig 6C: Genotype p=0.6712; Interaction p=0.7776). Paired pulse ratios are similar across all genotypes in both age points, indicating no change in probability of release (1-way ANOVA: young, Fig 5D: p=0.4967; old, Fig 6D: p=0.5801). The decay tau, or the amount of time after release for DA levels to return to baseline, is a product of release, reuptake and diffusion. These kinetics are majorly determined by DAT clearance of DA from near the release site. Here, GKI mice showed alterations at both age points after single pulse stimulation prior to drug application. In young and old mice, decay tau was significantly different between genotypes (1-way ANOVA, young, Fig 5E: F2,52=5.732, p=0.0056, Uncorrected Fisher’s LSD WT v Het t(52)=3.375, p=0.0014; old, Fig 6E: F2,64=3.720, p=0.0296, Uncorrected Fisher’s LSD WT v Het p=0.0244, Het v Homo p=0.0332) indicating slower DA transients in Het mice, potentially due to altered DAT reuptake, diffusion or release.  31  3.3.2 Quinpirole: D2 agonism alters repeated release in young GKI mice Peak response amplitudes decreased in all genotypes under D2 agonism via bath application of quinpirole (50nM). A stable reduction of 20-30% was observed in WT slices, whereas responses continued to depress in mutant slices over five consecutive stimuli in the continued presence of quinpirole. There was a significant interaction between pulse and genotype over the five ensuing peaks in quinpirole by 2-way ANOVA with WT not continuing a decrease in peak amplitude as robustly as Het and Homo mice (Fig 5F: Interaction F8,188=20343, p=0.0201, Uncorrected Fisher’s LSD Q4 WT v Homo p=0.0071, Q5 WT v Het p=0.0375, WT v Homo p=0.0226). This effect of quinpirole to invert the enhanced response to repeated stimulation seen in young Het and Homo mice (relative to WT littermates) was absent in old mice (Fig 6F: Interaction p=0.5200). The data likely indicate a transitional phase of a progressive phenotype, potentially indicative of nigrostriatal dysfunction. Alternatively, an emerging compensation with age might be a factor (although our behavioral data suggest otherwise). The amplitude of the quinpirole peaks were normalized to the first two peaks before drug wash-in (S1 & S2) to account for effects of repeated baseline and paired-pulse stimulation on peak responses.  3.3.3 GBR-12909: DAT inhibition alters DA release differently in young and old GKI mice Spatiotemporal limiting of DA is imperative in such a fine tuned system indicating the important role of DAT as the main clearance mechanism in keeping DA transiently available (Vaughan and Foster, 2013). DAT alterations have been shown to be an early and progressive indicator of DA dysfunction starting from subclinical time points (Nandhagopal et al., 2008). The application of DAT inhibitor GBR-12909 (1µM) had similar effects across all genotypes on the increase of single DA peak amplitude in young mice (Fig 5G: Kruskal-Wallis test: KW=0.3759, p=0.8287). 32  Similar increases in DA peak were seen in old mice, however in old Homo mice, a significantly reduced increase in peak height was observed (Fig 6G: Kruskal-Wallis test: KW=9.028, p=0.0110; Dunn’s multiple comparisons test: WT v Homo p=0.0116) potentially displaying a reduced sensitivity to DAT inhibition. DAT inhibition had a significant increase in decay tau of young, but not old, Het and Homo mice (young, Fig 5H: 1-way ANOVA: F2,57=3.803, p=0.0282; Uncorrected Fisher’s LSD: WT v Het p=0.0231, WT v Homo p=0.0146; old, Fig 6H: 1-way ANOVA: p=0.4167). Decay tau in young GKI animals, was altered under basal conditions and continued to be altered following the application of GBR-12909, contrary to our expectations. Staining for DAT in a subset of these mice revealed no significant difference in striatal DAT levels at either age (1-way ANOVA: young, Fig 5I: p=0.4665; old, Fig 6I: p=0.3585) ruling out difference in surface protein level, but not functionality differences. It is possible that DAT functions differently, working to mask tone and release under basal conditions, and exposed only when looking at reuptake in young animals. On the other hand, in old animals, basal decay is altered at baseline but normalized with the application of GBR-12909, which also uncovers release differences in the increase of DA peak amplitude compared to baseline for Het and Homo mice (Fig 6E, 6G). In the absence of altered DAT levels, differences, specifically longer, decay tau could be due to asynchronous release of DA. 33  fE P S P  P e a k  A m p litu d e100200300400500600700-1 .0-0 .50 .0S tim u lu s  In te n s ity  (u A )fEPSP peak norm. volley (N2/N1)N T  n = 1 5 (5 )O E  n = 1 6 (5 )fE P S P  P a ire d -p u ls e  R a t io20601001401802202603001 .01 .52 .0In te r -p u ls e  In te rv a l (m s )PPR (P2/P1)N T  n = 1 4 (5 )O E  n = 1 6 (5 )fE P S P  P e a k  D u r in g  T ra inP2/P1P3/P1P4/P10 .00 .51 .01 .5Peak Amp % ChangeN T  n = 1 5 (5 )O E  n = 1 6 (5 )*fE P S P  lo n g -te rm  d e p re s s io n01020304050600 .60 .81 .01 .2% baselineN