85 Characterizing Neuronal ECM in Culture Using HaloTag-HAPLN1

Faculty Mentor: Sungjin Park (Neurobiology, University of Utah)

In the brain, there exists an acellular structure in the extracellular space termed the extracellular matrix (ECM), which makes up about 20% of the brain volume in adult brains. Loosening of the ECM increases neuronal plasticity, which is essential for learning, implying ECM clustering inhibits neuronal plasticity. However, which cell types assemble the ECM in the brain and how clustered ECM regulates neuronal plasticity are not fully understood. In order to visualize the structures of the ECM and associated cells, the lab developed a novel tool that visualizes ECM deposition on live cells (HaloTag-HAPLN1: H-Link). Application of fluorescence-conjugated H-Link to the primary neuronal cultures showed clear heterogeneity of H-Link aggregations among neurons, ranging from dense heavy deposition on the neuronal surface to no signal at all. Because conventional ECM markers (WFA lectin, ACAN antibody staining) exhibit ECM deposition around specific inhibitory neurons like PV neurons, we tested if other neuron types assemble ECM clusters by quantifying and comparing H-Link population and intensity around inhibitory and excitatory neurons derived from the hippocampus and cortex of the neonatal rats. Blinded quantification of the H-Link population and intensity was done using ImageJ, and the measured mean intensity data was further analyzed using cumulative distribution. Results showed that a majority of the excitatory neurons in hippocampal cultures displayed H-Link clusters, whereas cortical excitatory neurons and cortical and hippocampal inhibitory neurons had a significantly smaller proportion of neurons displaying H-Link clusters. Cumulative analysis showed that inhibitory neurons showed more heterogeneity in H-Link intensity than excitatory neurons. This observation thus suggests that the ECM deposition on excitatory neurons may have a role in regulating neuronal plasticity.