Thesis (PhD)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Investigations into the neurobiology of obsessive-compulsive disorder (OCD) have provided useful insights into this prevalent and disabling disorder in recent decades. Encouraging advances have also been made in the pharmacological treatment of OCD. This has improved the quality of life for many who typically endure chronic unremitting symptoms. Despite the widespread use of first-line agents selective for the serotonergic system in OCD, relatively little is known about the neurobiology of treatment response, the specific components of the serotonin system involved in symptom modulation, and the overlapping and distinct brain regions impacted by alternative treatment options. Despite the advance that selective serotonin re-uptake inhibitors have been, a significant proportion of patients still fail to respond adequately to these agents, and alternative pharmacological interventions are required. The use of dopamine antagonists, a strategy which until recently has had only limited supporting data, presents one such alternative. Little however, is known about which subsets of patients are most likely to respond to these agents. In this thesis, I will present a series of six studies that use pharmacological treatments and single photon emission computed tomography (SPECT) to make contributions to three primary areas in OCD namely; neurobiology, treatment and the intersection of the two. First, I address OCD neurobiology by examining the impact of OCD on resting brain function. I then examine the effects of pharmacological challenge of the serotonin 1B receptor using sumatriptan on regional cerebral blood flow (rCBF) and clinical symptomatology. Second, I examine the intersection of neurobiology and treatment as I explore the changes in rCBF in response to treatment with inositol, a precursor of the phosphoinositol second messenger system. I then examine the distinct and overlapping effects on rCBF of treatment for 12 weeks with the selective serotonin re-uptake inhibitor (SSRI) citalopram across anxiety disorders. Third, I address treatment of OCD by examining the efficacy of controlled augmentation of serotonin re-uptake inhibitors with quetiapine, a dopamine antagonist, in treatment refractory OCD. I then combine this data with a second similar dataset to derive a predictive model for treatment outcome with quetiapine augmentation of SRIs. I demonstrate that rCBF in OCD differs significantly from normal controls, is correlated with severity in frontal brain regions, and remains an important line of investigation for OCD pathophysiology that has yet to fully delineated. Pharmacological challenge of the 5HT1B autoreceptor with the selective agonist sumatriptan results in heterogeneous behavioural and regional brain perfusion changes in OCD. Attenuation of pre-frontal perfusion following 5HT1B agonist administration is in line with the effects of SRIs. This work suggests that direct or indirect effects of SRIs on the 5HT1B receptor may be involved in mediating a clinical response in OCD. In the section exploring the intersection of neurobiology and treatment, I show that changes in rCBF partially parallel treatment response to SSRIs across a range of anxiety disorders. These data suggest that a degree of overlap exists in the neurobiology of treatment response or indeed core neurobiology across different anxiety disorders. I then show that effective treatment with inositol in OCD results in rCBF changes that are partially in line with the effects of SRIs on brain perfusion. These data support suggestions that second messengers may form part of the common pathway of action for effective anti-obsessional compounds. In the study in which we augmented SRIs with quetiapine, no advantage over placebo was found. This data has, however, recently been combined with similar data in meta-analyses and demonstrated a benefit over placebo. Finally, we found that patients who have failed fewer SRI trials, have more severe illness, and clinical dimensions with a putative dopaminergic underpinning, may derive preferential benefit from serotonin/dopamine antagonist augmentation of SRIs. Through this series of clinical treatment and functional brain imaging studies in OCD, I have contributed to the neurobiological understanding of OCD, and its treatment in refractory populations. In addition I have explored the intersection of these two domains using novel as well as conventional treatment across other anxiety disorders. Treatment and pharmacological challenges used, either directly or indirectly impacted the monoamine systems serotonin and dopamine and advanced our understanding of their involvement in symptom generation. Future work should focus on the functional intersection of brain function, treatment response, and functional genetic polymorphisms within the monoamine systems of the brain. / AFRIKAANSE OPSOMMING: Ondersoek na die neurobiologie van obsessief-kompulsiewe steuring (OKS) het in die afgelope dekades sinvolle bydraes gelewer tot die begrip van hierdie algemene en verminkende steuring. Bemoedigende vordering is ook in die farmakologiese behandeling van OKS gemaak. Dit het tot ’n verbetering in kwalitiet van lewe van meeste pasiënte