Thèses sur le sujet « Striatum Dynamics »

La mémoire procédurale est la mémoire des habitudes motrices. Les ganglions de la base (GB), un groupe de structures impliqué dans les fonctions motrices et cognitives, sont responsables de la formation de cette mémoire. Le striatum, principale structure d’entrée des GB, joue un rôle central dans le transfert de l’information entre le cortex et les autres structures sous-corticales, assurant ainsi la sélection et l’intégration de l’information corticale au sein de boucles fonctionnelles parallèles. Lors d’un apprentissage procédural, le comportement est tout d’abord dirigé vers un but, impliquant les boucles associatives et le striatum dorsomédial (DMS), pour ensuite évoluer vers un comportement habituel automatique, impliquant les boucles sensorimotrices et le striatum dorsolatéral (DLS). L’anatomie des circuits et la dynamique des réseaux striataux au cours de l’apprentissage procédural ont été bien décrites. Cependant, comment la mémoire procédurale est précisément encodée au niveau des réseaux corticostriataux (CS) reste inconnu.Dans mon travail de thèse, nous nous sommes intéressés à la caractérisation des dynamiques des réseaux CS impliqués dans l’apprentissage procédural et nous avons exploré l’existence de substrats neuronaux responsables de la formation de cette mémoire. Grâce à l’imagerie calcique ex vivo nous avons monitoré l’activité des réseaux CS durant les différentes phases d’apprentissage. Nous avons extrait et analysé les signaux calciques des neurones épineux moyens (MSN), les neurones de sortie du striatum. Afin de distinguer les MSNs des autres neurones striataux, nous avons développé un classifieur basé sur les réponses calciques des neurones et leur morphologie. Nous avons montré qu’il existe une réorganisation spécifique des réseaux DMS pendant la 1ère phase d’apprentissage moteur. L’activité dans le DMS est diminuée après un entraînement léger, avec une forte activité (HA) maintenue dans un petit groupe de cellules, et retournant à un niveau basal après un entrainement intense. Dans le DLS, la réorganisation est graduelle et localisée dans des ‘clusters’ d’activité (HA) après un entrainement intense. L’existence des cellules et clusters HA est directement corrélée à la qualité de l’apprentissage. Nous avons ensuite exploré les mécanismes sous-tendant cette réorganisation. Grâce à des enregistrements en patch-clamp nous avons examiné les propriétés des cellules et clusters HA et montré une augmentation du poids synaptique des afférences du cortex cingulaire sur les cellules HA dans le DMS après un entrainement léger. Des études de traçage anatomique ont montré des changements plus robustes dans le DLS avec une augmentation du nombre de projections du cortex somatosensoriel après entrainement intense. Une stratégie cFos-TRAP couplée à la chimiogénétique nous a permis d’inhiber spécifiquement les cellules et clusters HA, et montrer que cela affecte l’apprentissage moteur. Ceci montre la nécessité de ces cellules dans les premières et dernières phases de l’apprentissage moteur respectivement.Ensuite, notre but était d’explorer s’il existe des déficits d’apprentissage moteur dans une phase présymptomatique dans un modèle murin de la maladie de Huntington, et d’examiner l’association de ces déficits à des altérations au niveau des réseaux CS. Nous avons d’abord montré qu’il existait des déficits dans la dernière phase d’apprentissage dans ce modèle murin. Grâce à l’imagerie calcique ex vivo, nous avons observé une altération des réseaux du DMS et du DLS dans des conditions naïve ainsi qu’une absence de réorganisation des réseaux après l’apprentissage. Ainsi, ces résultats confirment l’importance de la réorganisation des réseaux pour l’apprentissage moteur.L’ensemble de ce travail offre de nouvelles perspectives quant au rôle des réseaux CS et leur réorganisation dans l’apprentissage moteur. La nécessité des cellules HA et des clusters ouvrent les portes du monde de l’engramme dans les réseaux striataux
Procedural memory is the memory of habits, involved in the acquisition and maintenance of new motor skills. The neural substrates underlying this memory are the basal ganglia (BG), a group of structures involved in motor and cognitive functions. The input nucleus of the BG is the striatum, earning it a central role in relaying information between the cortex and other subcortical structures, thus ensuring the selection and integration of cortical information within parallel functional loops. Procedural learning first follows a goal-directed behavior mediated by the associative loops, including the dorsomedial striatum (DMS), which is then transferred to an automatic behavior where habit is formed and mediated by the sensorimotor loops including the dorsolateral striatum (DLS). The anatomy and the evolution of the dynamics of the striatal networks has been well described during procedural learning, and the involvement of each striatal territory in a specific phase of learning established. However, how procedural learning is encoded at the level of the corticostriatal networks remains unknown.During my PhD work, we were interested in characterizing the dynamics of the corticostriatal networks involved in motor skill learning and determining the neural correlates responsible for the formation of this memory. We first used two-photon ex vivo calcium imaging to monitor the activity of the networks during the different phases of procedural learning. First we extracted the calcium responses of only medium spiny neurons (MSNs), the striatal output neurons. To distinguish MSNs from other striatal neurons, we developed a cell-sorting classifier based on the calcium responses of neurons and their morphology. We showed a specific reorganization of the DMS networks during the early phase, and the DLS during the late phase of motor skill learning. In DMS, the activity of the networks decreased after early training and returned to a basal level after late training. The main activity of the DMS networks was held by a group of highly active (HA) cells. In DLS, the reorganization of the activity was gradual and localized in small clusters of activity after late training. We then examined the properties of the HA cells in DMS and clusters in DLS. The existence of HA cells and clusters are directly correlated to the performance of the animals. Whole-cell patch-clamp recordings allowed us to characterize electrophysiological properties of HA bells and determine an increase of the synaptic weight of cingulate cortex inputs to HA cells in DMS after early learning. Anatomical tracing showed more robust changes in the DLS with an increase of the number of somatosensory projections to the DLS after late training. Using an AAV cFos-TRAP strategy coupled to chemogenetics, we inhibited HA and cluster cells, leading to impaired motor learning. These experiments thus highlighted the necessity of these cells in early and late phases of motor skill learning respectively.Next we wanted to explore if deficits in motor skill learning occur in a premotor-symptomatic phase of a mouse model of Huntington’s disease (HD), and if they would be associated to dysfunctions in the corticostriatal networks. We first showed deficits in the late phase of motor skill learning in a mouse model of HD. Using ex vivo two-photon calcium imaging, we explored the DMS and DLS networks and we observed an alteration of both networks in naïve HD animals and in addition, an absence of reorganization upon motor skill learning. These results confirm the importance of the reorganization of the networks in motor skill learning.Altogether, this work provides a new insight on the role of the corticostriatal networks and their reorganization in motor skill learning. The necessity of HA and cluster cells opens the door of the ‘engram’ world to the striatal networks

