Mark R. Mayford - US grants

DESCRIPTION (Adapted from applicant's abstract): Memory function is thought to involve changes in the strength of synaptic connections between neurons that are activated in the appropriate patterns during the learning process. The goal of the current proposal is to elucidate the role of Ca2+/calmodulin protein kinase II (CaMKII) in this type of activity-dependent synaptic plasticity and in memory formation. Stimulation of CaMKII in the hippocampus, a structure important for memory formation in both human beings and experimental animals, alters the ability of neurons to undergo two forms of activity-dependent synaptic plasticity: long-term potentiation (LTP) and long-term depression (LTD). A novel genetic approach will be used to express an activated Ca2+-independent mutant CaMKII transgene in both an anatomically and temporally regulated manner in order to test several hypotheses regarding CaMKII function in LTP and memory storage. In Aim 1 the effect of CaMKII activation on the stimulation frequency required to produce either LTP or LTD will be determined, and the underlying mechanism of any change will be explored. In Aim 2 the hypothesis that activated CaMKII exerts its effect on synaptic plasticity and memory by altering calmodulin availability will be tested. In Aim 3 the hypothesis that LTP in the 5-10 Hz frequency range is required for learning, memory consolidation and/or memory recall will be tested. Finally, in Aim 4 the role of the hippocampus in mediating the learning and memory defects will be determined. These experiments will provide insight into the role of CaMKII and LTP in memory formation, which might allow the development of pharmacological interventions for the treatment of various pathological conditions which affect memory.

Cellular damage caused by reactive oxygen species (ROS) is thought to play a role in numerous late onset diseases such as Parkinson's, Alzheimer's and cancer. In addition, enhanced organismal longevity itself has been associated with increased resistance to ROS and the free radical theory of aging postulates that it is the accumulation of ROS induced cellular damage that leads to the decline in various organ systems with age. A molecular understanding of the critical cellular responses to ROS has suffered from lack of a detailed genetic dissection of the process. Moreover, few animal models exist in which to test the impact of alteration in the cellular response to ROS at an organismal level. The current proposal uses screening in primary fibroblasts derived from mice carrying random ENU induced recessive mutations. The goal is to isolate single gene mutations that lead to either enhance or reduced resistance to ROS in the cultured fibroblasts. This approach allows for an unbiased genetic dissection of the cellular response to ROS induced damage and provides animal models with altered sensitivity to ROS, at least in one cell type, that are immediately available for aging studies. Although beyond the scope of this pilot grant, any heritable mutants will ultimately be positionally cloned to identify the molecular determinants of the observed phenotype.

DESCRIPTION (provided by applicant): A major difficulty in neurobiology is identifying functional neuronal pathways. Classical anatomical tracing techniques allow gross connectivity between brain regions to be determined. However, critical aspects of information storage and processing presumably reflect the activation of specific subpopulations of neurons in a given brain region in response to a given conjunction of sensory inputs. This project seeks to develop a system in transgenic mice that would allow the permanent marking of neurons that were active in a given brain region within a given time frame. The neurons would be marked with proteins that allow detection of the axonal projections of the marked neurons. In addition, any transgenically expressed protein could be targeted to these neurons. Finally, the relative level of circuit reactivation under various behavioral contingencies could also be examined with this system. The mice developed would have broad utility in studies of neuronal processing both in the study of normal brain function and in disease models.

[unreadable] DESCRIPTION (provided by applicant): Drug addiction has many components ranging from the immediate rewarding effects of the drug, escalation of drug intake, compulsive drug seeking and a tendency to relapse even many years after withdrawal, which can be the most difficult component to address from a clinical perspective. At its heart the changes in the addict's brain represent a long-lasting neuronal adaptation that must have an underlying cellular and molecular component. This has led some researchers to propose that mechanisms similar to those that mediate normal learning in memory are also involved in addiction. It has been known for many years that the consolidation of long-lasting memories requires new gene expression. This is a particularly attractive mechanism for mediating very long-lasting changes in neuronal function and work with transcription factors such as CREB and fos have supported the notion that addiction and normal memory may have common underlying molecular mechanisms. Transcriptional regulation is a complex process that requires not only the recruitment of transcription factors to the DNA but also specific modifications of chromatin structure. These epigenetic modifications are of critical importance and we have recently demonstrated using a mutant mouse that the histone acetyltransferase function of the transcriptional coactivator CBP is necessary for the development of normal long-term memory. Moreover, we showed that these behavioral deficits could be overcome using a histone deacetylase inhibitor currently in preliminary clinical trials. Since CBP is a major coactivator for CREB based transcription, which has been implicated in various addiction models, we postulate that CBP histone acetyltransferase function may be critical for long-lasting neuronal modulation in these paradigms as well. We will therefore examine behavioral sensitization of the psychomotor activating effects of cocaine, a nonassociative process, and the context-specificity of cocaine sensitization, an associative process in the CBP mutant mice. If deficits are obtained we will test the ability of histone deacetylase inhibitors to rescue these phenotypes. In this way we hope to expand our understanding of the transcriptional control of addictive mechanisms and identify new targets for potential therapeutic intervention. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Many psychiatric illnesses lead to alterations in mood, perception and memory. How activity in the brain leads to accurate perception and memory recall is a critical basic question in neuroscience that has been difficult to address directly. In this grant we develop an approach in mice that allows the genetic alteration of neurons based on their activity in response to a natural environmental stimulus or learning paradigm. We use this to introduce either the hM3Dq DREADD receptor or a variant of channelrhodopsin (ChEF) to allow the electrical stimulation of the labeled neurons either chemically or with light. In this way we can directly stimulate the ensemble of neurons activated naturally in response to a stimulus in the behaving animal to investigate the parameters required to produce a perception or memory. In preliminary studies we show that anatomically dispersed, and internally generated neural activity can be integrated into new memory. This is consistent with the idea that new memory does not form de novo but integrates with pre-existing schemas or relevant internal representations that may be active at the time of learning. Using ChEF we found that light stimulation of neurons in the retrosplenial cortex that were activated naturally with fear conditioning could produce a freezing response. This suggests that we are directly recruiting a component of the memory trace through the artificial stimulation of the correct pattern of neurons. We will extend these studies to investigate the parameters that control the integration of neural activity into new and existing memories during consolidation and reconsolidation. In addition, we will use local stimulation to directly test the optimal conditions (number of neurons, firing frequency) required for recruiting memory recall. These studies will provide the first direct test of the role of spatial and temporal patterns of neural activity in th generation of perceptions and memories. The data generated should advance our understanding of psychiatric disorders and aid in the creation of animal models in which to test treatments.

