Development Of Dopaminergic Neurons

Theneurotransmitter dopamine has just celebrated its 50thbirthday. The discovery of dopamine as a neuronal entity in the late 1950s and the notion that it serves in neurotransmission has been a milestone in the field of neuroscience research. This milestone marked the beginning of an era that explored the brain as an integrated collection of neuronal systems that one could distinguish on basis of neurotransm- ter identities, and importantly, in which one started to be able to pinpoint the seat of brain disease. The mesodiencephalic dopaminergic (mdDA) system, previously designated as midbraindopaminergic system, has received much attention since its discovery. The initial identification of dopamine as a neurotransmitter in the central nervous system (CNS) and its relevance to psychiatric and neurological disorders have stimulated a plethora of neurochemical, pharmacological and genetic studies into the function of dopamine neurons and theirprojections. In the last decade, studies on gene expression and development have further increased the knowledge of this neuronal population and have unmasked a new level of complexity. The start of the molecular dissection of the mdDA system has been marked by the cloning and characterization ofNurrl and Pitx3. These transcription factors were shown to have a critical function during mdDA development. These initial studies have been followed by the identification of many other proteins, which have a crucial function in the creation of a dopamine neuron permissive region, induction of precursors, induction of terminaldifferent- tion and finally maintenance of the mdDA neuronal pool.

This volume provides a variety of technical approaches to study dopamine system function and dysfunction. Chapters guide readers through dopamine release in ex vivo and freely moving animals, multi-recording devices for in vivo simultaneous single cell and population activity, in silico modeling of dopamine neurons activity, neuroanatomical approaches, unbiased stereology, ultrastructural analyses of dopaminergic neurons, and axonal innervation. Additionally, chapters also incorporate pharmacological tools to model neuropsychiatric diseases, novel behavioral paradigms to dissect dopamine's role in behavior, and functional imaging to follow human dopamine system development. In the Neuromethods series style, chapters include the kind of detail and key advice from the specialists needed to get successful results in your laboratory. Comprehensive and cutting-edge, Dopamine Neurotransmission aims to be a valuable resource for researchers in various disciplines.

The discovery of dopamine in 1957-1958 was one of the seminal events in the development of modern neuroscience, and has been extremely important for the development of modern therapies of neurological and psychiatric disorders. Dopamine has a fundamental role in almost all aspects of behavior: from motor control to mood regulation, cognition and addiction and reward, and dopamine research has been unique within the neurosciences in the way it has bridged basic science and clinical practice. Over the decades research into the role of dopamine in health and disease has been in the forefront of modern neuroscience. The Dopamine Handbook is the first single-volume publication to capture current progress and excitement in this dynamic research field.

Embryonic stem (ES) cells possess the capability to self-renew indefinitely and are capable of generating any cell of the three primary germ layers, making them an attractive source of material to investigate both basic physiological properties and neurodegenerative processes. Although ES cells can be directed into specific cell lineages, the differentiation of ES cells results in heterogenous cultures. To date, there are few differentiation protocols that produce homogenous populations of any desired cell type. Many methods have been used in an effort to obtain homogenous populations of cells; from forced expression of genes involved in developmental pathways, to FACS isolation of cells expressing markers of interest. There has been a considerable focus on generating homogenous populations of midbrain dopaminergic progenitors (or neurons) for Parkinson's disease which involves the degeneration of a specific population of midbrain dopaminergic neurons. In this thesis, I investigate the development of mouse embryonic stem cells into midbrain dopaminergic neurons using reporter cell lines. In the first experimental chapter, I investigate the expression of Lmx1a and Msx1; two key transcription factors implicated in dopaminergic neuronal development. I also examine the impact of the BMP, Shh and Wnt signalling pathways on dopaminergic neural differentiation. Activation of the BMP and Wnt pathways resulted in inhibition of neural induction and the expression of both Lmx1a and Msx1. In contrast, antagonising these signalling pathways increased the yield of tyrosine hydroxylase (TH) expressing neurons. Activating or inhibiting the Shh pathway did not affect Lmx1a, Msx1 or TH expression. These experiments show that early Lmx1a expression is not indicative of the number of dopaminergic neurons produced. Furthermore, many of the TH positive neurons derived from monolayer cultures were not of midbrain origin. In the following experimental chapter, I used immunocytochemistry and qPCR to characterise the population of cells expressing Lmx1a. The downstream targets of Lmx1a, Msx1 and Wnt1, and midbrain dopaminergic neuron markers, Lmx1b and En1, were significantly upregulated in Lmx1a positive cells. The Lmx1a positive fraction was enriched with neural progenitors, and give rise to highly neural cultures. However, the majority of neurons in the terminally differentiated cultures derived from Lmx1a positive cells were GABAergic. Immunocytochemistry identified these cells as forebrain GABAergic neurons with upper-layer identity. Furthermore, the isolated Lmx1a positive cells were not responsive to patterning cues, indicating that they were already committed towards a GABAergic neuron fate. To show that these Lmx1a+ progenitors could generate dopaminergic neurons I used an alternative differentiation paradigm, the PA6 co-culture method. Expression of Lmx1a in PA6 co-cultures was different from monolayer cultures; the percentage of Lmx1a positive cells increased throughout the differentiation period. In addition, PA6 co-culture derived TH positive cells were found to co-express Lmx1a, an occurrence that was uncommon in monolayer cultures.The ionotropic glutamate receptors on neurons derived on adherent monolayer and PA6 co-cultures were functionally characterised in the final experimental chapter. Previously, antagonism of ionotropic glutamate receptors has been reported to improve behavioural assay scores in Parkinsonian animal models (Johnson et al., 2009). Terminally differentiated monolayer cultures and PA6 co-cultures responded differently to stimulation with glutamate, AMPA kainate and NMDA. The ionotropic glutamate receptors of midbrain dopaminergic and GABAergic neurons derived from both culture systems were further investigated. An initial characterisation indicates distinct differences between the glutamate receptor populations in monolayer and PA6 co-cultures. It appears that monolayer differentiation generates AMPA expressing midbrain dopaminergic neurons in comparison to the NMDA receptors evident following PA6 differentiation. Interestingly, these differences in receptor expression appear restricted by culture method, rather than neuronal subtype, i.e. monolayer neurons expressed AMPA receptors, regardless of whether they were TH+ or GAD67+. Similarly both TH+ and GAD67+ neurons appeared to express NMDA receptors following PA6 differentiation. At present the significance of these findings is unknown. In addition, the effect of Wnt5a on cell responses to glutamate agonists was examined. Wnt5a was able to potentiate cell responses to sub-maximal concentrations of certain glutamate agonists depending on the differentiation paradigm performed.

This comprehensive reference provides a detailed overview of current concepts regarding the cause of Parkinson's disease-emphasizing the issues involved in the design, implementation, and analysis of epidemiological studies of parkinsonism.

The discovery of dopamine in 1957-1958 was one of the seminal events in the development of modern neuroscience, and has been extremely important for the development of modern therapies of neurological and psychiatric disorders. Dopamine has a fundamental role in almost all aspects of behavior: from motor control to mood regulation, cognition and addiction and reward, and dopamine research has been unique within the neurosciences in the way it has bridged basic science and clinical practice. Over the decades research into the role of dopamine in health and disease has been in the forefront of modern neuroscience. The Dopamine Handbook is the first single-volume publication to capture current progress and excitement in this dynamic research field.