L-glutamate (Glu) is the predominant excitatory neurotransmitter inthe mammalian central nervous system (CNS) and is associated with a widevariety of functions including motor behavior and sensory perception. Whilemicrodialysis methods have been used to record tonic levels of Glu, little isknown about the more rapid changes in Glu signals that may occur in awakeanimals. We have previously reported acute recording methods using anenzyme-based microelectrode array (MEA) with fast temporal resolution (800msec), that is minimally invasive and is capable of detecting low levels of Glu (andlt;0.2 ??M) in anesthetized animals with little interference from other analytes. Wehave made a series of modifications to the MEA design to allow for reliablemeasures in the brain of awake behaving rats. In these studies, wecharacterized the effects of chronic implantation of the MEA into the striatum andprefrontal cortex (PFC) of Fischer 344 and Long Evans rats. We measuredresting levels of Glu and local application of Glu for 7 days without a significantloss of sensitivity and determined that Glu measures due to exogenous Gluvaried between rat strain and brain region. In addition, we determined theviability of the recordings in the brains of awake animals. We performed studiesof tail-pinch induced stress which caused an increase in Glu in the striatum andPFC of Long Evans and Fischer 344 rats. Histological data show that chronicimplantation of our MEAs caused minimal injury to the CNS. Taken together, ourdata support that chronic recordings of tonic and phasic Glu can be carried out inawake rats reliably for 7 days in vivo allowing for longer term studies of Gluregulation in behaving rats.

Thesis (Ph. D. in Neuroscience)--Massachusetts Institute of Technology, Dept. of Brain and Cognitive Sciences, 2013.
Cataloged from PDF version of thesis. "February 2013."
Includes bibliographical references (p. 118-126).
Learning to direct behaviors towards goals is a central function of all vertebrate nervous systems. Initial learning often involves an exploratory phase, in which actions are flexible and highly variable. With repeated successful experience, behaviors may be guided by cues in the environment that reliably predict the desired outcome, and eventually behaviors can be executed as crystallized action sequences, or "habits", which are relatively inflexible. Parallel circuits through the basal ganglia and their inputs from midbrain dopamine neurons are believed to make critical contributions to these phases of learning and behavioral execution. To explore the neural mechanisms underlying goal-directed learning and behavior, I have employed electrophysiological and electrochemical techniques to measure neural activity and dopamine release in networks of the striatum, the principle input nucleus of the basal ganglia as rats learned to pursue rewards in mazes. The electrophysiological recordings revealed training dependent dynamics in striatum local field potentials and coordinated neural firing that may differentially support both network rigidity and flexibility during pursuit of goals. Electrochemical measurements of real-time dopamine signaling during maze running revealed prolonged signaling changes that may contribute to motivating or guiding behavior. Pathological over or under-expression of these network states may contribute to symptoms experienced in a range of basal ganglia disorders, from Parkinson's disease to drug addiction.
by Mark W. Howe.
Ph.D.in Neuroscience