T  n = 1 1 (5 )O E  n = 1 0 (5 )fE P S P  L T DfEPSP Amplitude (% change)NTOE0 .00 .51 .01 .5A B CD E Figure 1. Local electrical stimulation in acute striatal slices revealed subtle synaptic alterations, but normal long-term depression in LRRK2 hWT OE mice. Striatal field responses were unaltered in acute slices from 6-month LRRK2 hWT OE mice, relative to WT littermates. A) Field excitatory postsynaptic potential (fEPSP) peak amplitudes (normalized to afferent volley amplitude) were similar across increasing stimulus intensities. B) Paired-pulse ratios, calculated from two pulses in quick succession (20-300ms inter-pulse interval) were similarly unaltered in LRRK2 hWT OE mice, relative to controls. C) Rapidly 34  stimulated release during 100Hz tetanic stimulation (1sec) was decreased in OE mice when normalized to P1, relative to NT mice. D&E) One hour after HFS-induction of long-term depression, a stable lasting synaptic depression was expressed; there were no differences between genotypes in the percent peak amplitude change relative to baseline. NT = nontransgenic, OE = LRRK2 hWT overexpressor, fEPSP = field excitatory post-synaptic potential, *p < 0.05. Data are means ± SEM.  35  DA [norm WT]WT (n=9)Het  (n=6)Homo (n=5)0 .00 .51 .01 .5B a s a l D AADOPAC [norm WT]WT (n=9)Het  (n=6)Homo (n=5)0 .00 .51 .01 .5B a s a l D O P A CBHVA [norm WT]WT (n=9)Het  (n=6)Homo (n=5)0 .00 .51 .01 .5B a s a l H V ACDOPAC/DA ratioWT (n=9)Het  (n=7)Homo (n=6)02 04 06 0D O P A C  / D ADHVA/DA ratioWT (n=9)Het  (n=7)Homo (n=6)05 01 0 01 5 0H V A  /  D AE% of BLWT (n=6)Het  (n=6)Homo (n=4)05 01 0 01 5 02 0 02 5 0Q u in p iro lere s p o n s eGDOPAC+HVA/DA ratioWT (n=9)Het  (n=7)Homo (n=6)05 01 0 01 5 0D O P A C  +  H V AD AF5-HT [norm WT]WT (n=7)Het  (n=6)Homo (n=5)0 .00 .51 .01 .5B a s a l 5 -H TH5-HIAA [norm WT]WT (n=9)Het  (n=6)Homo (n=5)0 .00 .51 .01 .5B a s a l 5 -H IA AI5HIAA/5HT ratioWT (n=7)Het  (n=8)Homo (n=6)05 01 0 01 5 05 -H IA A  / 5 -H TJFigure 2. Microdialysis in the dorsolateral striatum of young LRRK2 GKI mice revealed no genotypic alterations in basal levels or turnover of monoamines and metabolites. By microdialysis basal tone of monoamines was unaltered in the dorsolateral striatum in 2-3 month LRRK2 GKI mice, relative to WT littermates. A-F) There were no alterations between genotypes in basal dopamine levels (A) or its metabolites DOPAC (B) and HVA (C). There were also no significant differences in ratio of metabolites to DA (D-F: DOPAC / DA, HVA / DA, metabolites combined DOPAC + HVA / DA). G) The D2 receptor agonist quinpirole (50µM) was infused into the brain by reverse microdialysis. There was no significant change from baseline with this drug, and it was not different between genotypes. There was no alteration in the levels of 5-HT (H), its metabolite 5-HIAA (I), nor the ratio between the two (J). Together the microdialysis data in young GKI mice suggest that with fifteen minute sampling intervals there are no alterations in monoamine tone even in response to D2 activation. Data are means ± SEM.  36  DOPAC/DA ratioWT (n=11)Het  (n=12)Homo (n=12)02 04 06 08 0D O P A C  / D ADHVA/DA ratioWT (n=11)Het  (n=12)Homo (n=12)05 01 0 01 5 02 0 02 5 0H V A  /  D AE% of BLWT (n=9)Het  (n=12)Homo (n=8)05 01 0 01 5 02 0 0Q u in p iro lere s p o n s eGDOPAC&HVA/DA ratioWT (n=11)Het  (n=12)Homo (n=12)01 0 02 0 03 0 0D O P A C  +  H V AD AF5HIAA/5HT ratioWT (n=11)Het  (n=12)Homo (n=12)051 01 52 05 -H IA A  / 5 -H TJDA [norm WT]WT (n=11)Het  (n=12)Homo (n=12)0 .00 .51 .01 .5B a s a l D AADOPAC [norm WT]WT (n=11)Het  (n=12)Homo (n=12)0 .00 .51 .01 .52 .02 .5B a s a l D O P A CBHVA [norm WT]WT (n=11)Het  (n=12)Homo (n=12)0 .00 .51 .01 .5B a s a l H V AC5-HT [norm WT]WT (n=11)Het  (n=12)Homo (n=12)0 .00 .51 .01 .5B a s a l 5 -H TH5-HIAA [norm WT]WT (n=11)Het  (n=12)Homo (n=12)0 .00 .51 .01 .52 .0B a s a l 5 -H IA AI Figure 3. Microdialysis in the dorsolateral striatum of old LRRK2 GKI mice revealed no genotypic alterations in basal levels or turnover of monoamines and metabolites. Microdialysis of the dorsolateral striatum LRRK2 GKI mice at ~12-months of age revealed no alterations relative to WT littermates. A-F) There were no alterations between genotypes in basal dopamine levels (A) or its metabolites DOPAC (B) and HVA (C). There were also no significant differences in ratios of metabolites to DA (D-F: DOPAC / DA, HVA / DA, metabolites combined DOPAC + HVA / DA). G) The D2 receptor agonist quinpirole (50µM) was infused into the brain by reverse microdialysis. There was no significant change from baseline with this drug, and it was not different between genotypes. There was no alteration in the levels of 5-HT (H), its metabolite 5-HIAA (I), nor the ratio between the two (J). Together the microdialysis data in old GKI mice suggest that with fifteen minute sampling intervals there are no alterations in monoamine tone even in response to D2 activation. Data are means ± SEM.  37  Glu [norm  WT]WT (n=5)Het  (n=4)Homo (n=6)0 .00 .51 .01 .5y o u n g  g lu ta m a teAGlu [norm  WT]WT (n=8)Het  (n=4)Homo (n=7)0 .00 .51 .01 .5o ld  g lu ta m a teBGABA [norm  WT]WT (n=7)Het  (n=4)Homo (n=6)0 .00 .51 .01 .5o ld  G A B ADGABA [norm WT]WT (n=1)Het  (n=4)Homo (n=2)0 .00 .51 .01 .5y o u n g  G A B AC Figure 4. Microdialysis in the dorsolateral striatum of young and old LRRK2 GKI mice revealed no genotypic alterations in glutamate and GABA tone.  