The overall goal of this work is to develop anatomical, functional, and molecular magnetic resonance imaging (MRI) techniques that allow non-invasive assessment of brain function and apply these tools to study plasticity and learning in the rodent brain. MRI techniques are having a broad impact on understanding brain. Anatomical based MRI has been very useful for separating gray and white matter and detecting numerous brain disorders. Functional MRI techniques enable detection of regions of the brain that are active during a task. Molecular MRI is an emerging area, whose major goal is to image a large variety of processes in tissues. The goal of this project is to translate MRI developments in all these areas to study system level changes that occur in the rodent brain during plasticity and learning. ? ? Aim 1: Over the past few years, we have completed studies in the rodent brain that acquired very high temporal and spatial resolution functional MRI (fMRI) to monitor changes in hemodynamics as a surrogate marker of electrical activity during forepaw stimulation. Work over the past year has been completed that has acquired very high signal to noise fMRI maps to determine if the borders of activated areas can be well defined. fMRI results can be compared to anatomical borders to get precise localization of functional and anatomical boundaries. Results indicate that when very high signal to noise fMRI maps are obtained that the border of the fMRI map becomes well defined. There are two spatial components of the fMRI signal in somatosensory cortex when either forepaw or hindpaw are stimulated. The major component corresponds to the normal representation expected based on anatomy. Interestingly the minor component extends well into the neighboring region and may represent known projections that send sub-threshold information into neighboring regions. To test this idea plasticity was induced in the forepaw representation by severing the nerves in the hindpaw on the same side of the body as fMRI maps of the forepaw are made. Interestingly after a few weeks there is a change in the fMRI map such that only a single major component exists that now extends into the hindpaw representation and ends in a region similar to the minor component of the normal fMRI response. This lends support to the notion that the minor component is delineating a neuronal response that is relevant to communication between regions. Future work will use electrophysiological measurements to probe the underlying neural activity responsible for these interesting fMRI results. Finally, attempts were made to map single digits of the rat forepaw and to test if fMRI could detect well known changes that occur after amputation of a digit early in life. If a differential imaging paradigm is used, single digits can be detected with fMRI and changes in representation due to loss of a digit can be detected. These are the first results that indicate well known changes in neural representations can be detected in the rodent brain and suggest fMRI strategies that enable quantitative delineation of the precise location of these changes.? ? Aim 2: Over the past several years we have demonstrated that manganese chloride enables MRI contrast that defines neural architecture, can monitor activity, and can be used to trace neural connections. Over the last couple of years we have completed the assignment of cortical layers detected using manganese enhanced MRI by comparison to histology and have demonstrated that functional anatomy of several cortical regions of the rodent brain can be defined in individual animals. In particular, clear cytoarchitectural boundaries can be detected between numerous brain areas enabling, for the first time, cytoarchitectural changes to be followed in individual brains over time. We have also demonstrated that activity in the olfactory bulb can be imaged to the level of single glomeruli using manganese enhanced MRI and we hypothesize and have obtained evidence that indicates the flow of neural information from the glomerular to mitral layer can be imaged. Finally, we have developed sensitive MRI techniques to monitor manganese levels and can now track neural connections from the olfactory bulb to the amygdala in individual animals and laminar specific connections from thalamus to cortex and laminar specific cortical-cortical connections. Indeed, neural track tracing with manganese enables delineation of columns and dysgranular regions between major representations in the cortex. Future studies will verify these exciting initial observations that contrast to the anatomy of columns in the brain can be obtained by MRI. ? ? Aim 3: Functional MRI studies were performed to measure changes in brain activation that occur after denervation of peripheral nerves. After severing the saphanous and sciatic nerve of one hindpaw, the good hindpaw is now able to cause activation of about 50% of the damaged hindpaw's cortical representation even though it is in the opposite brain hemisphere. Lesion experiments support the model that cortical-cortical communication via the corpus collosum is responsible for this plasticity. Similar results were obtained after severing the nerves that innervate the forepaw and looking at fMRI activation of the good forepaw. High resolution fMRI indicates that the activation in the damaged cortex is about 30% of the normal amplitude and occupies about 50% of the representation. Additionally the good cortex activation increases by a factor of about two. to understand the neural basis of these changes electrophysiology was performed. Consistent with the increased fMRI in the good cortex there was an increase in local field potentials and an increase in the number of single units that responded to stimulation from about 30% of cells to 60% of cells. Interestingly in the cortex ipsalateral to the good paw, no significant local field potential could be detected even though signifcant fMRI was detected. To the best of our knowledge this is the first time fMRI and local field potentials do not coreelate without using pharmacological manipulation. Single unit recordings found a number of cells that responded to stimulation on the ipsalateral side. The majority of cells had short action potential duration and juxtapositional labeling indicates that these cells are interneurons. Thus, the fMRI activation is attributed to increased interneuron activity without significant pyramidal cell activiation in this model of injury induced plasticity. This represents the first time in the intact brain where fMRI has been caused by interneuron acitivity. This has far reaching implications for the analysis of fMRI data. Furthermore, the fact that the ipsalateral cortex is in an increased state of inhibition has behavioral consequences which will be explored in the coming year. Finally, we have begun to use manganese enhanced MRI based neuronal track tracing to determine which connections are most likely to have changed in this model of plasticity.? ? Aim 4: We have begun to explore the use of advanced MRI tools for studying simple learning paradigms in the rodent. We have begun by examining changes in neural connectivity induced by simple fear conditioning. After pairing a specific odor with a shock, manganese enhanced MRI is used to trace connections from the olfactory bulb into the brain. Preliminary results indicate that the non-invasive, functional tracing afforded by manganese enhanced MRI enables changes in connectivity to the amygdala, cortex, and putamen to be detected after fear conditioning. Over the coming year, we will need to reproduce these results. Furthermore we will use fMRI techniques to determine if fear conditioning leads to changes in odor maps in the bulb.
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