L-glutamate is the predominant excitatory amino acid neurotransmitter inthe mammalian central nervous system. Prior aging studies have focusedprimarily on dopaminergic circuitry of the striatum, and data obtained studyingglutamate regulation in the striatum have been largely equivocal. Thesediscrepancies are due in large part to the limitations of microdialysis; while it isextremely sensitive to minute concentrations of analyte, it is lacking in terms ofthe temporal resolution necessary to study a neurotransmitter with rapid releaseand clearance kinetics such as glutamate. In order to address this matter, ourlaboratory has designed a ceramic-based multisite microelectrode with thecapability to detect and analyze fluctuations in extracellular glutamateconcentrations on a sub-second basis. These microelectrodes were utilized tostudy the phasic release and uptake dynamics of potassium-evoked glutamate inthe striatum of young (6 month), late-middle aged (18 month) and aged (24month) Fischer 344 rats. Our results showed a reduced glutamate clearancerate and an attenuated response to potassium depolarization in the corticostriatalprojections of aged animals in comparison to other age groups. In addition,average maximal glutamate release amplitudes were decreased in the striatumof aged animals. Pressure ejection of exogenous glutamate solution furtherconfirmed the decreased glutamate clearance ability of the aged striatum. Thesepotassium and exogenous glutamate data also highlighted a markeddorsoventral gradient in the striatum in terms of glutamate release and clearanceability. We further explored this phenomenon of age-related decreased glutamateuptake by coupling our in vivo technology with classical immunoblotting andbiotinylation techniques in order to investigate glutamate transporter regulation.Decreased glutamate clearance in the aged rats cannot be attributed to areduction in steady-state total transporter protein levels. Rather, our resultsindicate that reduced plasma membrane surface trafficking of GLAST in the agedstriatum may be partially responsible for this effect. Finally, we modified ourmicroelectrodes to study basal glutamate levels in the striatum of the aging,freely moving rat. This approach allowed us to study extracellular glutamateregulation free from the potential confounding variable of anesthesia. Our resultsdemonstrate that there is no significant alteration in basal glutamate levels inaging in the brain regions investigated. More importantly, this study validated theefficacy of the utilization of ceramic-based multisite microelectrodes for the studyof alterations in glutamate neurotransmission in the aging, freely moving rat, andit lays the foundation for future work correlating such changes with age associatedimpairments in motor function.

La regulació dopaminèrgica presinàptica és important per mantenir un equilibri homeostàtic dels nivells emmagatzemats de dopamina i el seu alliberament. Els canvis en la neurotransmissió de dopamina contribueixen a trastorns neurològics i psiquiàtrics. Treballs recents del nostre grup (Ma et al., 2015; González-Sepúlveda et al.,-presentada) van descriure importants efectes de diverses classes de fàrmacs dopaminèrgics sobre la síntesi de dopamina, inclosa la L-DOPA (emprada en Parkinson), la tetrabenazina (Huntington) i aripiprazol (esquizofrènia). En aquest estudi, vam confirmar i ampliar aquestes troballes i vam comparar els efectes dels antipsicòtics agonistes parcials D2R cariprazina i brexpiprazol, els pricostimulants amfetamina i metilfenidat i diversos altres compostos selectius i experimentals. L’estriat cerebral de rata va ser trocejat i incubat ex-vivo en presència o absència d’aquests fàrmacs a diferents concentracions. De manera espontània, la dopamina i la serotonina es van acumular al llarg del temps i van assolir nivells d’emmagatzematge gairebé màxims. Aquest enfocament experimental ens va permetre avaluar la seva síntesi i dinàmica d’emmagatzematge sota la influència d’agents farmacològics escollits. Els nostres resultats podrien ser útils per comprendre els mecanismes d’acció dels antipsicòtics, i podrien facilitar més investigacions amb models animals i assajos clínics mitjançant nous agents dopaminèrgics.
La regulación dopaminérgica presináptica es importante para mantener un equilibrio homeostático de los niveles almacenados y liberación de dopamina. Los cambios en la neurotransmisión de dopamina contribuyen a los trastornos neurológicos y psiquiátricos. Hallazgos recientes de nuestro grupo (Ma et al., 2015; González-Sepúlveda et al., presentado) describieron los fuertes efectos de varias clases de medicamentos dopaminérgicos en la síntesis de dopamina, incluida L-DOPA (utilizada en Parkinson), tetrabenazina (Huntington) y aripiprazol (esquizofrenia). En este estudio, confirmamos y ampliamos esos hallazgos y comparamos los efectos de los antipsicóticos agonistas parciales D2R cariprazina y brexpiprazol, las psicoestimulantes anfetamina y metilfenidato varios otros compuestos selectivos y experimentales. El estriado cerebral de rata fue troceado e incubado ex-vivo en presencia o ausencia de estos fármacos a diferentes concentraciones. Espontáneamente, la dopamina y la serotonina se acumularon con el tiempo alcanzando niveles de almacenamiento casi máximos. Este enfoque experimental nos permitió evaluar su dinámica de síntesis y almacenamiento bajo la influencia de los agentes farmacológicos elegidos. Nuestros resultados podrían ser útiles para comprender los mecanismos de acción de los antipsicóticos, y podrían facilitar la investigación futura con modelos animales y ensayos clínicos utilizando nuevos agentes dopaminérgicos.
Presynaptic dopaminergic regulation is important to maintain a homeostatic balance of dopamine stored levels and release. Changes in dopamine neurotransmission contribute to neurological and psychiatric disorders. Recent findings from our group (Ma et al., 2015; González-Sepúlveda et al.,-submitted) describe strong effects of several classes of dopaminergic drugs on dopamine synthesis, including L-DOPA (used in Parkinson), tetrabenazine (Huntington) and aripiprazole (schizophrenia). In this study, I confirm and extend those findings and compare the effects of D2R partial agonist antipsychotics cariprazine and brexpiprazole, the psychostimulants amphetamine and methylphenidate, and several other selective and experimental compounds. Rat brain striatum was minced and incubated ex-vivo in the presence or absence of these drugs at different concentrations. Spontaneously, dopamine and serotonin accumulated over time reaching near-maximal storage levels. This experimental approach allowed me to evaluate their synthesis and storage dynamics under the influence of chosen pharmacological agents. My results could be useful to understand the mechanisms of action of antipsychotics, and they could facilitate further research with animal models and clinical trials using new dopaminergic agents.