Microdialysis of the dorsolateral striatum in LRRK2 GKI mice at 2-3 months or over 12-months of age revealed no alterations between genotypes relative to WT littermates. A-D) There were no alterations between genotypes in basal levels glutamate in young (A) or old (B) GKI mice as well as a lack of genotypic alteration on basal GABA levels in young (C) or old (D) GKI mice. Together the microdialysis data suggest that with fifteen minute sampling intervals there are no alterations in basal tone of glutamate or GABA in GKI mice. Data are means ± SEM.  38  S tim u lu s  In te n s ity  (u A )Dopamine Peak (uM)0501002003000 .00 .20 .40 .6W T  1 8 (5 )H e t 1 2 (5 )H o m o  2 2 (9 )In p u t/O u tp u tADopamine (uM)WT 18(5)Het 13(5)Homo 22(7)0 .00 .20 .40 .6M a x . s in g le  p e a kBP1P2P3P4P50 .80 .91 .01 .11 .21 .3Peak Amplitude  (norm P1)e[DA]oS tim u lu s  N u m b e rR e p e a te d  s t im u la t io n ,b a s e lin eH o m o  n = 3 2 (1 1 )W T  n = 2 6 (8 )H e t n = 2 9 (1 0 )*###########CPPR, P2/P1WT 20(5)Het 13(4)Homo 22(7)0 .00 .10 .20 .30 .4B a s a l  P P RD B a s a l d e c a ydecay tau (ms)WT 20(5)Het 13(5)Homo 22(7)02 0 04 0 06 0 08 0 0**# #E% change single peakWT 18(6)Het 21(8)Homo 20(7)02 04 06 08 01 0 0G B R  s in g le  p e a k%  c h a n g eG S in g le  d e c a yG B R -1 2 9 0 9decay tau (ms)WT 17(6)Het 23(8)Homo 20(7)05 0 01 0 0 01 5 0 0*#HQ1Q2Q3Q4Q502 04 06 08 01 0 0Peak Amplitude  (norm S1&2)e[DA]oS tim u lu s  N u m b e rW T  n = 1 6 (5 )H e t n = 1 3 (5 )H o m o  n = 2 1 (7 )R e p e a te d  s t im u la t io n ,q u in p iro le*### , #FS tr ia ta l D A TFluorescence (a.u. -cc)WT n=5Het n=5Homo n=501 02 03 04 05 0IFigure 5. Fast-scan cyclic voltammetry in slices from the dorsolateral striatum of young LRRK2 GKI mice revealed alterations to repeated stimulation and decay under basal conditions, increased sensitivity to D2 agonism, and decreased sensitivity to DAT inhibition relative to WT littermates. 39  Fast-scan cyclic voltammetry was used to assess rapid DA release and reuptake in the dorsolateral striatum of acute brain slices from 2-3 month old LRRK2 GKI mice. A-B) No difference was revealed between genotypes in response to increasing stimulation (A) or the maximum amount of DA released (B). C) Over five single baseline stimulations with 2-minute inter-pulse intervals GKI mice increased DA peak response amplitude in contrast to the sustained decline seen in WT animals. D) Paired-pulse ratios, calculated from two pulses with a 4-second inter-pulse interval were unaltered in any genotype. E) After a single electrical DA release event, the decay tau was altered, with a slower return to baseline in Het mice when compared to WT littermates. F) The application of the D2 receptor agonist quinpirole (50nM) lead to a ~25% decrease in peak amplitude for all animals when compared to baseline release, with further sustained decreases in GKI mice over five stimulations relative to WT littermates. G-I) The dopamine transporter (DAT) was assessed with the application of DAT inhibitor, GBR-12909, which revealed similar effects on single event DA release (G), though the decay tau was significantly increased in GKI mice under these conditions (H). Staining in a subset of these mice revealed no alterations in striatal DAT levels by immunohistochemistry (I). Together, FSCV data reveal alterations in basal DA release and reuptake with differing effects of D2 agonism and DAT inhibition in response to LRRK2 GKI mutation. *p < 0.5 by 2-way RM ANOVA (C & F) or 1-way ANOVA (H), **p < 0.01 by 1-way ANOVA (E), #p < 0.5 and ##p < 0.01 by Uncorrected Fisher’s LSD (C, E, F, H). Data are means ± SEM.  40  S tim u lu s  In te n s ity  (u A )Dopamine Peak (uM)0501002003004000 .00 .10 .20 .3W T  1 5 (5 )H e t 1 8 (6 )H o m o  1 6 (5 )In p u t/O u tp u tADopamine (uM)WT 15(5)Het 18(6)Homo 16(5)0 .00 .20 .40 .6M a x . s in g le  p e a kB1 2 3 4 50 .91 .01 .11 .21 .3S tim u lu s  N u m b e rPeak Amplitude  (norm P1)e[DA]oH e t n = 3 4 (1 1 )W T  n = 3 8 (1 3 )H o m o  n = 2 2 (6 )R e p e a te d  s t im u la t io n ,b a s e lin eCPPR, P2/P1WT 18(6)Het 30(11)Homo 15(5)0 .0 00 .0 50 .1 00 .1 50 .2 00 .2 5B a s a l P P RD% change single peakWT 20(8)Het 9(5)Homo 21(6)02 04 06 08 01 0 0G B R  s in g le  p e a k%  c h a n g e*#GB a s a l d e c a ydecay tau (ms)WT 19(6)Het 33(11)Homo 16(5)02 0 04 0 06 0 08 0 0*ES in g le  d e c a yG B R -1 2 9 0 9decay tau (ms)WT 21(8)Het 12(5)Homo 21(6)05 0 01 0 0 01 5 0 02 0 0 0HR e p e a te d  s t im u la t io n ,q u in p iro le1 2 3 4 502 04 06 08 01 0 0S tim u lu s  N u m b e rPeak Amplitude  (norm S1&2)e[DA]oH e t n = 2 8 (1 0 )W T  n = 1 