Robert J. Calin-Jageman - US grants

DESCRIPTION (provided by applicant): Learning produces long-term changes in behavior, physiology, and gene expression. Although this principle is now well-documented across the animal kingdom, fundamental aspects of learning and memory remain unclear: 1) how physiological and genetic changes supporting long-term memory are integrated across a neural circuit. 2) if forgetting represents the decay of long-term memory processes or an active erasure process, and 3) how individual differences in memory acquisition and retention arise. To address these fundamental issues in learning and memory, we propose a simple set of experiments using long-term habituation of the Aplysia tail-elicited siphon-withdrawal reflex (T-SWR). Groups of animals will be exposed to long-term habituation training (5 blocks of 30 stimuli applied to one side of the tail, 30s ISI, 90 min between blocks). This training produces a long-lasting and unilateral decrease in T-SWR behavior. To determine the physiological correlates of LTH, trained animals will be anesthetized and reduced to a siphon+tail preparation, enabling physiological recordings from the T-SWR circuit during ongoing behavior. By comparing tail-evoked neural activity from the trained and untrained side of each animal, it will be possible to identify sites of long- term neural plasticity elicited by LTH training. After physiological recordings, neurons from trained and untrained sides of each layer of the T-SWR circuit will be physically isolated. To determine the molecular correlates of LTH, RNA from harvested cells will be extracted, reverse-transcribed, and profiled using microarrays. By repeating this same procedure with animals harvested 1 and 7 days after training, it will be possible to determine physiological and genetic correlates of a memory expression (1 day) and decay (7 days). In addition, within group analyses will enable identification of physiological and transcriptional factors that predict the considerable variability evident in the acquisition and decay of long-term habituation. Finally, LTH correlates identified with this approach will be experimentally manipulated to determine their casual role in the expression and decay of LTH memory. This project provides a tractable means to gain fundamental insight into the mechanisms of long-term memory in an experimental context that offers excellent opportunities for undergraduate involvement. PUBLIC HEALTH RELEVANCE: This project will explore the genetic and neural changes that mediate long-term memory for habituation, a simple form of memory that is shared across the entire animal kingdom. By studying the mechanisms of habituation in a simple model organism (Aplysia californica), it will be possible to gain fundamental insight into the processes by memories are stored and subsequently decay. Results may have implications not only for the treatment of memory disorders, but also for a variety of attentional processes thought to depend on habituation.

? DESCRIPTION (provided by applicant): Learning produces long-term changes in behavior, physiology, and gene expression. Although this principle is now well-documented across the animal kingdom, fundamental aspects of learning and memory remain unclear, including: 1) how the transcriptional changes that maintain long-term memory are sustained and 2) if forgetting is an active process or merely the passive decay of maintenance processes. We will address these issues through the study of long-term sensitization (LTS) in the marine gastropod Aplysia californica. Animals will receive long-term sensitization training, which produces a long-lasting but unilateral memory expressed as an increase in the duration of the tail-elicited siphon-withdrawal reflex (T-SWR). Changes in gene expression will then be measured via microarray when the memory is strongly expressed (1 day after training, Aim 1) or forgotten (1 week after training, Aim 2). How are memories maintained? (Aim 1) Identification of transcriptional changes that persist 1 day after training will provide insight into the maintenance of long-term memory. We will use this data to develop a model of memory maintenance. Specifically, we will utilize a bioinformatics approach (FIRE) to discover promoter motifs specific to LTS memory maintenance. qPCR will be used to confirm regulation of the functional clusters identified by microarray and bioinformatics. We will then use a neural analogue of sensitization to probe the functional significance of these learning-regulated motifs. Are memories erased or do they just fade away? (Aim 2) Comparison of transcriptional 7 days after training will reveal if forgetting is associated with the simple decline of maintenance-related changes (as predicted by passive decay models of forgetting) or with the induction of additional transcriptional changes (as predicted by active forgetting models). Our microarray findings will provide data in support of one of these two possible mechanisms. If passive decay is supported by our data, we will explore the time-course of transcriptional decay by comparing expression changes via qPCR at 3, 5 and 7 days after LTS training. If active forgetting is supported, we will use the same time-course to delineate the activation of forgetting-related transcripts. This project is based on strong pilot data and well-validated methodologies; it provides a tractable means to gain insight into the mechanisms of long-term memory in a straightforward experimental context that offers excellent opportunities for undergraduate involvement.