gelei wat normaalweg kronies en onophoudelike simptome moet verduur. Ten spyte van die uiteenlopende gebruik van eerste-linie behandeling wat spesifiek inwerk op die serotonien sisteem in OKS, is relatief min bekend oor die neurobiologie van respons op behandeling. So ook is min bekend oor; eerstens die spesifieke komponente van die serotonien sisteem wat betrokke is by simptoom modulasie, en tweedens die gedeeltelik samevallende en afsonderlike brein streke wat deur alternatiewe farmakologiese behandelings beïnvloed word. Ten spyte van die vooruitgang wat die selektiewe serotonien heropname inhibeerders tot gevolg gehad het, is daar nog altyd ‘n betekenisvolle proporsie van pasiënte wat nie voldoende respondeer op hierdie behandelings opsie nie. Dus word alternatiewe opsies benodig. Een so ‘n opsie is die klas dopamien reseptor blokkeerders wat tot onlangs min ondersteunende data gehad het. So ook, is min bekend oor die subgroepe van pasiënte wat die meeste voordeel uit hierdie alternatief sal trek. In hierdie proefskrif sal ek ‘n reeks van ses studies wat farmakologiese middels en enkel foton emissie rekenaar tomografie (EFERT) gebruik om ‘n bydra tot kennis in drie primêre areas van OKS te maak. By name; neurobiologie, behandeling, en die kruispunt van die twee. Eerstens spreek ek neurobiologie aan deur middel van ’n studie wat rustende brein bloed vloei (rBBV) in OKS ondersoek. Hierna ondersoek ek veranderings op rBBV en simptome na eenmalige toediening van ‘n serotonien 1B reseptor agonis, sumatriptan. Tweedens ondersoek ek die kruispunt van neurobiologie en behandeling deur die effek van behandeling met inositol, ‘n voorloper van die fosfoinositol tweedeboodskapper sisteem, op rBBV. Ek ondersoek dan die rBBV patroon van veranderinge in brein streke wat deur twaalf weke van behandeling met die selektiewe serotonien heropname inhibeerder citalopram in verskeie angversteurings bewerkstellig word. Laastens, spreek ek behandeling van OKS aan deur middel van ‘n gekontroleerde studie wat ondersoek instel na die effektiwiteit van die byvoeging van quetiapien, ‘n dopamien reseptor antagonis, tot serotonien heropname inhibeerders in behandelingsweerstandige OKS. Ek kombineer dan hierdie data met ’n soortgelyke datastel om ‘n model af te lei wat kliniese uitkoms vir hierdie behandelings opsie voorspel. Ek het gedemonstreer dat rBBV in OKS betekenisvol verskil van gesonde vergelykbare kontroles. Hierdie verskille het gekorreleer met ernstigheid van OKS in frontale brein streke. Dus bly hierdie tipe studies ’n belangrike rigting van ondersoek in OKS patofisiologie wat tot op hede nie tenvolle uitgewerk is nie. Eenmalige toediening van sumatriptan, het heterogene gedrags en rBBV veranderings in OKS tot gevolg gehad. Pre-frontale verhogings in rBBV voor behandeling is met 5HT1B sumatriptan toediening verminder, ’n effek wat in lyn staan met die effek van selektiewe serotonien heropname inhibeerders. Hierdie werk stel voor dat direkte of indirekte effekte van selektiewe serotonien heropname inhibeerders op die 5HT1B reseptore betrokke mag wees by die meganisme van behandelingsrespons in OKS. In die afdeling waarin ek die kruispunt van neurobiologie en behandeling ondersoek, demonstreer ek dat rBBV veranderings gedeeltelik oorvleuel met dié wat deur selektiewe serotonien heropname inhibeerders veroorsaak word in verskeie angsversteurings. Hierdie data stel voor dat oorvleueling in die neurbiologie van beide behandelingsrespons en kern neurobiologie van hierdie angversteurings ’n waarskynlikheid is. Ek wys ook dat effektiewe behandeling met inositol in OKS ook veranderings in rBBV bewerkstellig wat gedeeltelik in lyn staan met dié van die selektiewe serotonien heropname inhibeerders. Hierdie data ondersteun dus hipoteses van ‘n gemeenskaplike meganisme, wat tweede boodskapper sisteme insluit, wat in die behandelings respons van effektiewe anti-obsessionale middels betrokke is. Die finale deel van hierdie proefskrif handel oor behandeling van OKS. Ten spyte van die onvermoë om ‘n verskil tussen quetiapien en plasebo te demonstreer, het ons onlangs met hierdie data in ‘n reeks meta-analises wel ‘n voordeel vir hierdie intervensie getoon. Ten slote, het ons gevind dat (1) pasiënte wat minder kursusse selektiewe serotonien heropname inhibeerders gefaal het; (2) voor behandeling ‘n erger vorm van OKS gehad het, en (3) ook voordoen met simptoom dimensies wat oënskynlik ‘n dopaminerge basis het, die grootste waarskynlikheid toon om met quetiapien byvoeging tot selektiewe serotonien heropname inhibeerders te respondeer. Met hierdie reeks behandelings en funksionele breinbeeldings ondersoeke, lewer ek ‘n bydra tot die begrip van OKS. Spesifiek dra ek by tot die begrip van die neurobiologie, hantering van behandelingsweerstandige OKS asook die kruispunt van die twee. Farmakologiese middels wat ons óf eenmalig óf vir ‘n volle behandelingskursus toegedien het, het direkte of indirekte uitwerkings op die serotonien and dopamien sisteme gehad, en dus dra hierdie werk ook by tot kennis oor dié se betrokkenheid al dan nie in simptoom modulasie in OKS. Toekomstige werk in die area sal in die breë fokus op die kruispunt van breinfunksie, behandelingsrespons en funksionele genetiese polimorfismes van die monoamien sisteem.