9 (6 )H o m o  n = 1 6 (5 )FS tr ia ta l D A TFluorescence (a.u. -cc)WT n=5Het n=5Homo n=502 04 06 08 0I Figure 6. Fast-scan cyclic voltammetry in slices from the dorsolateral striatum of old LRRK2 GKI mice revealed no alterations in electrically evoked dopamine release, but with alterations to decay under basal conditions, while the inhibition of DAT revealed alterations to evoked DA release relative to WT littermates. Fast-scan cyclic voltammetry was used to assess rapid DA release and reuptake in the dorsolateral striatum of acute brain slices from LRRK2 GKI mice over 12-months of age. A-D) 41  No alterations were seen in single stimulated pulse DA release between genotypes under basal conditions in response to increasing stimulus intensity (A), the maximum amount of DA released (B). No alterations were observed between genotypes with repeated stimuli measured by five single stimulations with 2-minute inter-pulse intervals (C), or the paired-pulse ratio calculated from two pulses with a 4-second inter-pulse interval (D). E) After a single electrical DA release event, the decay tau was altered, with a slower return to baseline in Het mice when compared to WT littermates. F) The application of the D2 receptor agonist quinpirole lead to a ~30% decrease in peak amplitude for all genotypes when compared to baseline release, that was sustained over five stimulations. G-I) The dopamine transporter was assessed, and with application of DAT inhibitor GBR-12909 (1µM) a reduced increase in single event DA release was observed in Homo animals (G), though the decay tau was not different between genotypes under these conditions (H). Striatal DAT levels in a subset of these mice were no different by immunohistochemistry (I). Together, subtle differences in DA decay under basal conditions, normalized by DAT inhibition which in turn reveals release differences, indicates that alterations to DAT function are present in old LRRK2 GKI mice and differently than at a young age. *p < 0.5 by 1-way ANOVA (E) or Kruskal-Wallis (G), #p < 0.5 by Dunn’s multiple comparisons test. Data are means ± SEM.   42  Chapter 4: Discussion  4.1 LRRK2 and its role in PD, clues from hLRRK2 OE Prior to identifying how a specific point mutation can lead to such devastating neurodegenerative consequences, it is useful to understand the role of the protein in question under physiological conditions. Research on LRRK2 function has been done, and continues to be, in this regard in humans, mice, and other organisms. The LRRK2 hWT OE mice express 3-20 times more LRRK2 to enable the study of gain of function effects. Prior to this investigation, work in our lab showed that hWT OE mice are behaviorally hypoactive and exhibit long-term recognition memory impairments at 3-6 months, but had no alterations in basal striatal glutamatergic neurotransmission, as measured by whole cell patch clamp (Beccano-Kelly et al., 2015). Here, I have presented just one more piece of this puzzle: investigation of field plasticity in acute striatal slices prepared from six month old hWT LRRK2 OE mice. While transmission differences appear to be present in LRRK2 hWT OE synapses during high-frequency electrical stimulation (revealed by less facilitation during tetanic stimulation, Fig 1C), striatal LTD remained intact and was expressed to a magnitude similar to WT littermates (Fig 1D & E). The striatum is classically considered to control motor output, reward / goal-mediated motor learning, and spatial recognition memory (Cragg, 2006; Cui et al., 2013; Darvas and Palmiter, 2009; Gerfen and Surmeier, 2011; Paladini and Roeper, 2014). OE mice exhibited behavioral alterations attributable to the striatum (hypoactivity, spatial recognition memory impairments) but no basal synaptic transmission alterations, indicating that these measures do not always correlate.  To further probe synaptic transmission, short-term synaptic plasticity at striatal inputs was examined by investigating synaptic facilitation. In brief: the ratio of two temporally adjacent 43  stimuli can inform upon the probability of release of a neuron. Stimulating within the dorsolateral striatum, informs about not just glutamatergic, but also the effects of dopamine, interneuron, and retrograde endocannabinoid signaling upon presynaptic short-term plasticity (Akopian and Walsh, 2007; Bamford et al., 2004; Kreitzer, 2002; Yin and Lovinger, 2006). Striatal stimulation while recording from SPNs in OE mice showed reduced facilitation, indicating impaired short-term plasticity at these synapses (Beccano-Kelly et al., 2015). As this was not seen when stimulating from the corpus callosum (solely glutamatergic input) this indicated that other neurotransmitters were involved with this alteration in synaptic short-term plasticity as a result of a threefold increase in LRRK2 protein. Corticostriatal glutamate release is negatively regulated by the activation of D2 receptors (D2R) on the axon terminal, meaning that release of glutamate from the first stimulation can (via subsequent DA release) reduce the amplitude of the successive glutamatergic responses (Akopian and Walsh, 2007; Bamford et al., 2004; Ford, 2014).  