John Freeman - US grants

The overall objective of the proposed research is to identify the cellular functions and mechanisms of regulation of GAP43, the most prominent of a recently discovered class of growth associated proteins whose synthesis is selectively enhanced during periods of axonal growth and regeneration in retinal ganglion cells and other CNS neurons. We have obtained preliminary evidence suggesting that GAP43 expression is dually regulated by transcriptional as well as by post-transcriptional mechanisms, and postulate that retinal ganglion cells constitutively express the gene for GAP43, but fail to upregulate GAP43 levels following injury due to inhibitory factors, associated with contact with non-neuronal cells, conveyed by retrograde transport from the nerve terminal to the nucleus. We propose experiments designed to provide definitive information about the cellular function of this protein, to identify molecules that control its expression, and to identify the mechanisms by which its expression is regulated post-transcriptionally. Specific residues associated with known functional domains of GAP43 will be modified by oligonucleotide-directed site-specific mutagenesis. These include sequences associated with membrane attachment, phosphorylation by protein kinase C (pKC) and by casein kinase II (CKII), and GTP-G-O protein binding. Resulting functional effects will be measured in growth cones of rat retinal ganglion cells, including transport targeting, motility (using quantitative time-lapse video microscopy), intracellular calcium levels (using fura-2 imaging), and calcium channel conductance (using patch clamp analysis). Retrograde regulatory mechanisms will be investigated using blockers of axonal transport and quantitative 2D gel autoradiography. Experiments are designed to determine whether retrogradely transported molecules endocytosed from or modified by contact with mature oligodendrocytes in the optic nerve regulate the expression of GAP43, and if so, to isolate and test the regulatory molecules. The regulation of GAP43 message will be analyzed using quantitative RNA hybridization analysis and nuclear run-on assays to examine GAP43 message quantitatively. This will determine the extent to which GAP43 mRNA is regulated post-transcriptionally by alterations of message stability and translation efficiency during development and following optic nerve injury. Understanding the functions and molecular genetic control of this protein is likely to provide important insights more generally into the mechanisms of axonal growth, and in particular, might provide information critical to attempts to promote optic nerve regeneration and repair in the mammalian CNS. This information is of paramount importance in understanding the development of the visual system, and its response to injury.

Recent work has led to the discovery of a class of growth-associated proteins, called GAPs, whose expression is selectively enhanced in regenerating and developing neurons of some species. The purpose of this investigation is to study the cellular role of these growth-associated proteins, and in particular to determine whether tje abo;otu pf a meirpm tp regenerate depends on its ability to induce their synthesis. There are five related objectives. The first objective is to determine, by means of a limited phylogenetic survey, whether these proteins appear in CNS neurons lacking the capability to regenerate, or whether they occur only in growing or regenerating neurons. Two very sensitive and powerful techniques will be employed to detect and quantitate these proteins: computer-analyzed 2-dimensional gel electrophoresis, and immunological techniques. The second objective is to determine the cellular localization of GAPs, using EM immunocytochemical localization methods. The third objective is to characterize the major distinguishing characteristics of GAPs, and the fourth is to investigate the cellular functions of GAPs, by correlating the kinetics of GAP expression with different phases of axonal growth in developing and regenerating systems. The last objective is to determine how the expression of GAPs is regulated. We plan to study the effects of several likely regulatory molecules on GAP synthesis, including nerve growth factor (NGF) and cyclic AMP. The site of regulation (transcriptional, translational, of post- translational) will be determined with inhibitors of RNA and protein synthesis. Finally, specific changes in GAP messenger RNA during regeneration will be observed using a cell-free synthesis system. The significance of these studies lies in their promise to reveal some molecular mechanisms which control axonal growth. These are of paramount importance in understanding the development of the nervous system, and its response to injury.