The dorsolateral striatum (DLS) of the basal ganglia plays a critical role in action selection and motor control. The DLS receives cortical and thalamic afferents, which are extensively modulated by monoaminergic inputs, such as dopamine and serotonin (5-HT). Dopamine and 5-HT act as circuit neuromodulators by activating both stimulatory (Gs) and inhibitory (Gi) protein-coupled receptors that regulate synaptic mechanisms of plasticity. On a system level, 5-HT signal has been classically associated with learning of negative events, acting as an opponent of dopamine regulation of rewarding processes. Recent evidence has challenged this view, suggesting that 5HT signaling can synergize with dopamine signaling to shape reward-guided behavior. However, the molecular and synaptic correlates of this behavioral role of 5-HT at striatal circuits remain to be established. To address this hypothesis, we investigated the role of serotonergic signaling in regulating the strength of glutamatergic synaptic connections to the Medium Spiny Neurons of the direct pathway (dMSNs), which mediate movement, reward and reinforcement. Specifically, we focused on the regulation of distinct forms of long-term synaptic plasticity that depend on both the relative timing of a neuron output and an input spike (Spike timing-dependent plasticity, STDP), and on the pattern of neuronal stimulation (high-frequency stimulation, HFS). Upon a STDP protocol, the chemo-genetic inhibition of 5-HT release resulted in a long lasting depression (STDP-LTD) of glutamatergic afferents to the dMSNs of the DLS. The synaptic effects of chemo-genetic inhibition of 5-HT release were recapitulated by the pharmacological inhibition of the Gs-coupled 5-HT4 receptor subtype (5-HT4R). This form of LTD was independent from presynaptic CB1 receptor (CB1R) activation, it showed a postsynaptic locus of expression, and it was associated with an increased dendritic Ca2+ signal. We obtained similar results upon HFS; antagonism of 5-HT4R resulted in a CB1R independent form of HFS-LTD, which was associated with enhanced dendritic Ca2+ levels. Collectively, these data provide molecular and synaptic insights on the neuromodulatory role of 5-HT at striatal circuits. Dysfunctional serotonergic modulation of striatal circuits has been associated with repetitive behaviors in obsessive-compulsive disorders (OCD). Thus, elucidating how 5-HT4R manipulation affects aspects of reward-guided behavior, and how this is causally relevant for defined cognitive processes implicated in action control, could facilitate the development of new pharmacological approaches to treat OCD symptoms.