In OE mice the D2R antagonist remoxipride reversed this short-term plasticity alteration by significantly increasing PPRs to levels comparable to NT mice in both D1 and D2 expressing SPNs, indicating presynaptic D2 effects on stimulated DA release are involved in tuning of striatal glutamate (Beccano-Kelly et al., 2015). Further indication of aberrant D2R signaling in LRRK2 OE mice was confirmed when presynaptic D2R activation with pramipexole reproduced behavioral hypoactivity in NT mice that mimicked untreated OE behavior (Beccano-Kelly et al., 2015). Alterations in short-term plasticity of LRRK2 OE suggested there might be alterations to long-term plasticity (Fig 1). Ultimately, a threefold increase in LRRK2 protein had no effect on striatal LTD (Fig 1D & E). As mentioned, subtle alterations were seen in re-release during HFS (Fig 1C) and in decreased DA tone by microdialysis (Beccano-Kelly et al., 2015). Though no alterations were seen in LTD, this does 44  not rule out alterations to long-term potentiation (LTP). Tonic versus phasic firing of dopaminergic neurons can differently influence LTP and LTD induction, favored by the activity of lower DA affinity D1 and higher affinity D2 receptors, respectively (Lovinger, 2010). It is also possible that this 6-month age point is in a transition phase, or HFS was too intense, either of these could occlude subtle genotypic differences. Another, more sensitive technique that could be used to tease out LRRK2 differences is spike-timing dependent plasticity where timed pairing  is used to induce LTP or LTD with pre- and postsynaptic neuronal elements either before, during, or after glutamate release (Lovinger, 2010; Surmeier et al., 2007; Yagishita et al., 2014). Though this study informs about how extra LRRK2 can alter synaptic transmission, it does not inform upon physiologically relevant PD causing mutations.    4.2 LRRK2 G2019S-induced alterations to the DA system In LRRK2 G2019S knock-in mice presented here, no differences in striatal DA tonus were observed over long (fifteen minute microdialysis) sampling intervals, however when assessed on a more rapid timescale (voltammetry), differences started to emerge. Previous studies of both GKI and an OE mouse model have shown a deficit in extracellular DA at 12 months of age by microdialysis with reduced exploratory behavior in OE mice (Melrose et al., 2010; Yue et al., 2015). Despite a strong trend in 12 month old mice, no significant reduction in basal neurotransmitter levels was seen here by microdialysis, even in response to D2 agonism by quinpirole (Fig 2, 3, 4). Microdialysis is conducted over 15 minute sampling intervals of the extracellular space, and so subtle alterations to neurotransmission due to minute kinetic exocytotic packaging / release differences or involving compensatory mechanisms may not be 45  noticed. Ratios of neurotransmitter to metabolites can inform about turnover and synthesis alterations, however we did not observe extracellular alterations here either. The alterations seen by FSCV potentially implicate D2 autoreceptor function or altered DA recycling / vesicle packaging (Fig 5& 6). Under basal conditions with a standard two minute IPI to allow for electrode sensitivity recovery and DA repackaging, peak DA response amplitude decreased slightly in young WT mice over time. Contrastingly, responses in slices from both Het and Homo mice increased and were resistant to this run-down, being significantly increased on the third, fourth, and fifth stimulation, relative to WT (Fig 5C). Thus, it appears that transgenic GKI mice are immune to basal synaptic run-down or fatigue when examined by repeated stimulation at two minute intervals. Such alterations to DA release under basal conditions could be due to decreased number or functionality of inhibitory D2 autoreceptors and their downstream signaling cascades. Previous studies on OE mouse models of hWT and G2019S LRRK2 (BAC mice) found that D2 autoreceptor mediated feedback was normal, though there was a trend towards increased D2 receptor expression in transgenics (Melrose et al., 2010). A separate LRRK2 knock-in model, carrying the R1441C knock-in mutation, found no alterations in basal levels of STR DA but impaired DA neurotransmission and D2R-mediated functions (Tong and Shen, 2012; Tong et al., 2009). To investigate this in our mice, the D2 receptor agonist quinpirole was applied for protracted periods (10-20 minutes), resulting in a mostly stable ~20% reduction in WT evoked DA peak by 10 minutes of exposure, as would be predicted by activation of D2 and its subsequent inhibitory downstream effects on adenylyl cyclase, cAMP, and PKA. This reduction was similar in Het and Homo slices, yet increased exposure time and repeated stimulation inverted the basal effect; DA release in both Het and Homo GKI mice continued to decrease in 46  peak amplitude over the subsequent five stimulation events, whereas WT littermate release remained relatively stable (Fig 5F).  