The reinforcing properties of nicotine depend partly on cholinergic projections from the pedunculopontine tegmental (PPTg) and laterodorsal tegmental (LDTg) nuclei to midbrain dopamine neurons in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Neuronal activation was investigated using Fos expression in these areas following acute (0, 0.1, 0.4, 0.8mg/kg) or chronic systemic nicotine (0, 0.1, 0.4, 0.8, 1.0mg/kg given once per day for 5 days). We also examined co-localization of Fos expression in bNOS and TH positive neurons to determine what populations of neurons were activated by nicotine. Acute nicotine resulted in dose related Fos expression, with the biggest increase seen after 0.4mg/kg nicotine, but no co-localization occurred with bNOS in the PPTg/LDTg. Surprisingly, nicotine also failed to activate midbrain dopamine neurons. After animals were sensitized to nicotine there was a similar dose response curve in Fos expression, but the biggest increase was seen after 0.8mg/kg nicotine. Chronic nicotine, like acute, also preferentially activated non-cholinergic neurons in the LDTg and PPTg and non-dopamine neurons in the SNc and VTA. Further experiments looked at the mechanisms of Fos expression after nicotine administration. Fos expression in the LDTg/PPTg and SNc/VTA was suppressed after d-amphetamine, despite an increase in locomotor activity, suggesting that the increased Fos expression after chronic nicotine was not simply due to the locomotor activating effects of sensitized nicotine. Blocking autoreceptors in the dopaminergic midbrain by haloperidol pre-treatment did not increase Fos expression in dopamine neurons indicating that the inhibitory mechanism was not dependent on local autoreceptors. Novel methods of visualising and lesioning GABA neurons in the mesopontine tegmentum and midbrain were also examined. The data suggest that the mechanisms by which dopamine is involved in the pharmacological actions of passively administered nicotine are more complex than was first thought and that the role of non-dopamine neurons in the VTA (possibly GABA or glutamate containing) are also important.

Heterozygous mutations in the glucocerebrosidase (GBA) gene represent the most common risk factor for Parkinson’s disease (PD), a disease in which midbrain dopaminergic neurons are preferentially vulnerable. However, the mechanisms underlying this association are still unknown, mostly due to the lack of an appropriate model of study. In this thesis, we aimed at elucidating the role of heterozygous GBA mutations in PD using a specific human induced pluripotent stem cell (hiPSC)-based model of disease. First we developed a protocol for the efficient differentiation of hiPSCs into dopaminergic cultures, and extensively characterized the derived dopaminergic neurons which expressed multiple midbrain relevant markers and produced dopamine. Next we screened a clinical cohort of PD patients to identify carriers of GBA mutations of interest. Using for the first time hiPSCs generated from PD patients heterozygous for a GBA mutation (together with idiopathic cases and control individuals) we were able to efficiently derive dopaminergic cultures and identify relevant disease mechanisms. Upon differentiation into dopaminergic neuronal cultures, we observed retention of mutant glucocerebrosidase (GCase) protein in the endoplasmic reticulum (ER) with no change in protein levels, leading to upregulation of ER stress machinery and resulting in increased autophagic demand. At the lysosomal level, we found a reduction of GCase activity in dopaminergic neuronal cultures, and the enlargement of the lysosomal compartment in identified dopaminergic neurons suggesting a decreased capacity for protein clearance. Together, these perturbations of cellular homeostasis resulted in increased release of α-synuclein and could likely represent critical early cellular phenotypes of Parkinson's disease and explain the high risk of heterozygous GBA mutations for PD.