Les ganglions de la base (GB) sont principalement connus pour leurs fonctions motrices, mais présentent également des fonctions non motrices. Sans surprise, il a été montré qu’ils sont impliqués dans des troubles moteurs tels que la maladie de Parkinson ou les dystonies. Des études récentes suggèrent que les GB jouent également un rôle prépondérant dans des maladies “non-motrices” telles que l’épilepsie d’absence , qui est une épilepsie généralisée non convulsive. Dans l’ensemble de ces dysfonctions des GB, les symptômes sont accompagnés de différents patrons oscillants d’activité neuronale souvent synchronisés entre les différents noyaux des GB, le cortex et d’autres aires cérébrales. Comment les GB peuvent-ils favoriser ou soutenir ces différentes activitées oscillantes?Des expériences récentes ont montré le rôle clé joué par les GB dans l’épilepsie d’absence et remettent en question le point de vue traditionnel selon lequel les circuits thalamo-corticaux sont responsables des crises d’absence. Nous proposons une nouvelle théorie selon laquelle les rétroactions opérées par les GB sur l’activité corticale rend le réseau bistable et entraîne les patrons d’activité oscillante qui apparaîssent pendant les crises. Notre théorie est compatible avec l’ensemble des résultats expérimentaux connus et elle prédit qu’un input excitateur transitoire sur le cortex peut terminer prématurément les crises d’absence. Nous présentons ici des résultats préliminaires en accord avec cette prédiction.De multiples fréquences des oscillations d’activité sont observées dans la maladie de Parkinson au sein des GB, telles que les fréquences correspondant aux tremblement des membres ou encore les oscillations béta. Nous montrons que notre model peut générer des oscillations à différentes échelles temporelles qui coïncident avec les fréquences des oscillations dans la maladie de Parkinson. Notre théorie peut rendre compte des oscillations observées dans la maladie de Parkinson et dans l’epilépsie d’absence dans un cadre théorique unifié et suggère deux scénarios pour expliquer les multiples fréquences des oscillations d’activité, à la fois pathologiques et fonctionnelles
The Basal Ganglia (BG) are thought to be involved primarily in motor but also in non-motor functions. Unsurprisingly, the BG are shown to be involved in motor dysfunctions such as Parkinson's disease or dystonia. More recent studies suggest the key role of the BG in "non-motor" diseases such as absence epilepsy which is a generalized non-convulsive epilepsy. In these diseases, symptoms accompany various oscillatory patterns of neural activity often synchronized across the BG, cortex and other brain areas. How can the BG support these different kinds of oscillatory patterns?Recent experiments have highlighted the key role of the BG in absence seizures and question the traditional view in which thalamocortical circuits underlie absence seizures. We propose a novel theory according to which the feedbacks of cortical activity through BG make this network bistable and drive the oscillatory patterns of activity occurring during the seizures. Our theory is compatible with virtually all known experimental results and it predicts that well-timed transient excitatory inputs to the cortex advance the termination of absence seizures. We report preliminary experimental results consistent with this prediction.Multiple oscillatory frequencies are observed in Parkinsonian BG such as the frequencies of the limb tremor and the beta oscillations. We show that our model can generate oscillations with multiple timescales which resemble Parkinsonian oscillations. Our theory can model the oscillations in Parkinson's disease and absence epilepsy in a unified framework and points to two scenarios to explain multiple frequencies of pathological and functional oscillations

Assessing costs and benefits associated with different options that vary in terms of reward magnitude and uncertainty is an adaptive behaviour which allows us to select an optimal course of action. Previous studies using reversible pharmacological inactivations have shown that the basolateral amygdala (BLA) to nucleus accumbens (NAc) pathway plays a key role in promoting choice towards larger, riskier rewards. Neural activity in the BLA and NAc show distinct, phasic changes in firing prior to action initiation and following action outcomes. Yet, how temporally precise patterns of activity within BLA-NAc circuitry influence choice behaviour is unclear. We assessed how optogenetic silencing of BLA projection terminals in the NAc altered action selection during probabilistic decision making. Rats that received intra-BLA infusions of an AAV encoding for the inhibitory opsin eArchT were well-trained on a probabilistic discounting task, where they chose between a smaller/certain reward and a larger/riskier reward, with the probability of obtaining the larger reward changing from 50% to 12.5% across two separate blocks of trials. During testing, discrete 4-7 second pulses of light were delivered via fiber optic ferrules into the NAc to suppress activity within BLA terminals during specific task events; during the period prior to choice or during the outcome immediately following a choice. Silencing activity of BLA inputs to the NAc prior to choice reduced selection of the more preferred option, suggesting that at this time, activity within this pathway biases choice towards more preferred rewards. Silencing during reward omissions increased risky choice during the low-probability block, indicating that activity in this circuit after non-rewarded actions serves to modify subsequent choice behaviour. In contrast, silencing during rewarded outcomes did not reliably affect choice behaviour. Collectively these data demonstrate how patterns of activity in BLA-NAc circuitry convey different types of information that guide optimal action-selection in situations involving reward uncertainty.
Arts, Faculty of
Psychology, Department of
Graduate