Growth of organ primordia during post-gastrula morphogenesis, along with cell shape change and cell differentiation, leads to the form and shape of the developing tissue. Although cell replication occurs throughout the organ forming period in most tissues, its role in generating the force for forming the three dimensional shape of the tissue such as the evagination of a tube from an epithelial sheet, is not well understood. Factors that have hindered comprehension of the potential of cell division for shape generation include the previous inability to experimentally separate mitotic pressure from other shape generating processes and the lack of a system in which the process can be followed in situ. In the proposed study the role of cell replication in generating both the number and pattern of cells in the epithelium of a segment, as a stimulant for cell differentiation of specific cell types within the epithelium, and as a motive force in the shape change of the epithelium during limb bud formation will be investigated using larvae of the brine shrimp, Artemia. The simplicity of the tissue organization of these larvae makes this organism ideally suited for this study. The cells involved in the limb bud and segment forming regions are few in number, clearly defined, not surrounded by other cells, and have well timed growth periods. Analysis of the experiments will be carried out using both video-based image analysis and immunocytochemistry. The following hypotheses will be tested: 1) Cell replication occurs in a spatio-temporally directed manner that results in a set pattern of cells and regions of unequal cell density. 2) Attainment of this pattern leads to the differentiation of arthrodial membrane and tendonal cells in which microtubules are involved in cell shape change. 3) Deformation of the epithelium and evagination of the thoracopod limb bud result form unequal cell density, the presence of differentiated arthrodial membrane cells, and region-specific apolysis. 4) Maintenance of the form and shape of the limb bud involves continued patterned cell replication, new cuticle formation, and tendonal cell differentiation. The role of microtubules and microfilaments in the evagination process will also be determined. Some sections of the study will provide preliminary evidence necessary for future studies on regulation of cell replication during development of segmental structures.

The purpose of this project is to implement Phase 1 of the Geospace General Circulation Model (GGCM). The development of a GGCM is the primary goal of the GEM (Geospace Environment Modeling) program. Phase 1 of the GGCM development is designed to provide the user community with detailed simulation results for a selected set of idealized IMF and solar wind dynamic pressure conditions. The conditions to be simulated were chosen by the GGCM steering committee. The Magnetospheric Specification Model (MSM) has been an important tool in modeling the magnetosphere for several years. It is an operational component of the Air Force 55th Weather Squadron's air weather service. In the past few years. Improvements have been made to the MSM by including the Rice Field Model (RFM). This proposal would use the combination of MSM and RFM to implement Phase 1 of the GGCM for the GEM community.

DESCRIPTION (provided by applicant): The proposed research project is the continuation of an analysis of developmental changes in the central nervous systems that underlie the ontogeny of eyeblink classical conditioning in rats. The cerebellum and its synaptic connections with the pontine nuclei and inferior olive are essential components of the eyeblink conditioning neural circuitry. The mossy fiber projection from the pontine nuclei to the cerebellum forms the input pathway for the conditioned stimulus (CS) and the climbing fiber pathway from the inferior olive to the cerebellum forms the unconditioned stimulus (US) pathway. The initial findings of this project demonstrate that the ontogeny of eyeblink conditioning is correlated with developmental changes in stimulus-elicited and learning-related neuronal activity in the cerebellum. The input pathways to the cerebellum also undergo significant developmental changes. One of the most striking developmental changes in the input pathways is the development of neural feedback from the cerebellum to the inferior olive. Cerebellar feedback regulates the input pathways and thereby regulates the induction of learning-specific plasticity in the cerebellum. The initial studies suggest that the ontogeny of eyeblink conditioning is due to the development of interactions between the cerebellum, pontine nuclei, and inferior olive. The findings of the initial experiments of this project are promising, but additional studies are required to determine the origins and mechanisms of the aforementioned developmental changes in the cerebellum and its interactions with brainstem nuclei. The first specific aim of the proposed project will examine the anatomical development of the CS and US pathways. The second specific aim will examine the physiological mechanisms underlying the developmental changes in the CS pathway using electrical stimulation of the pontine nuclei, reversible inactivation of the cerebellum and red nucleus, and behavioral methods. The third specific aim will examine the mechanisms underlying the development of the US pathway using electrical stimulation of the inferior olive, pharmacological manipulations of the inferior olive, and behavioral methods. The fourth specific aim will examine developmental changes in the induction of neural plasticity within the cerebellum using in vivo and in vitro neurophysiological techniques. Elucidating the neural mechanisms underlying the ontogeny of eyeblink conditioning may lead to the discovery of general principles concerning the relationship between neural and behavioral development. In addition to the basic research goals of this project, the results of the proposed studies may lead to a better understanding of the functional pathology associated with various developmental disorders that affect the nervous system including fetal alcohol syndrome, exposure to environmental neurotoxins, infantile autism, and Down's syndrome.

