Statement of Expectations
W. Kaya Erbil
As a potential participant of the Marine Biological Laboratory (MBL) Neurobiology course, I aspire to gain familiarity with methods of cellular and molecular neurobiology and to acquire an introduction to modern methods that facilitate the study of neural and glial events in the central nervous system (CNS). More specifically, I hope to interact with experts in the field of neurobiology and learn how they approach research problems, design experiments, and integrate experimental results into existing knowledge in order to apply their intellectual methods and approaches to my work. I also look forward to discussing science with the other students and developing potentially career-long scientific friendships.
Relevance to future research goals
As outlined in Statement of Goals, one of my present interests is the elucidation of cellular and molecular mechanisms of neurodegeneration. In particular, I would like to learn more about events that occur during the progression of Parkinson’s disease (PD) in humans and in animal models of PD. I find the PD research literature to be one of the most fascinating in the field of neurobiology. The chemical and genetic tools in the field are thoroughly developed and well understood, which allows the more subtle underpinnings of the disease to be investigated. For example, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) rapidly lesions dopaminergic neurons (1). Second, the study of PD is a paradigm for neural circuits in the CNS. The unique wiring of the substantia nigra to the rest of the circuit in the basal ganglia provides balance and gives synchrony. Loss of dopaminergic innervation in PD deranges the flow of information through basal ganglial circuits, and changes in synaptic plasticity occur in the striatum following dopaminergic neuron death in PD models (2, 3).
In the following paragraphs, I give more specific details of topics I hope to learn more about during in the Neurobiology course. I utilized the 2008 Neurobiology course syllabus and the 2009 course description as a reference for the topics that may be explored in the course. I have also explored the literature composed by the corresponding instructor of each section to illustrate potential topics of discussion outside of the classroom.
During the Electrophysiology segment of the program, I hope to learn about three specific techniques that will facilitate my research into molecular and cellular events in the basal ganglia. First, whole cell recordings from specific cell types in the basal ganglia are now made easier because a new generation of transgenic bacterial artificial chromosome (BAC) mice are available that express green fluorescent protein (GFP) under the control of cell-type specific promoters (4-6). In my future research into mechanisms of neurodegeneration, whole cell recordings from cells in the basal ganglia of these mice may reveal new aspects of brain function and dysfunction. I look forward to the opportunity to conduct an independent research project utilizing the skills learned in Electrophysiology. For example, during the 2008 course with Michael Haüsser of University College, London I might have performed whole cell recordings from substantia nigra neurons, an activity for which Haüsser’s expertise (7) would have been particularly helpful. Second, as previously mentioned, loss of dopaminergic innervation to the striatum leads to changes in synaptic plasticity in animal models of PD (2, 3). Thus, careful study of mechanisms of synaptic plasticity in the brain is essential to understanding the progression of PD. This program consistently draws experts in synaptic plasticity with whom I would be delighted to have scientific discussions. For instance, one lecturer in 2008 was Julie Kauer of Brown University, an expert in dopaminergic synaptic plasticity and addiction (8). Third, the complex anatomy of the basal ganglia presents difficulty in isolating specific subpopulations of cells for experimental analysis and impedes progress in developing a comprehensive understanding of the mechanisms and function of the neural circuits in this brain region. Light-activated ion channels are one experimental approach that may facilitate improving our understanding of the neural circuitry of this region (9). Thus, I am looking forward to lectures such as the one delivered in 2008 by Karl Deisseroth, whose laboratory at Stanford University is actively developing this new technology (10, 11).