La synapse glutamatergique assure la majeure partie de la transmission excitatrice du cerveau et des changements de sa force constituent un corrélat cellulaire des processus d’apprentissage et de mémoire. Ces processus adaptatifs nécessitent souvent l’activation des récepteurs ionotropiques au glutamate de type NMDA (NMDAR) et l’influx calcique dans le compartiment postsynaptique qui suit leur ouverture. Jusqu’alors, l’activation des voix de signalisations sous-jacentes était considérée comme le seul mécanisme essentiel à la plasticité synaptique. Il est apparu récemment que les NMDAR diffusent à la surface des neurones, assurant un remodelage dynamique de leur distribution. La possibilité que la dynamique de surface des NMDAR joue un rôle déterminant dans les propriétés plastiques des synapses a donc émergé. Au cours de ma thèse, je me suis intéressé à cette problématique à l’aide d’approches d’imagerie dynamique à haute-résolution (ex. suivi de nanoparticules uniques, FRAP) et d’outils moléculaires de haute spécificité (ex. ligand biomimétique, x-link de récepteurs via les anticorps). J’ai dans un premier temps étudié la dynamique de surface des NMDAR endogènes au cours de la plasticité synaptique au sein de réseaux neuronaux hippocampiques in vitro. Mes résultats révèlent que l’induction de la potentialisation à long terme (LTP) des synapses glutamatergiques s’accompagne d’une redistribution latérale des NMDAR de surface dans la région postsynaptique. De façon remarquable, la réduction de la diffusion de surface des NMDAR via des anticorps commerciaux, mais aussi des anticorps purifiés de patients atteints d’encéphalite auto-immune, ciblant des épitopes extracellulaires des NMDAR, bloque la LTP. Dans un second temps, je me suis intéressé à la régulation de cette dynamique des NMDAR. En collaboration avec le groupe de Stéphane Oliet (CRI, INSERM), nous avons découvert qu’une redistribution rapide de surface des NMDAR s’opère différemment sous l’effet des co-agonistes du récepteur, la glycine et la D-sérine, et cela de façon dépendante des sous-unités GluN2A/GluN2B des NMDAR. De plus, j’ai démontré que l’interaction directe entre les NMDAR et les récepteurs dopaminergiques D1 membranaires contrôle la distribution des deux types de récepteurs aux abords de la synapse et module la plasticité synaptique. L’ensemble de ces données indique que la dynamique de surface des NMDAR est régulée par la présence d’un neuromodulateur, la dopamine, et de co-agonistes, contrôlant de façon dynamique la fenêtre plastique des synapses. / Glutamate synapse mediates most synaptic excitation in the brain and changes in its strength constitute a cellular basis for learning and memory processes. These adaptive properties often require ionotropic glutamate NMDA receptor (NMDAR) and the calcium influx in the postsynaptic compartment following their opening. So far, the activation of the subsequent signaling pathways was considered as the only mechanism essential for synaptic plasticity. It recently appeared that NMDAR diffuse at the neuronal surface, dynamically shaping their distribution. Whether the NMDAR surface dynamics and its potential regulators play an instrumental role in the plastic properties of synapses emerged thus as a possibility. During my PhD, I tackled this question using a combination of high resolution imaging techniques (e.g. single nanoparticle tracking, FRAP) and high specificity molecular approaches (e.g. biomimetic ligand, antibody based receptor cross-link). First, I studied surface dynamics of endogenous NMDAR during synaptic plasticity on hippocampal neurons in vitro. My results reveal that the induction of glutamate synapse long-term potentiation (LTP) is accompanied by a lateral redistribution of surface NMDAR within the postsynaptic area. Strikingly, reducing the surface diffusion of NMDAR using both commercial and purified antibodies from autoimmune encephalitis patients targeting extracellular epitopes of the NMDAR prevents LTP. Second I investigated whether NMDAR dynamics were regulated. In collaboration with Stephane Oliet’s group (CRI, INSERM), we uncovered that rapid surface redistribution can also be achieved differentially using the NMDAR co-agonists, glycine and D-serine, in a GluN2A/GluN2B NMDAR subunit dependent manner. In addition, I demonstrated that the direct interaction between NMDAR and dopamine D1 receptor at the membrane controls both receptors distribution in the synaptic area and modulates synaptic plasticity. Altogether, these data indicate that the NMDAR surface dynamics is regulated by ambient neuromodulators such as dopamine and co-agonists, dynamically controlling then the plastic range of synapses.

Why is motivation important? The answer is simple to most of us: it is what makes people push forward and act. Intrinsic motivation is the kind of motivation that arises from within a person, making her or him strive towards a goal for no other reward than the feeling it will bring. Additionally, this kind of motivation has shown correlations with enhanced learning, creativity, performance, optimal development, and well-being. While intrinsic motivation has long been a topic within the field of psychology, the neural correlates underlying it have only recently become of interest for researchers, and studies have shown some interesting but also contradictory findings. Therefore, the aim of this literature review thesis is to investigate the neural correlates of intrinsic motivation further. Firstly, a background review of motivation in general and intrinsic motivation in particular is presented, focusing on concepts such as the self-determination theory, flow, and cognitive evaluation theory. This is followed by a chapter on motivation- and intrinsic motivation from a neuroscientific perspective, concerning concepts such as the reward system, the undermining effect, and studies examining the neural correlates of intrinsic motivation. These studies show that there was activity in several different areas when participants were intrinsically motivated. However, a frequent pattern of activity in dopaminergic pathways involving the striatum and the prefrontal cortex (PFC) was detected in most studies, indicating the involvement of these areas in particular when a person is intrinsically motivated.

