Development of HaloTag-Based Tools for Recording Synaptic Dynamics

Abstract

Synaptic transmission and plasticity are fundamental processes that underlie inter-neuronal communication, learning, and memory. However, current methods for tracking synaptic changes in vivo have limited spatial resolution, imaging depth, and imaging volume. This dissertation presents two novel HaloTag-based molecular labeling techniques designed to map presynaptic activity and postsynaptic plasticity in situ in live animals. The first technique, Extracellular Protein Surface Labeling in Neurons (EPSILON), uses a pulse-chase strategy with membrane-impermeable dyes to selectively track α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) exocytosis, a key indicator of synaptic potentiation. By labeling newly surface-exposed AMPARs at genetically targeted synapses, EPSILON provides high-resolution maps of synaptic plasticity in vivo. In hippocampal CA1 pyramidal neurons, EPSILON was employed to investigate the relationship between AMPAR exocytosis and cFos expression during contextual fear conditioning. This study revealed a strong correlation between synaptic potentiation and immediate early gene activation, suggesting a synapse-level mechanism for the formation of memory engrams. Additionally, EPSILON uncovered spatial patterns of synaptic plasticity, including preferential potentiation in perisomatic dendrites and localized clustering of potentiated spines, highlighting the structured organization of synaptic modifications during learning. The second technique enables time-resolved tracking of presynaptic vesicle release, in vitro and in vivo. This method uses HaloTag fusion constructs of presynaptic proteins, such as synaptophysin and vesicular glutamate transporter 1, to selectively label active presynaptic terminals upon neurotransmitter vesicle exocytosis. In cultured neurons, optogenetic stimulation or chemical depolarization significantly increased HaloTag dye labeling on presynaptic boutons, demonstrating the method’s sensitivity to presynaptic activity. In live mice, this technique was used to map presynaptic activity under optogenetic stimulation or sensory deprivation, revealing distinct patterns of presynaptic vesicle release dynamics in response to neural activity changes in vivo. Furthermore, sequential labeling with multiple dyes confirmed that this approach enables multiplexed tracking of synaptic activity across different time points, providing a powerful tool for studying functional synaptic connectivity in naturalistic conditions. Together, these two techniques offer complementary approaches for investigating the dynamics of synaptic activity and plasticity. Unlike conventional real-time imaging methods, which are limited by optical access and restricted fields of view, these HaloTag-based approaches enable synaptic events to be recorded in situ and read out ex vivo, allowing for comprehensive analysis of synaptic modifications throughout the brain. Future applications of these methods may include simultaneous tracking of presynaptic and postsynaptic changes, brain-wide mapping of synaptic modifications, and integration with advanced imaging, proteomic, or transcriptomic approaches. By enhancing the ability to study synaptic changes in vivo with high precision, this dissertation contributes to a deeper understanding of the molecular mechanisms underlying functional neural circuits in the brain.