Appendix 2: Movie Clips is supplied as a Zip Archive and will need to be unzipped before viewing. The mechanism of force transfer from contracting sarcomeres to the membrane and endomysium of striated muscle fibres is unclear. The caveolar complex array in striated muscle membranes is a local concentration of cholesterol, sphingomyelin, signalling molecules and the protein caveolin-3. Immunofluorescence microscopy of caveolin-3 in the membrane reveals a regular pattern of fluorescent nodes arranged in longitudinal and transverse rows. The primary aim of this study was to analyse this pattern and how caveolin-3 behaves during contraction. Dynamic imaging and Fast Fourier Transforms (FFTs) were used to study force transmission across the fibre membrane. This pattern was studied in frozen sections of both shortened and rest-length striated muscle fibres. Direct and FFT measurements of spacings between these nodes demonstrated significant reductions in longitudinal measurements in shortened muscle when compared to rest-length muscle. Caveolin-3 nodes lay in register with underlying actin bands in both muscle states, and co-localised with dystrophin. Caveolin-3 was not detectable in C2C12 myoblasts. During differentiation expression became detectable at 2 days. Caveolin-3 was present during myoblast fusion, before forming the regular pattern on the membrane from days 4-5. Fibres became contractile after 5-6 days of development. By 12 days, muscle fibres are 1-2 mm long, multinucleated myotubes with evidence of the caveolin-3 immunofluorescence pattern seen in mature fibres. Knockdown of caveolin-3 expression greatly reduced the number of differentiated myotubes at 12 days. This pattern was not demonstrated in contracting myotubes, possibly owing to lack of permeability to the antibody. The results are consistent with the hypothesis that the force of contraction is transferred across the whole membrane rather than at fibre distal ends.

The influence of forest canopy disturbance (FCD) on La Crosse virus (LACV), leading cause of US pediatric arboviral encephalitis, is critical to understand in landscapes where forests are periodically harvested. Southwestern Virginia is part of an emerging focus of this interior forest bunyavirus. I investigated how the temperate forest mosquito community, LACV vectors, and the LACV amplifying vertebrate host (chipmunks) were impacted by logging. This research was conducted across an experimental FCD gradient (from least to most disturbed: contiguous control, fragmented control, clearcut, and high-leave shelterwood (SW)). Using gravid traps, I found that the mosquito community was resilient to logging with no significant difference in diversity or community composition across treatments. Mean number of female mosquitoes caught per trap-night declined with disturbance. FCD significantly affected the abundance of vector species in different ways. The primary LACV vector, Aedes triseriatus, and the recent invasive Ae. japonicus declined with logging. Other vectors (Ae. albopictus, Ae. canadensis, and Ae. vexans) thrived with logging. Culex pipiens/restuans was affected by disturbance but had no treatment preference. A mark-recapture study revealed that chipmunk abundance and LACV seroprevalence were greatest on the SW. In sync with Ae. triseriatus abundance but in contrast to the chipmunk results, mosquito LACV detection was significantly greater on unlogged sites. Surprisingly, LACV was detected in Ae. japonicus and Cx. pipiens/restuans. In a follow-up study, I isolated LACV from field-collected Ae. japonicus. Although LACV was previously isolated from Cx. pipiens, the vector competence was unknown. Therefore, I examined the vector competence of Cx. pipiens and Cx. restuans. Although poor vectors, I did detect LACV in the saliva of both species. An additional experiment found that nutritionally-stressed Cx. restuans were better vectors than those in the control group, indicating that environmental stressors (e.g., FCD) may alter the ability of accessory vectors to spread LACV. The influence of FCD on LACV is complex. Because logging decreases Ae. triseriatus abundance, human LACV risk is likely lowered by decreased transovarial vertical transmission. However, high chipmunk seroprevalence on disturbed sites suggest horizontal transmission with accessory vectors plays a larger role in LACV risk on recently logged sites.
Ph. D.

Clustering of single-cell RNA sequencing data is often used to show what states and subtypes cells have. Using this technique, striatal cells were clustered into subtypes using different clustering algorithms. Previously known subtypes were confirmed and new subtypes were found. One of them is a third medium spiny neuron subtype. Using the observed heterogeneity, as a second task, this project questions whether or not differences in individual neurons have an impact on the network dynamics. By clustering spiking activity from a neural network model, inconclusive results were found. Both algorithms indicating low heterogeneity, but by altering the quantity of a subtype between a low and high number, and clustering the network activity in each case, results indicate that there is an increase in the heterogeneity. This project shows a list of potential striatal subtypes and gives reasons to keep giving attention to biologically observed heterogeneity.