Ce manuscrit détaille la stratégie utilisée par nos laboratoires pour identifier de nouvelles molécules neuroprotectrices pour le traitement curatif de la maladie de Parkinson (MP). La MP est une maladie neurodégénérative caractérisée par des symptômes moteurs invalidants qui résultent de la dégénérescence des neurones dopaminergiques (DA) des noyaux gris centraux. Précédemment, nos laboratoires ont synthétisé et identifié au cours d’un criblage 3 molécules chefs de file qui possèdent un effet neurotrophique sur les neurones DA embryonnaires. Des études préliminaires d’ADMEtox nous ont permis de sélectionner la molécule SF41, un dérivé 6-aminoquinoxaline, pour une première évaluation de l’effet neuroprotecteur in vivo de nos molécules. En effet, cette molécule est bien tolérée chez l’animal et, administrée par voie orale, elle est capable de traverser la BHE. SF41 a montré un faible effet protecteur vis-à-vis des fibres DA dans un modèle animal de la MP. Dans le but d’augmenter l’activité neurotrophique de cette molécule, une 50ène de dérivés de seconde génération ont été synthètisés et criblés in vitro dans un modèle de mort spontanée des neurones DA. Ce criblage nous a permis d’identifier 5 molécules lead plus puisssantes et efficaces que SF41. Ces molécules, qui possédent les mêmes propriétés physico-chimiques que SF41, pourraient également atteindre le système nerveux central et ainsi conduire à un effet neuroprotecteur marqué dans un modèle animal de la MP. De plus, ces molécules possèdent un profil pharmacologique intéressant car elles sont capables d’empêcher la mise en place de mécanismes qui peuvent potentiellement contribuer à la mort des neurones DA dans la MP (stress oxydant, stress médié par les astrocytes, dyshoméostasie calcique, stress médié par la diminution en facteur trophique…). Une étude préliminaire avec la molécule PAQ, l’une de ces 5 molécules, a permis d’obtenir un effet neuroprotecteur dans un modèle in vivo de la MP qui semble supérieur à celui de la molécule SF41. Ces résultats encourageants nous donnent bon espoir d’obtenir la preuve de concept de l’activité neuroprotectrice de nos dérivés 6-aminoquinoxaline. / This manuscript describes the strategy used by our laboratories to identify new neuroprotective molecules for the therapy of Parkinson disease (PD). PD is a neurodegenerative disease characterized by disabling motor symptoms resulting from the degeneration of dopaminergic (DA) neurons of the basal ganglia. Previously, our laboratories have synthesized and identified in a screening 3 lead compound which exhibited a neurotrophic effect on embryonic midbrain DA neurons. Preliminary ADMEtox studies allowed us to select the molecule SF41, a 6-aminoquinoxaline derivative, for a first in vivo evaluation of the neuroprotective effect of our molecules in an animal model of PD. Indeed, SF41 is well tolerated in animals and is able of crossing the BBB after oral treatment. SF41 showed a weak protective effect on DA fibers in an animal model of PD.In order to increase the neurotrophic activity of this molecule, around fifty second generation derivatives were synthesized and screened in vitro in a model of spontaneous death of DA neurons. This screening allowed us to identify five lead compounds more powerful and effective than SF41. These molecules, which possess the same physico-chemical properties that SF41, could also reach the central nervous system and lead to a marked neuroprotective effect in an animal model of PD. In addition, these molecules have an interesting pharmacological profile because they are able to prevent the establishment of mechanisms that can potentially contribute to the death of DA neurons in PD (oxidative stress, stress mediated by astrocytes, calcium dyshomeostasis, stress mediated by trophic factor deprivation...).A preliminary study with the molecule PAQ, one of these five molecules, yielded a neuroprotective effect in animal model of PD that seems higher than with SF41. These encouraging results give us hope to achieve proof of concept of the neuroprotective activity of our 6-aminoquinoxaline derivatives.

