Eva Fifkova, MD, PhD - US grants

Plasticity of the brain that allows its function to change adaptively in response to experience is one of the most important properties of the central nervous system. Plastic changes, occurring on synapses, are probably the most important ones because they may alter communication between neurons. Hippocampus proper and the dentate fascia are regions which show a number of age related changes. A loss of perikarya and synaptic contaats, regression of dendritic spines and dendrites were described to varying degrees in both regions. Since also a dendritic growth has been observed, which implies synapse sprouting or relocation, the ultrastructure of the neuropile will be studied in both regions. There are indications that a loss or alteration of synaptic plasticity in the hippocampus may account for a number of problems in memory and performance associated with senescence. In view of this, the proposed experiments are attempting to identify the possible cytological and cytochemical substrates which could explain changes in plastic reactions of an aging brain. Various forms of synaptic plascticity suggest that they may have a common denominator which could be linked to actin. This contractile protein is known to be present in neurons similarly to other nonmuscle cells. Actin filaments in neurons may have a dual role: as support providing elements and as a substrate for plastic reactions. With this assumption in mind, we propose to investigate the organization of actin filaments in dendritic spines, dendrites and axon terminals in the hippocampal formation of senescent rats. Of special interest will be the relation of actin filaments to: a) the plasma membrane, the pre-and postsynaptic membrane specializations and to the postsynaptic density; b) other cytoplasmic organelles, notably to the spine apparatus, smooth endoplasmic reticulum, microtubulues and synaptic vesicles. Actin microfilaments will be identified using actin's affinity to the S-1 subfragment of myosin. The spatial relation of microfilaments will be studied in stereomicrographs taken with the high voltage electron microscope. Because properties of actin filaments are critically affected by free cytoplasmic Ca++, the intracellular distribution of this ion will be studied using cytochemical calcium precipitation techniques either in freeze-substituted or aldehyde fixed tissue. The distribution of this cation will be correlated with the fine structure of hippocampal neurons under different conditions of excitation in aged rats and controls.

Alcohol has been for a long time a major health problem which affects a significant proportion of the population. This proposal is designed to study the effect of chronic ethanol administration on the ultrastructural, cytochemical and electrophysiological characteristics of glutamatergic synapses of the dentate fascia in lines of mice that were selectively bred for a differential CNS sensitivity to ethanol: the long-sleep (LS), the short-sleep (SS) and the heterogeneous (HS) mice. The target region will be the dentate fascia of the hippocampus because after chronic ethanol consumption a loss of dentate granule cells and dendritic spines was observed, together with a shrinkage of the dentate molecular layer. This indicates that dendrites, dendritic spines as well as axons and axon terminals of the perforant path are affected. A measure of the functional normalcy of the perforant path-dentate synapse will be its capacity to generate long-term potentiation upon tetanic stimulation of the perforant path. The importance of this experiment is not only in testing the normalcy of the synaptic system but also in the implication of the long-term potentiation in mnemonic functions. Its absence could be related to the damage of mnemonic processes observed following chronic ethanol consumption. The synapses of the dentate fascia use glutamate as a transmitter. In vitro glutamatergic synapses show an increase in glutamate binding accompanied by a considerable Ca++ displacement from synaptic membranes. In addition significantly lower accumulation of glutamate in synaptic vesicles was shown in the LS than in the SS mice. Therefore, ultrastructural parameters like the size and density of synaptic vesicles, the size of the postsynaptic density which harbors the synaptic receptors will be measured. Since biochemical changes observed in the synapses might not be coded in the ultrastructure but in the ultrastructural cytochemistry of the synapse, the distribution of Ca++ and organization of actin filaments will be studied employing the recently developed cytochemical methods. It is hoped that these multidisciplinary studies on the effect of chronic ethanol administration in lines of mice which differ in sensitivity to ethanol may be important in elucidating questions of practical and theoretical importance to the alcohol research.