Throughout this dissertation I examine the α-pulse coupled Leaky Integrate and Fire network as a relevant model accounting for biological observations in the neuroscience’s field. In doing so, I study in detail the role of the structural properties (parameters) of the α-LIF model in shaping the dynamics in three experimentally inspired applications: First I provide a theoretical substrate to understand the role of sparse connectivity in generating non-trivial dynamics in excitatory networks, akin to the Giant Depolarizing Potentials (GDP) in developing hyppocampal networks. I then analyze the importance of inhibition as the origin of irregular spiking and perturbation amplification of balanced neural networks, which are prototypical of cortical dynamics. Finally I describe how sparse connectivity, inhibition and synaptic dynamics can provide the conditions of emergence of cell assembly dynamics, which are related to the encoding mechanism observed in the striatum.

Time is a fundamental dimension of the environment. The ability to estimate the passage of time is essential for both learning and performance of adaptive behavior in natural situations. Yet, how this ability is implemented in the brain is poorly understood

碩士
國防醫學院
解剖學研究所
84
Heterotransplantion various types of embryonic tissue to mammalian brain has long been as a tool to investigate basic biological issues and applications in central nervous system. Previously, scientists using embryonic olfactory bulb inserted to central nervous system that indicated grafting olfactory bulb neurites could navigate and elongate in host tissue. However, adult tissue is being much less to know their capabilities. In order to explore this question, a study for heterotopically transplanted was made. Adult olfactory bulb derived from Spraque-Dawley rats was allografted into the striatum in adult male host. At different time points such as 7, 30, 120 days after surgery for major experiments, animals were sacrificed and the host brain tissue was analysed to evaluate progressive changes onto the graft through the use of light and electron microscopy. TH immunohistochemistry was used to determine whether neurons of graft tissue may survive in the host CNS environment. Nerve path tracer horseradish peroxidase (HRP) was injected into the striatum to determine the various patterns of nerve innervation in comparison with those of phenomenon between before and after grafting. Electron microscope was used to observe that ultrastructure changes in graft tissue at different time points after grafting. To date, data suggest that neurons of graft tissue still survive at least 120 days after grafting. Striatal nerve innervation display extraordinarily isolated phenomenon following grafting compared to that patterns exhibited before grafting; particularly at the time point 30 days after grafting. Certain amounts of neurons, non-myelinated nerve fibers, myelinated nerve fibers, incoming vessels as well as conspicuous synapses appear within and/or surrounding graft region, suggesting significantly dynamic interactions occurred in this area at the series time tested under EM observation.