Decreases in D2 autoreceptor expression (or hypoactive functionality) could account for decreased D2 mediated feedback-inhibition (Fig 5C) under basal conditions, however exaggerated decreases in DA peak amplitude with agonism by quinpirole would suggest hypersensitivity and increased feedback-inhibition (Fig 5F). Increased DA release may be due to mutation dependent alterations in the vesicle release cycle but masked by DAT such that availability is increased (Fig 5C; similar to increased availability of glutamate/GABA in neuronal cultures; Beccano-Kelly et al., 2014). If so, then progressive decrease in response to D2 agonism could be in response to a combination of drug and increased endogenous DA release, acting as a second hit on D2 autoreceptors and further decreasing repeated DA release (Fig 5F). The insensitivity to release fatigue in young GKI mice is absent in old GKI mice (Fig 6C & F), potentially indicative of a once hyperactive system having normalized due to aging, compensation, or metabolic necessity, along a similar timeframe as the normalizing of motor behavior in these mice. This is likely to still be a transitional phase, preceding more deleterious alterations. Cognitive impairment (at 6 months) precedes a decrease in grooming at 12 months in Homo mice, indicating that the system is still in flux and due to the subtlety of this model, the enrichment of our mice, and different lifespans, we expect that full effects of the mutation are delayed to later time points.       Although basal evoked DA response peaks were unaltered, there was a significant increase in DA response decay. This could be due to increased DA release, but with slower or irregular release kinetics. Alternatively, impedance in DA reuptake could also lead to prolonged extracellular DA after release, and thus an increase in decay tau. The primary extracellular clearance mechanism for DA is through the DA transporter: DAT. Immunohistochemical 47  staining and fluorescence confocal microscopy in slices prepared from the same animals used for voltammetry from the hemisphere that received microdialysis revealed no difference in striatal DAT levels at either age (Fig 5I & 6I) consistent with previous reports (Melrose et al., 2010). Lack of alteration in protein levels does not rule out differences in function, therefore to investigate DAT function, the DA competitive binding inhibitor GBR-12909 was applied (Andersen, 1989). Rather than normalizing the decay differences between genotypes, blocking DAT exaggerated the differences between young mutant mice compared to WT littermates (Fig 5H), indicating that the basal increase in decay tau in these animals is not due to reduced DAT functionality. This suggests that there may be elevated DA release in young GKI mice that is only observed as increased decay times due to highly efficient DAT (or altered release timing). DAT inhibition resulted in a trend towards increased peak and a highly significant increase in decay tau. Together the data show that the amount of DA released in striatal GKI slices by single pulse stimulation (if considered as DA charge) is elevated and maintained (upon repeated stimulation / release) at a higher level than WT littermates. In the intact system (in vivo) DA release occurs spontaneously from 3 - 8 Hz and in burst patterns (Grace and Bunney, 1984a, 1984b). Perturbations to the timing of this system would likely have a compounding effect over time leading to phenotypic changes in behavior (hyperactivity and anxious phenotypes are seen at a young age) and likely oxidative stress, as DA is a rapid oxidizer and can cause toxic reactive oxygen species (Cohen, 2006). In slices from old GKI animals there were no significant differences in evoked DA decay constants in the presence of GBR-12909, relative to WT slices; indicating that in the absence of DAT function (produced by normal levels of DAT, Fig 6I) DA release is no longer increased by the mutation. Blocking DAT lead to increased DA peak amplitude in all genotypes at both ages 48  as expected, however old Homo mice were significantly less increased when compared to WT littermates (Fig 6G). LRRK2 is thought to be involved in synaptic vesicle storage and recycling (Beccano-Kelly et al., 2015, 2014; Piccoli et al., 2011) and here GKI mutation leads to augmented DA release that is not attenuated by DAT inhibition, indicating other mechanisms are affected and play a role in this alteration from early stages (2-3 months, and potentially earlier) in response to this mutation. As diffusion and degradation are also other mechanisms by which DA is cleared from the synapse, differences in levels of MAO or COMT (predictably a reduction) could lead to longer decay tau, which was not tested here directly, though an alteration in the ratio of DA to metabolite(s) in the dialysate would be expected if this were the case (not seen, Fig 2 & 3 D-F).  