Imaging is of particular importance to the study of glia in the brain because these cells typically do not conduct electric currents (12), rendering electrophysiological methods nearly useless. Imaging methods are also helpful in studies of neurons, where a myriad of molecules work in concert to facilitate electrical signal transduction. Molecular imaging modalities such as fluorescent reporters provide a means to study these events. Proficiency in imaging also requires skill with microscopy. As a potential participant in the program, I hope to obtain in-depth training in a variety of modern methods of microscopy and an introduction to molecular reporters of brain cell activity. Some specific approaches covered in the 2008 Neurobiology course that are potentially applicable to studying mechanisms of neurodegeneration in PD are as follows. First, the reactive nitrogen species (RNS) nitric oxide (NO) is produced by the enzyme nitric oxide synthase (NOS), two isoforms of which are dependent on cellular concentrations of calcium (Ca2+) (13, 14). Thus, Ca2+ imaging, which has been discussed during previous years’ courses, is playing an increasingly important role in the study of NO dependent signal transduction in brain cells. For example, a recent study by Ian Forsythe of the University of Leicester showed through combined NO and Ca2+ imaging at the Calyx of Held that NO can serve as a “volume transmitter” capable of modulating the activity of a population of neurons within a radial volume of tissue (14). Second, modern optical imaging techniques provide an avenue for directly observing the functional states of cells. To this end, I am hoping that Jeff Lichtman of Harvard University will be in attendance at the course, as he has in recent years. Lichtman approaches the study of the brain as a “naturalist” and applies and develops new imaging approaches to observe the cells of the nervous system in action. For example, Lichtman has observed axonal degeneration and regeneration of spinal cord neurons following trauma in transgenic GFP labeled mice (15, 16). Third, I am delighted by the prospect of interactions with experts in neuronal/glial imaging. For example, in 2008, Yi Zuo of UC Santa Cruz, delivered a lecture entitled “Confocal: barrel ex and NMJ, neuron/glia imaging”. Recently, Zuo imaged glia specifically labeled with fluorescent proteins at the neuromuscular junction (17). These analyses demonstrated the accumulation of immune cells such as macrophages at sites of injury. Similar experiments may provide new insights in the mechanism of neurodegeneration in PD if they can be accomplished in the context of the CNS.
Modern molecular biological approaches such as molecular genetics and biochemistry facilitate the study of the fundamental components of brain: proteins, nucleic acids, sugars, and lipids. I would be delighted by the opportunity to learn the following cutting edge techniques presented in the MBL course entitled Neurobiology. First, I would like to learn to employ the single cell polymerase chain reaction (PCR) of RNA transcripts. This technique was recently used by James Surmeier of Northwestern University to illuminate molecular and cellular mechanisms of neurodegeneration in the basal ganglia of rodents (18, 19). For example in one study, Surmeier’s group showed that dopaminergic neurons of the substantia nigra pars compacta (SNc) are pacemaking cells that, as assayed by single cell PCR, express a rare L-type Ca2+ channel with a Cav1.3 subunit (19). Insertion of L-type Ca2+ channels containing pore forming Cav1.3 subunits occurred with advancing age in the animals (19). The insertion of this channel was correlated with increased susceptibility of dopaminergic neurons to lesions by neurotoxins that cause Parkinsonism in mice (19). Second, electroporation of chick embryos may provide a new higher-throughput vertebrate system for modeling certain neurodegenerative diseases. For example, using electroporation of chick embryos, the research group of Christopher Shaw of King’s College London showed that wild type and mutant variants of TAR DNA binding protein (TDP-43) gain a toxic function and cause neural apoptosis and developmental delays (20). Mutant TDP-43 variants that were originally identified in human patients with amyotrophic lateral sclerosis (ALS) led to neurodegenerative defects of greater magnitude in the chick embryo, thus suggesting a pathophysiological link between TDP-43 and ALS (20). I look forward to the opportunity to learn how to utilize chick embryos in neurobiology research, as I may be able to set up a similar system for PD in my postdoctoral research in the Lindquist laboratory. Third, I look forward to possible interactions with Susan Ackerman of the Jackson Laboratory because of her recent work to characterize neurodegenerative phenotypes in mice. For instance, Ackerman’s paper entitled “The harlequin mouse mutation down-regulates apoptosis-inducing factor” (21) fascinates me because it illustrates how careful analysis of protein structure can provide new insights into molecular function in mouse models of neurodegeneration. Based on crystallization studies, the authors point out that the protein fold of apoptosis-inducing factor (AIF) is similar to that of glutathione reductase, an antioxidant enzyme capable of scavenging hydrogen peroxide (22). Over-expression of AIF in cerebellar granule cells suppressed cell death when the cells were challenged by exogenous hydrogen peroxide (21). Furthermore, Ackerman’s group observed a link between oxidative stress and abnormal cell cycle reentry in the harlequin mouse suggesting a cellular mechanism by which oxidative stress is linked to cell death in neurons (21). Lastly, C. elegans models of neurodegenerative diseases such as PD are now available (23). These models have recently been employed to identify and characterize pathways of cell death (24, 25). Eric Jorgensen of the University of Utah recently attended the MBL course entitled Neurobiology and reviewed dopamine function in the nervous system of C. elegans (26). I hope to be able to talk to Jorgensen about the possibility of studying PD in the worm.
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