[unreadable] DESCRIPTION (provided by applicant): Inhibitory conditioning is a central component of theoretical models of associative learning but its neural basis has not been fully elucidated. Conditioned inhibition is generally thought to be due to the development of an association between a conditioned stimulus (CS) and the omission of an unconditioned stimulus (US). The proposed project will initiate a research program to examine the neural mechanisms of inhibitory classical conditioning. The eyeblink conditioning preparation will be used in these experiments in order to compare the neural mechanisms of inhibitory conditioning with the previously identified neural mechanisms of excitatory conditioning. The experiments of Specific Aim 1 will investigate the role of the cerebellar cortex in conditioned inhibition using excitotoxic lesions, unit recording, and reversible inactivation. In the experiments of Specific Aim 2, pharmacological inactivation will be used to assess the roles of several possible sites within the neural circuitry underlying the acquisition and expression of excitatory eyeblink conditioning in the acquisition of conditioned inhibition. The experiments of Specific Aim 3 will determine whether conditioned inhibition involves neural systems that are distinct from the neural systems that mediate excitatory conditioning. The fundamental importance of inhibitory learning underscores the need to study its neural mechanisms. The proposed research project will provide important initial steps toward determining the neural mechanisms of inhibitory classical conditioning. A successful analysis of the neural mechanisms of inhibitory classical conditioning may lead to the application of this experimental approach to the study of excitatory and inhibitory processes that occur in other learning situations. The results of these studies may also lead to a better understanding of the functional pathology associated with various psychiatric disorders that involve deficits in behavioral inhibition.

DESCRIPTION (provided by applicant): The cerebellum and its connections with other parts of the brain act as functional loops that underlie various aspects of motor control, learning, attention, language, working memory, and emotion. Disruption of these functional loops may be the basis for symptoms of schizophrenia, bipolar disorder, autism, fragile X syndrome, and other disorders. The current proposal is designed to examine sensory inputs to the cerebellum that are necessary for learning and to determine whether the cerebellum sends feedback to these sensory inputs during learning. Pavlovian eyeblink conditioning will be used as the method for assessing cerebellar learning. Previous findings from this project identified the neural pathway necessary for auditory eyeblink conditioning, which includes the medial auditory thalamus (MAT) and its projections to the pontine nuclei. MAT neurons exhibit learning-related changes in activity during eyeblink conditioning that are hypothesized to be driven by feedback from the cerebellum. The first aim of the current proposal is to identify the neural pathway(s) necessary for visual eyeblink conditioning and to determine whether the visual thalamus also shows learning-related activity using reversible inactivation and high-density neuronal recording methods. Aim 2 is to determine whether learning-related activity in the thalamus is driven by the cerebellum or its downstream target nuclei by recording neuronal activity in the thalamus while inactivating the cerebellum and its target nuclei. Aim 3 will investigate the mechanisms underlying cross-modal facilitation of cerebellar learning using high- density neuronal recording methods. Aim 4 is to determine the roles of auditory and visual areas of the cerebral cortex in cerebellar learning using reversible inactivation. The proposed project will significantly increase knowledge about the nature of cerebellar interactions with sensory areas of the brain that underlie associative learning. Findings from this project could be used to develop methods for treating various symptoms caused by pathology in cerebellar interactions with other brain areas.