Dissertação de mestrado em Genética Molecular
Altered mitochondrial dynamics has been implicated in the pathogenesis of several neurodegenerative disorders, including Huntington’s disease (HD). Sirtuins, NAD+-dependent lysine deacetylases, have emerged as important cellular targets that can interfere with mitochondrial biogenesis, fission/fusion, motility and mitophagy. Among them, sirtuin 3 (SIRT3) is particularly relevant, being the main deacetylase located in mitochondria. Here we evaluated the influence of SIRT3 on mitochondrial dynamics using striatal cells derived from HD knock-in mice (STHdhQ111/Q111) versus wild-type cells (STHdhQ7/Q7). Increased mitochondrial fragmentation was observed in untransfected HD cells. Indeed, STHdhQ111/Q111 cells exhibited an overall decrease in the levels of mitochondrial fusion proteins (Mfn2, OPA1) and an increase in fission-related Fis1. Drp1 (also involved in mitochondrial fission) was preferentially accumulated in the mitochondrial fraction of HD cells. Increased LC3-II/I ratio, which evaluates autophagosome formation, was observed in STHdhQ111/Q111 cells. Moreover, the autophagy adaptor p62 was found to be decreased in mutant cells. Parkin and PINK1, two markers of mitophagy, were also assessed. Untransfected HD cells exhibited lower levels of both proteins. No significant changes were detected in phosphorylated Parkin (required for its enzymatic activation and mitochondrial translocation). These data suggest that PINK1/Parkin-dependent mitophagy is impaired in HD striatal cells. Overexpression (OE) of SIRT3 reduced the unbalance between fission/fusion by decreasing the protein levels of Fis1 in STHdhQ7/Q7 and STHdhQ111/Q111 cells, and Drp1 accumulation in mitochondria in STHdhQ111/Q111 cells. Concordantly, an increased number of mutant cells presenting tubular mitochondria was observed after SIRT3OE. An additional significant increase in LC3-II/I ratio was observed in STHdhQ111/Q111-SIRT3 cells, indicative of macroautophagy activation. Data suggest that enhanced SIRT3 levels restore mitochondrial morphology in mutant cells by reducing mitochondrial fission, with additional activation of macroautophagy.1
Alterações na dinâmica mitocondrial têm sido relacionadas com diversas doenças neurodegenerativas, incluindo a doença de Huntington (DH). As sirtuínas são deacetilases de lisinas dependentes de NAD+ que demonstraram ter um papel importante no re-estabelecimento do equilíbrio entre biogénese e fissão/fusão mitocondrial, e mitofagia. De todas, a sirtuína 3 (SIRT3) destaca-se por ser a deacetilase predominantemente localizada na mitocôndria com maior número de alvos proteícos. Neste trabalho avaliou-se o efeito da SIRT3 na dinâmica mitocondrial recorrendo ao uso de células estriatais derivadas de murganhos knock-in para a DH (STHdhQ111/Q111) versus células ‘wild-type’ (STHdhQ7/Q7). As células mutantes não transfetadas apresentaram um aumento da fragmentação mitocondrial. De facto, as células STHdhQ111/Q111 apresentaram um decréscimo dos níveis proteícos de Mfn2 e OPA1, duas proteínas envolvidas na fusão mitocondrial, e um aumento de Fis1, uma proteína relacionada com a fissão mitocondrial. Verificou-se ainda uma acumulação preferencial da Drp1 (também envolvida na fissão mitocondrial) na fração mitocondrial das células STHdhQ111/Q111. Embora se tenha observado um aumento do rácio LC3-II/I (que avalia a formação de autofagossomas) nas células STHdhQ111/Q111, os níveis do adaptador autofágico p62 encontraram-se diminuídos. Células mutantes não transfetadas apresentaram ainda uma redução dos níveis de Parkina e PINK1, dois marcadores do processo mitofágico. Contudo, não se observaram diferenças significativas nos níveis da forma fosforilada da Parkina (indicador da sua ativação enzimática e translocação para a mitocôndria). Estas evidências sugerem alterações deste processo mitofágico nas células mutantes. A sobre-expressão de SIRT3 reduziu o desequilíbrio entre fissão/fusão ao diminuir os níveis de Fis1 nas células STHdhQ7/Q7 e STHdhQ111/Q111, e a acumulação da Drp1 na mitocôndria nas células STHdhQ111/Q111. Consequentemente, observou-se um aumentou do número de células mutantes com mitocôndrias tubulares. Verificou-se ainda um aumento significativo do rácio LC3-II/I nas células STHdhQ111/Q111- -SIRT3, indicativo de uma ativação da macroautofagia. Em conclusão, o aumento dos níveis de SIRT3 permite restaurar a morfologia mitochondrial em células mutantes ao reduzir a fissão mitocondrial, conduzindo ainda à ativação da macroautofagia.
Fundação para a Ciência e Tecnologia (FCT) - Project reference: EXPL/BIM-MEC/2220/2013;
This work was funded by FEDER funds through the Operational Programme Competitiveness Factors - COMPETE;
Fundação Luso-Americana para o Desenvolvimento (FLAD);
Gabinete de Apoio à Investigação, funded by FMUC and Santander Totta Bank;
Center for Neuroscience and Cell Biology (CNC) is supported by projects PEst-C/SAU/LA0001/2013-2014 and UID/NEU/04539/2013.

The aim of this work is to define the function of I-band titin as a dynamic element in parallel whit the array of myosin motors using fast half-sarcomere level mechanics on intact fibres of frog muscle in the presence of 20 uM para-nitro-blebbistatin (PNB)

碩士
國立清華大學
系統神經科學研究所
98
Behavioral and neuronal activity can be influenced by reward information through dopamine-modulated synaptic plasticity. Recent primate experiments (Lauereyns, Watanabe, Hikosaka 2002) using biased saccade task (BST) have demonstrated that the activity of caudate nucleus (CD) neurons are stronger and the behavioral responses are faster when the target of a saccadic eye movement indicates a reward than when it does not. It has been suggested that the observation can be explained by the following mechanism: The co-activation of the pre- and post synaptic neurons facilitates the synapses when dopamine is presented, but depresses the synapses when dopamine is absent (Hikosaka 2007; Hikosaka, Nakamura 2005). However, whether the proposed mechanism is sufficient to produce the observed behavioral and neuronal changes has not been tested. To address this problem, we built a spiking neural circuit model (Lo & Wang 2006) which includes a cortical module (Cx) that processes the visual stimulus, a basal ganglia module that employs the inhibitory control over eye movements and a superior colliculus module that drives the eye movements. By implementing the dopamine-induced plasticity in the cortico-striatal synapses, a pathway that has been shown to be a major target of dopaminergic neurons, we found that the previously proposed mechanism is not likely to be sufficient and additional neuronal interactions are needed for reproducing the observations. To address this issue, we propose a spick-timing dependent plasticity (STDP) mechanism based on the latest observations of STDP in the brain slices of rodent striatum. The proposed mechanism is able to reproduce the observations. We further explored the neural circuit model with several possible scenarios of synaptic dynamics and proposed experiments that might help to identify the detailed mechanism underlying the observed neuronal and behavioral changes in the biased reward condition.