Sample-size planning is currently the Achilles heel of biomedical research. Many fields of preclinical research routinely rely on inadequate samples, leading to waste, excessive false-positives, and inflated effect sizes. These practices persist because researchers often eschew sound sample-size planning for easy but invalid approaches. To help address this critical issue, this project will produce a self-guided online course that will help trainees exchange poor sample-size determination practices for sound approaches. The course will a) help redirect researchers away from erroneous approaches by making clear their severe negative consequences, b) will dispel common misconceptions that discourage sound sample-size planning, and c) will provide exercises with instant feedback to build self-efficacy for independent sample-size planning. The materials will be produced for a modest budget, will incorporate guidance from a team of renowned methodological experts, and will be anchored in a proven behavioral-change approach. The course will provide a needed supplement to current materials, which do not adequately teach sample-size determination. A thoughtful piloting plan will enable extensive feedback from diverse stakeholders to ensure strong impact not only on knowledge but also on attitudes and actual competency. Dissemination will be supported through partnerships with professional societies and the extensive social media reach of the participating guest lecturers. Funding this proposal can have a strong positive impact on rigor and reproducibility in preclinical research.

PROJECT SUMMARY/ABSTRACT: This project will investigate the neurobiology of forgetting in a model of memory with known clinical relevance. Through study of long-term sensitization memory in Aplysia californica we have found that forgetting is accompanied by changes in gene expression that could work to actively degrade stored memories (Conte et al., 2017). However, even when forgetting seems complete, there are latent traces of initial learning that persist, including both rapid-relearning (savings) and continued changes in gene expression (Perez et al., 2018). We have integrated our results into a dual-process model that suggests 1) that encoding produces both synaptic and structural changes that foster recall, 2) that an active forgetting processes erodes the synaptic changes, impairing recall, but 3) that a structural memory trace persists through epigenetic mechanisms to support rapid re-learning (Patel et al., 2018). We will test 3 key predictions of this model to provide new insights into the neurobiology of forgetting: ? Is forgetting an active process? Forgetting may not be a passive decay of stored information but may reflect instead intrinsic neuronal mechanisms that actively work to disrupt maintenance mechanisms. If so, it should be possible to experimentally manipulate the forgetting process. We will attempt to speed and slow rates of forgetting by manipulating neuromodulatory signaling from FMRFamide, a peptide transmitter. FMRFamide functions as a memory suppressor and our previous work shows it is strongly and persistently up-regulated as forgetting occurs. ? What is the nature of savings memory? Savings is the rapid acquisition of information which seemed to have been forgotten. Savings suggests that some trace of the original memory remains intact, but the molecular nature of savings remains unclear. We will characterize the transcriptional correlates of savings memory using microarray and qPCR. Our results will indicate if savings represents a lower threshold for learning or a recovery of an existing but latent memory trace. ? Do epigenetic mechanisms contribute to memory persistence? As LTS forgetting occurs, the initial transcriptional changes evoked by learning collapse (Perez et al. 2018a). Remarkably, 7 transcripts remain strongly regulated for weeks after LTS recall has fully decayed, potentially mediating savings memory (Patel et al., 2018). We will follow up promising pilot data by testing if learning alters patterns of DNA methylation. We will be able to test two specific neuron types of known behavioral relevance for changes during memory maintenance and forgetting. The research proposed in this renewal will further advance a generative line of inquiry into the fundamental mechanisms of forgetting while providing exceptional opportunities for undergraduate involvement.