DESCRIPTION (provided by applicant): The proposed project is designed to examine the neural mechanisms underlying the ontogeny of cerebellar learning using short delay and trace eyeblink conditioning procedures. Previous findings from this projected showed that the ontogeny of delay eyeblink conditioning is highly correlated with developmental changes in the induction of neuronal plasticity within the cerebellum. A substantial body of evidence now indicates that the development of auditory input to the pontine nuclei plays a critical role in the ontogeny of cerebellar learning. The current proposal significantly extends these findings to elucidate the nature of developmental changes in auditory input to the pontine nuclei in Aim 1. It will also test the more general hypothesis that cerebellar learning depends on the development of sensory input to the pontine nuclei, not on the development of cerebellar plasticity mechanisms in Aim 2. That is, the cerebellum is capable of learning early in development but only with early developing sensory systems (olfactory, gustatory, and somatosensory), whereas cerebellar learning emerges later when late developing sensory systems (auditory and visual) are used. The scope of the project will be extended to examine the development of mechanisms underlying trace eyeblink conditioning, a cerebellar learning paradigm that requires the hippocampus and cortical projections to the pontine nuclei (Aim 3). New methods for recording neuronal activity in rat pups with moveable tetrodes will be used to examine the development of thalamic, pontine, and hippocampal activity during eyeblink conditioning. Nothing is known currently about the ontogeny of learning -related activity in the thalamus or hippocampus. Thus, the findings of these experiments will be novel and significant.

? DESCRIPTION (provided by applicant): Memory deficits are found with many neurological disorders and the breakdown of interactions between memory systems can be particularly debilitating. Different memory systems typically combine and interact to influence everyday behavior. The amygdala and cerebellum have traditionally been viewed as essential players in different types of memory, emotional and motor memory, respectively. These memory systems have been viewed as largely independent. However, recent evidence suggests that the amygdala facilitates cerebellar learning. Our preliminary data indicate that reversible inactivatio of the amygdala in rats severely impairs acquisition and retention of eyeblink conditioning, a type of cerebellar learning. Moreover, amygdala inactivation reversibly impairs the development of learning-related neuronal activity in the cerebellum. This proposal is designed to elucidate the mechanisms underlying interactions between the amygdala and cerebellum. Aim 1 is to determine how the amygdala influences cerebellar function during eyeblink conditioning using reversible inactivation of the amygdala and multi-electrode neuronal recording in the cerebellum. Aim 2 is to determine whether or not the amygdala interacts with the cerebellum through the conditioned stimulus input pathway to the cerebellum using reversible inactivation of the amygdala, axonal tracing, electrical brain stimulation, and multi-electrode neuronal recording. Aim 3 is to determine whether or not amygdala memory consolidation is necessary for facilitating cerebellar learning using protein synthesis inhibition and NMDA receptor blockade in the amygdala and multi-electrode neuronal recording in the pontine nucleus. This project would provide an unprecedented analysis of amygdala-cerebellum interactions.

Learning is how organisms adapt to changes in their environment and involves the coordination of neural systems mediating cognition, emotion, and motor control. The major goal of the proposed research program is to elucidate the neural circuit mechanisms underlying interactions between cognitive, emotional, and motor systems during associative learning. Interactions between these neural systems are particularly important because the context and emotional significance of stimuli provide essential information for acquisition and performance of motor responses. The breakdown of interactions between cognitive, emotional, and motor systems in various neurological disorders can therefore have devastating consequences for learned behaviors. The prefrontal cortex, amygdala, and cerebellum play significant roles in cognition, emotional responses, and motor learning, respectively. The proposed research program constitutes a comprehensive analysis of cerebellar interactions with the amygdala and prefrontal cortex during associative motor learning. Our general conceptual framework is that the cerebellum receives inputs from the amygdala and prefrontal cortex via the pons regarding which stimuli are important and when they occur, and the cerebellum then sends error-driven feedback to these forebrain systems to facilitate learning about important events. This conceptual framework takes into account the bidirectional relationship between the cerebellum and the relevant forebrain systems as well as interactions between forebrain systems. Multi-site electrophysiology, pathway-specific optogenetics, and precise behavioral analyses will be combined to investigate circuit-level interactions between the cerebellum, amygdala, and prefrontal cortex during associative learning and extinction (inhibitory learning) training. The proposed studies would significantly advance understanding of the neural circuit mechanisms underlying cerebellar interactions with the forebrain. This would be a substantial contribution to the field because it has been known that the cerebellum must interact with the forebrain in many contexts that are crucial for everyday life such as learning, memory, planning, control of emotions, and communication, but very little is known mechanistically about how the cerebellum interacts with the amygdala and prefrontal cortex.