Brenda L. Bloodgood - US grants

Learning requires the conversion of transient experiences into long-lasting changes in neural circuitry. Animal behavior triggers changes in gene expression in small populations of neurons and behaviorally induced genes regulate synapses and neuronal morphology. Yet, it is unclear if changes in gene expression are the cause of behavioral plasticity, or the consequence. This project will develop a new genre of fluorescent reporters that enable the visualization and manipulation of endogenous transcription factors in individual neurons, in real time, and within the brain of behaving animals. During the award period, candidate reporters will be made that recognize six different transcription factors. These reporters will have widespread utility for investigating the molecular mechanisms that support learning in vivo and analysis of populations of neurons that are active during a learning paradigm. The development of these reporters includes ongoing training of undergraduate, graduate, and postgraduate scientists. Student training is optimized with guidance from the CREATE STEM Success Initiative on the UCSD campus.

Inducible transcription factors (ITFs) translate signals that last milliseconds or seconds into changes in cellular function that may persist for hours, days, or longer. This project will develop genetically encoded transcription factor reporters (GETFaRs) that are designed to visualize or manipulate an ITF. GETFaRs are based on molecular scaffolds, engineered through a process of synthetic affinity maturation of camelid nanobodies (Nbs) which bind the endogenous ITF. The Nb protein will be fused to a fluorophore or DNA modifying enzyme, allowing users to visualize or manipulate endogenous transcription factors. A degradation signal (degron) will be incorporated into the Nb near the ITF binding site. Consequently, GETFaRs will be constitutively expressed and rapidly degraded in the cytoplasm. When the ITF is expressed, the GETFaR-ITF interaction will mask the degron, stabilizing the complex. The ITF's nuclear localization signal will translocate the complex into the nucleus, resulting in stabilized GETFaRs that accumulate in the nucleus and stoichiometrically reflect ITF expression. Candidate GETFaRs will be validated in vitro using standard biochemical and imaging techniques and in vivo using two photon imaging of neurons in head fixed mice. Optimal GETFaRs will enable research that 1) monitors or manipulates transcriptional states during learning, 2) studies the emergence of ensembles of co-active neurons within a circuit, 3) probes the dynamics of chromatin and nuclear organization, and 4) analyzes the genome of defined populations of neurons responding to complex, natural stimuli.

? DESCRIPTION (provided by applicant): Lasting memories require synaptic modifications as well as communication between the synapse and the nucleus where activity-dependent gene expression is initiated. Elucidating the cellular mechanisms that allow active synapses to rapidly communicate to the nucleus is an outstanding challenge for neurobiology. Current models of synaptic-nuclear communication focus on the movement of second messengers or proteins between these distant cellular compartments or the propagation of action potential or ER evoked calcium waves into the nucleus. While translocation of molecules from the synapse to the nucleus clearly occurs, it is relatively slow and cannot account for the rapidity of experimentally observed nuclear responses to synaptic activity. Moreover, synaptic activity has been demonstrated to rapidly trigger nuclear events independent of action potential generation and when the ER is depleted of calcium. This proposal will test the hypothesis that synaptic activation can depolarize the ER membrane generating an electrical signal that propagates throughout the cell to the nucleus. The ER and nuclear membranes are polarized, have a high membrane resistance, and contain voltage and calcium gated ion channels - biophysical features that support the generation and propagation of electrical signals. ER-mediated electrical signaling would have privileged access to voltage gated channels in the nuclear envelop initiating or facilitating nuclear calcium influx. This idea will be tested by targeting genetically-encoded fluorescent voltage sensors to the ER membrane in order to image real time changes in ER membrane potential in response to synaptic activation. Individual or small clusters of synapses that contain ER will be activated with two-photon glutamate uncaging allowing the precise stimulation of synapses that are in closest proximity to the ER. Complimentarily, channel rhodopsin will be targeted to the ER membrane to determine if ER membrane depolarization is sufficient to trigger nuclear calcium events. If successful, the results of this study will redefine the biological role of the ER, establish a new mode of intercellular cellular communication, resolve a longstanding question in neurobiology, and develop imaging tools that are broadly useful to the neuro- and cell biology communities.

In neurons, membrane depolarization leads to the expression of immediate early gene transcription factors (IEG-TFs), including NPAS4, that regulate programs of gene expression associated with plasticity. IEG-TFs are widely used as tools to identify task-relevant neurons in vivo, yet it is unclear if these proteins are induced in response to changes in the action potential (AP) output or synaptic inputs (EPSPs) to the neuron. Even less is known about whether APs and EPSPs can lead to distinct patterns of gene regulation and cellular phenotypes. This information ?transfer function? is an essential component of how neurons monitor and regulate their own activity. In the specific case of NPAS4, an IEG-TF that regulates excitatory-inhibitory (E-I) balance, studying this transfer function will provide valuable insight into the mechanisms underlying neurodevelopmental and psychiatric disorders that stem from dysregulation of E-I balance. We have developed an acute hippocampal slice preparation from the mouse that allows us to independently evoke APs or EPSPs, from defined populations of inputs, within the context of an intact circuit. We propose investigating the activity requirements for NPAS4 expression and the divergent genomic and synaptic regulation that follows from each type of stimulus. We have used this approach to demonstrate that APs and EPSPs lead to NPAS4 expression with distinct spatio-temporal profiles and have extensive preliminary results characterizing the unexpected underlying mechanisms. Using the methods developed for this proposal, in combination with electrophysiology, optical, and sequencing techniques, we are poised to determine how APs and EPSPs differentially impact activity-dependent gene regulation and synapse function. This proposal is a significant departure from how IEG-TFs are typically studied. The execution of these aims will yield important new insights into the mechanics of activity-dependent gene regulation in neurons and how this biology is disrupted in disorders of the brain such as Autism Spectrum Disorders and schizophrenia.

Project Summary An increasing number of undergraduates (~30% in the UC System) enter research universities after 2+ years in community colleges. Transfer students are more likely to be underrepresented minorities (URMs) or first-generation college students, and they seldom continue on to the PhD. Although many of them express interest in research, they have little to no access to such experiences at community colleges. Soon after matriculating at universities, they tend to opt out of scientific research careers, particularly the idea of continuing to graduate school. In response to this need and in the interest of increasing diversity in our population of neuroscience trainees, here we propose a program to provide transfer students with the skills, mentorship, and research experience to improve their chances of success in a research career. Our proposal builds on the START program, which for three years brought cohorts of URMs and first- generation transfer students to campus in the summer before they matriculated at UCSD. The START program, and others like it, have resulted in improved outcomes for these students. Via a partnership with local community colleges in collaboration with Academic Enrichment Programs (AEP), the STARTneuro program will begin by identifying interested students before they apply to UCSD. Accepted participants will then engage in a 10-week summer bridge program, with intensive and immersive full-day lab training in neuroscience techniques, from physiology to gene expression and function. Each student will also design and implement an independent research project based on the summer modules. During the academic year, students will meet regularly with faculty, be shepherded into lab internships, and be mentored in applying for a summer research scholarship the following summer. A core group of faculty who have demonstrated success mentoring undergraduates will provide the research experience and stewardship necessary to ensure that participants can succeed in scientific research beyond college.