Since the first successful derivation of human embryonic stem cells (hESC), rapid progress has been attained in the development of strategies in differentiation of these cells into various neural lineages, with the fundamental objective of using these cells for replacement and repair of damaged neuronal circuits in the central nervous system (CNS). Of particular interest are midbrain dopaminergic (mDA) neurons, which play a central role in regulation of voluntary movement. Degeneration or loss of function of mDA neurons in the nigrostriatal pathway is associated with Parkinson disease (PD). Stromal-Derived Inducing Activity (SDIA) is recognized as one of the most efficient methods in restricting ESC differentiation to a dopaminergic lineage, and refers to the property of mouse stromal cell lines such as PA6 or MS5 to cause ESC to differentiate to DA neurons. Although this strategy has been extensively used to generate mDA neurons from hESC, the biochemical nature of SDIA is yet unknown.  In the present study mDA neurons were generated from the BG01V2 hESC line by SDIA. To examine whether SDIA exerts its effect directly on hESC and is responsible for early dopaminergic induction, neural progenitor cells (NPC) were enyzmatically isolated from the co-cultures and allowed to differentiate in feeder-free conditions. The isolated cells were committed to a mesencephalic neural lineage, and were capable of maintaining their phenotype and developing into postmitotic mDA neurons in feeder-free conditions. The mDA neurons showed neuronal excitability and dopamine transporter function. The in vitro proliferation and differentiation of the NPC was also investigated by a BrDU incorporation assay. Next, the maintenance of cellular memory and capacity for proliferation of the mesencephalic NPC was assessed. The NPC could be expanded in vitro by five-fold as neurospheres for up to two weeks while retaining their DA differentiation potential, but did not retain a stable phenotype over extended periods of time. Preliminary transplantation experiments of neurospheres in striatal lesioned animals indicated, however, that these cells could survive and conserve their phenotype in vivo. To gain additional insight into the biochemical role of SDIA in early dopaminergic induction of hESC, the separate contributions of cell surface activity and secreted factors were examined. The data revealed that the PA6 cell surface activity promoted cell survival and was mainly responsible for enhanced neurogenesis of hESC, whereas secreted factors provided DA lineage-specific instructions. In order to identify the soluble factors responsible for the DA phenotype-inducing component of SDIA, the gene expression profile of PA6 cells was compared to that of cell lines lacking the DA-inducing property. A number of soluble factors known to be associated with CNS development that were highly expressed in PA6 cells were identified as potential DA differentiation-inducing candidates. These differentially-expressed genes included stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1 (EFNB1). When these factors, termed SPIE, were applied to the hESC, they induced dopaminergic neuronal differentiation of hESC line, BG01V2 and other karyotypically normal hESC lines in vitro. Thus, it appears that SPIE comprises the DA phenotype-inducing property of SDIA. This may provide a simple and direct means of differentiating hESC to form DA neurons in a single step, without a requirement for co-culture, animal cell lines, or animal products. / QC 20100916

The mechanisms by which antipsychotics (neuroleptics) produce their therapeutic effects in schizophrenia are largely unknown. Although neuroleptic efficacy is attributed to central dopamine D2 and/or serotonin 5-HT2 receptor antagonism, clinical improvements in schizophrenia are not seen until two or three weeks after daily neuroleptic administration. The mechanisms underlying the neuroleptic response must therefore occur downstream from initial receptor blockade and be a consequence of chronic neurotransmitter receptor blockade. The goal of the present study was to use neuroleptics with varied dopamine vs. serotonergic receptor blocking profiles to elucidate some of these intracellular post receptor mechanisms. Since the final steps of both dopamine and serotonin synthesis require the enzyme aromatic L-amino acid decarboxylase (AADC), the effects of neuroleptics on AADC gene (mRNA) expression were examined in PC12 cells and compared to their effects on the synthetic enzyme tyrosine hydroxylase (TH) and ' c-fos' (an early immediate gene [IEG]) mRNA. The neuroleptics examined did not significantly regulate AADC mRNA in PC12 cells, and only haloperidol upregulated TH and 'c-fos' mRNA. Later studies in rats showed that acute neuroleptic administration increased ' c-fos' mRNA, whereas the immunoreactivity of a related IEG (delta FosB) was increased upon chronic treatment. These studies and a subsequent dose response study demonstrated that upregulation of both 'c-fos' mRNA and delta FosB immunoreactivity was most prominent in dopaminergic projection areas including the striatum and nucleus accumbens. Because it has been suggested that neuroleptic treatment might prevent neurodegeneration in schizophrenia, the effects of neuroleptics on the mRNA expression of neuroprotective target genes of delta FosB were examined both ' in vivo' and 'in vitro'. These genes included brain-derived neurotrophic factor (BDNF), the neuroprotective enzyme superoxide dismutase (SOD), and the low affinity nerve growth factor receptor (p75). While dopamine D2 blockade unfavorably regulated BDNF and p75 mRNA, 5-HT 2 blockade either had no effect on or favorably regulated BDNF, SOD, and p75 mRNA. Thus, although little about the contribution of serotonergic blockade in the neuroleptic response was determined, dopaminergic blockade regulated IEG's and several of their target genes. Future studies will be needed to understand the role of 5-HT2 receptor blockade in the neuroleptic response.