DESCRIPTION (provided by applicant): A key question in attempts to understand brain function is how does the electrical activity of neurons give rise to specific perceptions or memories? What is the neural representation of the external world or of past events? While there is a basic understanding of brain circuits at the macroscopic level between defined anatomical regions and to a lesser extent locally within brain regions, there are currently no techniques that allow the identification or manipulation of neuronal ensembles that represent a specific external stimulus or event. The primary goal of this proposal is to improve and extend upon a genetic approach that we have developed that uses the cfos promoter and the tetracycline system to allow the introduction of long lasting genetic tags into active neuronal ensembles (Reijmers et al. 2007; Matsuo et al. 2008). We will develop and validate transgenic mouse lines that allow the direct electrical and biochemical manipulation of environmentally activated neuronal ensembles. If successful, these mice will provide a tool that should be generally useful throughout a wide range of neuroscience disciplines. PUBLIC HEALTH RELEVANCE: The human mind is made up of specific bits of knowledge and memories that are integrated within a relational network. Many neurological and neuropsychiatric disorders lead to a disruption of memories or of the cognitive framework in which these memories exists. In this proposal we develop mouse models that allow us to genetically alter, and thus manipulate electrically and molecularly, neurons that make up specific memories. These tools should be useful in dissecting the underlying circuit structure of specific memories, how they are accessed, and how they are disrupted in disease models.

DESCRIPTION (provided by applicant): Information encoding in the brain is thought to be reflected in the pattern of activation of excitatory neurons in response to a given stimulus. This suggests that, in essence, a neural cell type is defined by the various stimuli and conditions that recruit its electrical activity. Alterations in activity in specific brain regions are associated wth a variety of neurological and psychiatric diseases and the pharmacological interventions to treat these diseases alter activity in specific circuits. The cellular and molecular changes that underli complex cognitive functions such as learning and memory are likely to occur at critical specific points in the circuits activated by the relevant stimuli. A great deal of effort in neuroscience is focused on defining these activated circuits however, currently available techniques are limited to discrete brain areas, lack cellular specificity, or provide a record of activity at only a singl time point preventing the identification of consistent patterns of network activation from noise or the identification of network changes over time in response to intervention. The approach that we will develop in this grant uses a single florescent marker to identify neural activity patterns t two independent time points. This provides a number of advantages over existing technology including, the ability to analyze the brain using high throughput automated imaging techniques, to identify specific cell populations in brain slices based on their activation patterns in the whoe animal for electrophysiological, morphological, or molecular studies, and the ability to apply FACS sorting techniques to the isolation of individual nuclei for epigenetic studies. The two time points at which activity is reported can be separated by at least one week allowing the analysis of circuit changes and target cell populations that are responsive to prolonged behavioral or pharmacological intervention. This should be useful in identifying the critical changes in the brain in response to these therapies.

DESCRIPTION (provided by applicant): Information encoding in the brain is thought to be reflected in the pattern of activation of excitatory neurons in response to a given stimulus. This suggests that, in essence, a neural cell type is defined by the various stimuli and conditions that recruit its electrical activity. Alterations in activity in specific brain regions are associated wth a variety of neurological and psychiatric diseases and the pharmacological interventions to treat these diseases alter activity in specific circuits. The cellular and molecular changes that underli complex cognitive functions such as learning and memory are likely to occur at critical specific points in the circuits activated by the relevant stimuli. A great deal of effort in neuroscience is focused on defining these activated circuits however, currently available techniques are limited to discrete brain areas, lack cellular specificity, or provide a record of activity at only a singl time point preventing the identification of consistent patterns of network activation from noise or the identification of network changes over time in response to intervention. The approach that we will develop in this grant uses a single florescent marker to identify neural activity patterns t two independent time points. This provides a number of advantages over existing technology including, the ability to analyze the brain using high throughput automated imaging techniques, to identify specific cell populations in brain slices based on their activation patterns in the whoe animal for electrophysiological, morphological, or molecular studies, and the ability to apply FACS sorting techniques to the isolation of individual nuclei for epigenetic studies. The two time points at which activity is reported can be separated by at least one week allowing the analysis of circuit changes and target cell populations that are responsive to prolonged behavioral or pharmacological intervention. This should be useful in identifying the critical changes in the brain in response to these therapies.