Plastic properties of the brain that allow its function to change adaptively in response to experience are one of the most important functions of the CNS. Electrophysiological phenomena, like long-term potentiation, inwhich the response of the stimulated pathway is modified by its preceding activity, are likely to underlie neuronal plasticity. The dentate fascia yeilds hours, even days lasting potentiation following brief tetanic stimulation of its entorhinal afferents. Electron microscopy of the dentate fascia neuropile shows a long-lasting volume increase of the dentate dendritic spines on which the stimulated pathway terminates. Since such a change can profoundly affect electrical properties of the neuronal elements involved, it has been postulated that the dendritic spine enlargement might be the underlying mechanism of long-term potentiation. Furthermore, in stimulated preparations, dentate perikarya show structural changes which indicate an enhancement of protein synthesis. Experiments are, therefore, proposed which would bring conclusive evidence that structural changes observed in the dentate perikarya and dendritic spines are the underlying mechanism of long-term potentiation. This should be brought about by experiments in which both phenomena will be simultaneously observed and simultaneously suppressed by a protein synthesis blocking drug - Anisomycin. Such an experiment will furthermore provide support for the protein hypothesis as a mechanism of spine enlargement. Since the time course of long-term potentiation is comparable to that of a learned response, it has been assumed that long-term potentiation and memory formation may have a common mechanism. It follows that by establishing the mechanism of long-term potentiation one could come close to the identification of the neural mechanism of memory trace formation. Indeed, in a classical conditioning paradigm, spine enlargement similar to that observed following tetanic stimulation has been found. Should the dendritic spine enlargement in the dentate be viewed as a general neural mechanism of learning and memory, it has to occur in other behavioral tasks as well. Experiments are, therefore, proposed which will include a reward magnitude shift paradigm and aversive conditioning.

Plasticity of the brain that allows its function to change adaptively in response to experience is one of the most important properties of the central nervous system. Plastic changes, occurring on synapses, are probably the most important ones because they may alter communication between neurons. Hippocampus proper and the dentate fascia are regions which show a number of age related changes. A loss of perikarya and synaptic contaats, regression of dendritic spines and dendrites were described to varying degrees in both regions. Since also a dendritic growth has been observed, which implies synapse sprouting or relocation, the ultrastructure of the neuropile will be studied in both regions. There are indications that a loss or alteration of synaptic plasticity in the hippocampus may account for a number of problems in memory and performance associated with senescence. In view of this, the proposed experiments are attempting to identify the possible cytological and cytochemical substrates which could explain changes in plastic reactions of an aging brain. Various forms of synaptic plascticity suggest that they may have a common denominator which could be linked to actin. This contractile protein is known to be present in neurons similarly to other nonmuscle cells. Actin filaments in neurons may have a dual role: as support providing elements and as a substrate for plastic reactions. With this assumption in mind, we propose to investigate the organization of actin filaments in dendritic spines, dendrites and axon terminals in the hippocampal formation of senescent rats. Of special interest will be the relation of actin filaments to: a) the plasma membrane, the pre-and postsynaptic membrane specializations and to the postsynaptic density; b) other cytoplasmic organelles, notably to the spine apparatus, smooth endoplasmic reticulum, microtubulues and synaptic vesicles. Actin microfilaments will be identified using actin's affinity to the S-1 subfragment of myosin. The spatial relation of microfilaments will be studied in stereomicrographs taken with the high voltage electron microscope. Because properties of actin filaments are critically affected by free cytoplasmic Ca++, the intracellular distribution of this ion will be studied using cytochemical calcium precipitation techniques either in freeze-substituted or aldehyde fixed tissue. The distribution of this cation will be correlated with the fine structure of hippocampal neurons under different conditions of excitation in aged rats and controls.

spectrin; actins; synaptic vesicles; hippocampus; synapses; conditioning; neural plasticity; growth /development; electrophysiology; calcium metabolism; aging; neurofilament; dendrites; neuroanatomy; protein structure; immunoelectron microscopy; electrostimulus; laboratory rat; animal age group; crosslink;

This is a request for a Research Career Development Award Level II. The long-term objectives of this research are to study the effect of chronic ethanol exposure and withdrawal from this exposure on neuronal plasticity. Because neuronal plasticity allows for adaptation to any stimuli and conditions the nervous system is exposed to, it represents one of the most important properties of the nervous system. Animal experiments suggest that chronic ethanol consumption affects neuronal plasticity which could reduce the capacity of the brain to adjust adequately to afferent stimuli. Such deficiencies could be in turn responsible for pathological brain changes observed in chronic alcoholics. Our previous work suggests that the neuronal cytoskeleton, because of its versatility, is well suited to being involved in the mechanism of neuronal plasticity. In addition, the neuronal cytoskeleton is also likely to be involved in ethanol-induced morphological changes in neurons. Therefore, this proposal is designed to study the mechanism underlying morphological changes after chronic ethanol exposure and withdrawal from this exposure . Ethanol-induced morphological changes were shown prevailingly in dendrites, dendritic spines, and synaptic contacts. Since microtubules are responsible for maintaining dendritic and axonal arborization and actin filaments for maintaining dendritic spines and synaptic contacts, it is possible that alcohol-induced alteration of these cytoskeletal components constitutes the mechanism of morphological changes observed following chronic ethanol exposure and withdrawal. To establish the nature of these changes, we shill determine possible modifications in the composition of microtubules and actin filaments. I. With quantitative immunoblotting, relative levels of actin and tubulin polymerization will be established in whole brain preparations together with posttranslational modifications of alpha-tubulin. II. With immunoelectron microscopy, the microtubule associated proteins (MAP 2 and MAP tau) and posttranslational modifications of tubulin will be studied in dendrites and axons together with the organization of actin filaments. III[. With conventional electron microscopy, the density of microtubules in dendrites and axons and the microtubule spacing in dendrites will be determined with conventional electron microscopy in selected brain regions. Alcohol-sensitive (LS-IBG ) mice will be used in these experiments. Although the proposed experiments deal with basic question of the chronic effect of ethanol on the molecular composition of the neuronal cytoskeleton, the results could be of practical importance, leading ultimately to improvements in therapeutic interventions.