Neural stem (progenitor) cells (NPCs) from fetal tissue are an ideal transplantable cell source. They divide rapidly, are able to generate cells of all three neural lineages and do not divide uncontrolled once transplanted into a host organism. To obtain large quantities of cells for transplantation strategies and to eliminate primary cell contaminations, long periods of in vitro cultivation are necessary. Mouse NPCs are a crucial tool for further investigations of neural stem cells because they make the employment of transgenic animals in vivo and cells in vitro possible. So far only short-term expanded fetal mouse NPCs have been shown to generate dopaminergic neurons and it is not clear whether this was due to differentiation or a result of increased survival of primary dopaminergic neurons. The aims of the thesis were to characterize mouse fetal NPCs, to establish the long-term expansion of fetal mouse NPCs and the generation of dopaminergic neurons in long-term expanded fetal mouse NPCs, to investigate the signaling mechanisms involved in the differentiation of mouse fetal NPCs towards the dopaminergic phenotype and to compare short and long-term expanded NPCs. Long-term expanded fetal mesencephalic NPCs could be grown under suspension and adherent culture conditions and showed self- renewing capacity as well as markers typical for NPCs. They could be differentiated into the three major cell types of the nervous system, but suspension NPCs had a larger potential to generate neurons than adherently grown NPCs. Signaling cascades involved in this process were p38 and Erk1/2 mediated. Long-term expanded NPCs did not have the potential to generate neuronal sub-types. Importantly, they did not generate dopaminergic neurons. Mouse fetal NPCs from three different developmental stages (E10, E12, and E14) were employed but were not able to differentiate into dopaminergic neurons using factors known to stimulate in vitro dopaminergic specification. When cultivated in vitro for short periods, fetal mesencephalic NPCs were able to generate dopaminergic neurons. By eliminating all primary Th- positive neurons, FACS-sorting of NPCs proved a de novo generation of dopaminergic neurons, because after cultivation and differentiation of Th- depleted cell solutions dopaminergic neurons were present in the culture. However, these newly generated neurons failed to incorporate BrdU, making a generation without cell division from precursors probable. The precursor population of short cultures differed from long-term expanded cultures suggesting an ‘aging’ effect of in vitro conditions. IL-1 was a potent inducer of the dopaminergic neuronal phenotype in short-term expanded in vitro cultures and was expressed in vitro as well as in vivo at E14. Several important conclusions concerning fetal mouse stem cell behavior could be drawn from the results of this work: Firstly, the results showed for the first time that in fetal mouse mesencephalic NPCs dopaminergic neurons differentiate from precursors without cell division, therefore consuming those progenitors. Therein fetal mouse NPCs differ significantly from rat and human NPCs or respond differently to the same in vitro conditions that need to be optimized for fetal mouse NPCs. Secondly, less committed precursors find appropriate conditions to proliferate but not to generate the more committed DA precursors that are able to generate dopaminergic neurons. The hallmarks of stem cells, self-renewal and multipotentiality, seem to be part of a delicate balance, that, when unsettled, goes in favor of one side without the possibility of returning to the previous status. Further research should focus on two coherent issues: the isolation of more pure populations of progenitors and the more precise characterization of progenitor populations to find out which in vitro conditions need to be provided to keep the balance between proliferation and differentiation potential. The knowledge gained about stem cells this way would help establish cell sources for transplantation strategies. / Stammzellen sind ein wichtiges Werkzeug für regenerative Therapien im Bereich der neurodegenerativen Erkrankungen wie der Parkinson’schen Erkrankung. Ein besonderer Vorteil von Stammzellen gegenüber dem bereits zur Transplantation verwendeten Primärgewebe, ist ihre Fähigkeit zur fortlaufenden Zellteilung, so dass ausreichende Mengen zur Transplantation zur Verfügung stehen. Der Vorteil von fetalen neuralen Stammzellen (fNSZ) ist ihre genomische Stabilität, die dazu führt, dass bei Transplantationen keine Tumore entstehen. Dennoch ist der Großteil ihrer Eigenschaften und Potentiale noch unbekannt und die optimalen Wachstumsbedingungen für eine lange in vitro Kultur und optimale Differenzierung in dopaminerge Neuronen müssen erforscht werden, um bessere Transplantate herzustellen. Insbesondere Stammzellen der Maus sind für die Forschung von immenser Wichtigkeit, da sie die Arbeit mit transgenen Tieren ermöglichen. Die Zielsetzungen dieser Arbeit waren die Charakterisierung der fNSZ der Maus, die Langzeitexpansion und die anschließende Differenzierung in dopaminerge Neurone. Die Signalkaskaden der frühen Differenzierung und die Unterschiede von kurz- und langzeitkultivierten Stammzellen wurden untersucht. Es konnte gezeigt werden, dass fNSZ der Maus nach Langzeitkultivierung in alle Zelltypen des zentralen Nervensystems, also Neuronen und Glia differenzieren und die dabei aktivierten Signalkaskaden p38 und Erk1/2 vermittelt sind. Das Differenzierungspotential zu neuronalen Subtypen (also auch zu dopaminergen Nervenzellen) verloren diese fetalen Stammzellen unter Kulturbedingungen schnell. Das steht im Gegensatz zu fetalen Stammzellen aus Ratte oder dem Menschen, die auch nach langer Kultivierung ihr dopaminerge Potential erhalten. Nur nach Kurzzeitkultivierung waren dopaminerge Neurone nachzuweisen, die jedoch nicht durch Zellteilung aus Vorläuferzellen hervorgegangen waren. Die Eliminierung aller primären Neurone aus der Mittelhirnisolation durch FACS-sorting von Th-Gfp transgenen Mäusen bewies die de novo Generation der dopaminergen Neurone aus Vorläuferzellen ohne Zellteilung während der Kultivierung der Stammzellen. Diese Ergebnisse zeigten, dass in fetalen mesenzephalen NSZ der Maus dopaminerge Neurone von spezialisierten Vorläuferzellen differenzieren, wodurch diese der Kultur verloren gehen. Weniger spezialisierte Vorläuferzellen finden Bedingungen, die ihre Kultivierung ermöglichen, sind aber nicht in der Lage, spezifischere Vorläuferzellen zu bilden. Die Markenzeichen von Stammzellen, Selbsterneuerung (durch Zellteilung) und das Potential, die Zelltypen des Nervensystems zu generieren, scheinen fein balancierte Zustände zu sein, die bei einer Störung nicht wiederherzustellen sind. Die Ergebnisse dieses Projektes sind von großer Bedeutung für die Forschung zur Zellersatztherapie der Parkinson’schen Erkrankung, deren ultimatives Ziel es ist, eine sichere und verlässlich expandierbare Zellquelle zu etablieren, die fähig ist, in dopaminerge Neurone zu differenzieren. Solche Stammzellen würden Bemühungen um Transplantationsstrategien für neurodegenerative Erkrankungen unterstützen und vorantreiben.