Another possible mechanism for increased DA decay times could be altered release timing. In addition to SPNs, thalamostriatal axons also synapse on to cholinergic interneurons (ChIs), which in turn synapse onto nigrostriatal dopamine axons (Surmeier and Graybiel, 2012; Threlfell et al., 2012). Synchronized activity in ChIs, controlled by thalamostriatal glutamatergic inputs, causes ACh release which agonizes nAChR on DA axons and generates release of DA from nigrostriatal terminals, which dominates over ascending nigral action potentials (Threlfell et al., 2012). This mechanism of release resulting from local thalamostriatal terminal release, interneuron activation, and then DA release from nigral terminals requires two extra synaptic events in series (then direct stimulation and release from nigrostriatal terminals). This thalamo-interneuron-nigral activation, if augmented in GKI slices, may also account for temporal differences to intrastriatally-evoked DA release. ChIs have previously been implicated in movement disorders, and their functional impact is known to rise as intrastriatal DA levels are depleted (Pisani et al., 2007; Shen et al., 2007). DA has also been potentially implicated in 49  modulating the activity of ChIs through D2 and D5 receptors with D2 receptors suppressing the pacemaking activity of interneurons and diminishing acetylcholine release (Gerfen and Surmeier, 2011). This data add to an increasing body of evidence that at early stages GKI mice exhibit alterations to DA release that are consistent with increased release. We see similar trends as other labs towards reduced DA tone in older animals, and significantly less DA release in the presence of DAT inhibition in old homozygous mice. This is strikingly similar to asymptomatic human LRRK2 carriers for whom there are indications of an early increase in DA turnover that is not related to DAT (Sossi et al., 2010). Contrary to late stage DA depletion, alterations to the DA system are being recognized in human mutation carriers and genetically modified models as increases at early stages. These indications are vital to understanding the etiology and pathophysiology of PD. If DA is increased prior to classical late stage depletion and degradation of the nigrostriatal DA system, treatments should be designed accordingly. Targeting this augmentation of DA in LRRK2 mutation carriers prior to motor symptom presentation could potentially delay disease onset and progression. Disease modifying targets and timelines can be tested with LRRK2 GKI mice as we are defining early and later stage phenotypes including early motor hyperactivity and anxiety (prior to 3 months of age, unpublished) and later hypoactivity, loss of self-care (grooming), and cognitive deficits in these animals. Changes seen over these time points in GKI mice probably signify the transition to onset of pathological processes prior to overt loss of DA (as we see no nigral degeneration up to 20 months (Yue et al., 2015)).  50  4.3 Limitations and future directions It is important to remember, that the nigrostriatal system examined here does not act in isolation. The striatum is a gateway of inputs from many different brain regions with various neurotransmitters, receptors, and signaling pathways within and to / from other brain areas that we are still learning about. As dopamine and its metabolites were the main focus of this thesis, glutamate and GABA analysis was secondary, meaning that samples were stored in a way to maximize monoamines and endured an extra round of freezing and thawing prior to HPLC analysis of the amino acid neurotransmitters. It would be potentially interesting for further experiments to add to the work that has been started in this regard, especially with the knowledge of alterations in STR glutamatergic transmission in this model (unpublished) and others (Beccano-Kelly et al., 2014). It is also possible that the lack of alterations seen in regard to glutamate and GABA but also the monoamines and metabolites is due to the subtlety of this model and the environmental enrichment our mice get throughout their lifespans. The subtlety of this genetically faithful knock-in model compared to something like an OE or KO model can obstruct genotypic differences or vary them from lab to lab with slight alterations in procedures.  Although the LRRK2 G2019S knock-in model is arguably the most physiologically relevant due to endogenous mutation expression patterns and levels, ultimately, mice are not people and acute slices are not whole brains. Mice do not develop Parkinson’s disease spontaneously, or in the same way as humans even with the same genetic mutation. These mice do not show nigral degeneration or similar motor phenotypes as humans, one reason potentially being that they just don’t live as long. These mouse models and studies can inform upon the natural and mutant role of affected proteins and downstream signaling effects, which may be 51  present in prodromal disease states ultimately informing on biomarkers and disease-modifying therapeutic strategies. Due to similarities between idiopathic and sporadic Parkinson’s disease, hopefully what is gleaned from these genetically faithful mouse models can and will translate over to therapeutic knowledge for this and other neurodegenerative and/or motor diseases.  