The mechanism by which ethanol exerts its effect on brain morphology is not well understood. Therefore, this proposal is designed to study the mechanism underlying morphological changes after chronic ethanol exposure and withdrawal from this exposure. Our experiments and those reported in the literature suggest that morphological changes occur prevailingly in dendrites, dendritic spines, and synaptic contacts. Since microtubules are responsible for maintaining dendritic and axonal arborization and actin filaments are responsible for maintaining dendritic spines and synaptic contacts, it is possible that alcohol-induced alteration of these cytoskeletal structures constitutes the mechanism of morphological changes observed following chronic ethanol administration. In order to establish the nature of these changes, we shall determine possible modifications in the composition of microtubules and actin filaments in individual neuronal compartments. We shall also determine whether mutual interactions between microtubules and actin filaments and their respective associated proteins are affected. To this end, we have designed the following experiments. Experiment I. With quantitative immunoblotting, we shall establish the relative levels of polymerized and nonpolymerized tubulin and actin and the relative levels of posttranslationally modified alpha-tubulin (acetylated and tyrosinated) in the whole brain. Experiment II. With immunoelectron microscopy, we shall study: a) relative levels of posttranslational modifications: acetylation and tyrosination of (alpha-tubulin subunit in axons and dendrites, respectively; b) distribution of microtubule associated proteins (MAP 2 and MAP tau); c) colocalization of MAP 2 and MAP tau with tyrosinated and acetylated microtubules in dendrites and axons, respectively; d) distribution of actin filaments in dendritic spines and their colocalization with actin regulatory proteins (myosin and MAP 2); e) colocalization of actin and MAP 2 in the postsynaptic density (PSD) of axosomatic and axospinous synapses. Experiment III. With conventional electron microscopy, we shall determine in selected brain regions the: a) density of microtubules in dendrites and axons; b) microtubule spacing in dendrites. Alcohol-sensitive (LS[IBG]) mice will be used in these experiments.

The mechanism by which ethanol exerts its effect on brain morphology is not well understood. Therefore, this proposal is designed to study the mechanism underlying morphological changes after chronic ethanol exposure and withdrawal from this exposure. Our experiments and those reported in the literature suggest that morphological changes occur prevailingly in dendrites, dendritic spines, and synaptic contacts. Since microtubules are responsible for maintaining dendritic and axonal arborization and actin filaments are responsible for maintaining dendritic spines and synaptic contacts, it is possible that alcohol-induced alteration of these cytoskeletal structures constitutes the mechanism of morphological changes observed following chronic ethanol administration. In order to establish the nature of these changes, we shall determine possible modifications in the composition of microtubules and actin filaments in individual neuronal compartments. We shall also determine whether mutual interactions between microtubules and actin filaments and their respective associated proteins are affected. To this end, we have designed the following experiments. Experiment I. With quantitative immunoblotting, we shall establish the relative levels of polymerized and nonpolymerized tubulin and actin and the relative levels of posttranslationally modified alpha-tubulin (acetylated and tyrosinated) in the whole brain. Experiment II. With immunoelectron microscopy, we shall study: a) relative levels of posttranslational modifications: acetylation and tyrosination of (alpha-tubulin subunit in axons and dendrites, respectively; b) distribution of microtubule associated proteins (MAP 2 and MAP tau); c) colocalization of MAP 2 and MAP tau with tyrosinated and acetylated microtubules in dendrites and axons, respectively; d) distribution of actin filaments in dendritic spines and their colocalization with actin regulatory proteins (myosin and MAP 2); e) colocalization of actin and MAP 2 in the postsynaptic density (PSD) of axosomatic and axospinous synapses. Experiment III. With conventional electron microscopy, we shall determine in selected brain regions the: a) density of microtubules in dendrites and axons; b) microtubule spacing in dendrites. Alcohol-sensitive (LS[IBG]) mice will be used in these experiments.