Binge alcohol consumption is a rising concern in the United States, especially among adolescents as during this developmental period alcohol use is usually initiated and has been shown to cause detrimental effects on brain structure and function. These findings have been established through the use of binge models in animals, where animals are repeatedly administered high doses of ethanol typically over a period of three or four days. While such work has examined the effects of a four-day and repeated three-day binge, there has been almost no work conducted aimed at investigating the long-term behavioral and neurochemical and/or functional consequences of repeated binge pattern administration during adolescence relative to adulthood on later ethanol-induced behavior and neurochemistry in adulthood. The present set of experiments aimed to examine the dose-response and age-related differences induced by repeated binge pattern ethanol administration during adolescence or adulthood on voluntary ethanol consumption (Aim 1), changes in ethanol metabolism following ethanol pretreatment (Aim 2) and mesolimbic dopamine functionality (Aim 3) in adulthood. In both experiments, adolescent and adult male rats were intragastrically administered ethanol (0.5, 1.0 or 2.0 g/kg/ig) or isovolumetric water on postnatal days (PND) 28-31, PND 35-38 and PND 42-45 for adolescent rats and PND 60-64, PND 67-70 and PND 74-77 for adult rats. In both experiments all rats underwent fourteen days of abstinence (PND 46-59 or PND 78-91, respectively). Subsequently, in Experiment 1, all rats underwent voluntary ethanol consumption procedures, in which animals were exposed to 10% ethanol combined with decreasing saccharin concentrations across days from PND 60-82 for adolescent-exposed rats and PND 92-114 for adult-exposed rats. Finally, on PND 83 and PND 115, respectively, all animals were challenged with 2.0 g/kg ethanol and trunk blood samples were collected at 60 and 240 minutes post-injection. Results indicate there was a significant increase in voluntary ethanol intake in adolescent ethanol-exposed rats pretreated with 2.0 g/kg relative to their adult ethanol-pretreated counterparts. Faster ethanol metabolism was observed in adolescent rats pretreated with 2.0 g/kg during adolescence relative to adolescent-exposed rats pretreated with 0.5 g/kg and adults pretreated with 2.0 g/kg. For Experiment 2, all rats underwent surgery (PND 60 for adolescent-exposed and PND 92 for adult-exposed rats). From PND 61-64 for adolescent-exposed and PND 93-96 for adult exposed rats, all animals underwent recovery from surgery. Finally, all rats underwent in vivo microdialysis on PND 65 for adolescent-exposed and PND 97 for adult-exposed rats, with K+ (100 mM) infused into the ventral tegmental area and accumbal dopamine overflow assessed in the nucleus accumbens septi. The results from Experiment 2 indicate lasting changes in mesolimbic dopamine functionality with a trend for decreased potassium-stimulated dopamine overflow in the nucleus accumbens septi in adolescent-ethanol pretreated rats and a trend for increased potassium-stimulated dopamine overflow in adult ethanol-pretreated rats. The results from the present set of experiments show the dose-dependent impact of binge-pattern ethanol exposure during adolescence on subsequent ethanol consumption and ethanol metabolism in adulthood. These findings indeed determine adolescence as a period of vulnerability to the long-term changes in ethanol consumption relative to similarly-exposed adult male rats. Importantly, the results of Experiment 2 indicate an alteration in the functionality of the mesolimbic pathway in adulthood following adolescent binge pattern ethanol exposure, which demonstrates a long-term depression in mesolimbic dopamine functionality following adolescent binge pattern ethanol exposure.