4.3.1 DAT/D2 quantification, functionality Some work has been done and presented here on the expression levels and functionality of DAT, and less so for D2 receptors, specifically D2 autoreceptors. Alterations in the expression or actions of any of these important regulators of DA neurotransmission due to LRRK2 mutation could have strong implications for the health of these metabolically taxing DA neurons that are so vulnerable in Parkinson’s disease. Although nigral degeneration of DA neurons does not happen in the GKI model, alterations to homeostatic mechanisms could provide clues to their demise in humans.   4.3.2 Individual stimulation of striatal inputs Optogenetics is a rapidly growing technique in neuroscience that when paired with virally targeted gene expression can result in laser controlled activation of an isolated subset of neurons. This allows for the study of one type of neuron or pathway within a milieu such as the striatum helping to distinguish between the many parts of the whole. Channelrhodopsin-2 (ChR2) is a light activated channel, which when activated by blue light, allows the passage of cations and the subsequent depolarization of the cell without directly firing those around it (Boyden et al., 2005; Guru et al., 2015). Using targeted viral expression, ChR2 can be expressed in a subset of neurons leading to isolated stimulation in contrast to whole field electrical stimulation. Injections into the SNpc with viral expression of ChR2 targeting dopaminergic neurons would allow for stimulation of solely nigrostriatal afferents in acute striatal slices. This would 52  allow for the separation, helping to parse out the role of the dopaminergic system from the glutamatergic system in striatal plasticity. Working backwards from sole dopaminergic activation, thalamostriatal and/or cholinergic interneuron (ChI) stimulation could be controlled in a similar, light-dependent fashion to investigate effects on DA neurotransmission due to LRRK2 mutation. One hypothesis for the increased decay tau seen in FSCV experiments (Fig 5 & 6) is the activation of cholinergic interneurons. Previous experiments have used optogenetic control of thalamostriatal neurons to evoke DA release by way of ChIs (Threlfell et al., 2012). The light controlled activation of ChR2 expressing thalamostriatal axons trigger ChIs which in turn trigger DA release via activation of nicotinic acetyl choline (ACh) receptors (nAChRs) (Threlfell et al., 2012). If this ChI activation is causing a delay in DA release due to the extra synapse from thalamostriatal to ChI to DA neuron (versus corticostriatal to DA), this could explain the increase in basal decay tau seen in Het mice (Fig 5 & 6). Alternatively, this possibility could be investigated by pharmacologically blocking nAChRs (DHBE, 1uM) or mAChRs (atropine, 2uM) (Threlfell et al., 2012) and testing for the continued presence of increased decay times of DA transients in GKI slices. Laser stimulation of one subset of neurons can be combined with electrical stimulation and pharmacology to parse out the function of various systems in isolation or in concert.   4.4 Conclusions LRRK2 G2019S mutation is the single most common genetic risk factor for Parkinson’s disease and leads to increased kinase activity with subtle effects on the timing of dopaminergic transmission in the nigrostriatal system. Although no differences were seen in monoamine, metabolite, glutamate, or GABA release by microdialysis at young or old age in GKI mice, more 53  temporally sensitive investigation revealed subtle release augmentation in mutants. Increases in DA release with repeated stimulation, and increased decay constant in mutants by fast-scan cyclic voltammetry suggest that from an early age (<3 months) release kinetics are altered and respond differently to D2 receptor agonism, but release increase is not affected by DAT. At later age points (~12 months) release is still augmented as seen by increased decay constant, however D2 receptors and DAT are functioning differently, as seen by pharmacological manipulation effects that do not match those in young animals. LRRK2 is thought to be involved in synaptic vesicle storage and recycling (Beccano-Kelly et al., 2015, 2014; Piccoli et al., 2011) and GKI mutation in mice leads to augmented DA release from an early age that is not attenuated by DAT inhibition, indicating other mechanisms (e.g. altered release / vesicle cycling) are affected and play a role in this alteration from early stages (<3 months, and potentially earlier) in response to this mutation. 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