Abstracts

Rationale: The goal of our work is to better understand the underlying mechanisms of Dravet syndrome (DS). One gene linked to DS is SCN1B, which encodes the protein β1. β1 plays multiple roles in the development and physiology of neurons, including acting as an ion channel auxiliary subunit. Normal expression levels and subcellular distribution of ion channels in neurons is vital for synaptic integration, action potential generation, and neural processing. Previous work has shown that the subcellular localization, surface stability, and physiology of voltage-gated sodium and potassium channels is altered in Scn1b knockout (KO) neurons. Our focus is understanding how mutations to the SCN1B gene can impact HCN channel localization and the role this plays in the neural dysfunction in DS. Methods: Here, we hypothesize that loss of Scn1b causes a disruption to the subcellular distribution of HCN1 channels, which typically exhibit a gradient of increasing expression from proximal to distal dendrites. We used juvenile (postnatal day 14-18) Scn1b KO and wild-type (WT) littermate mice.  We stained coronal hippocampal slices with primary antibodies for HCN1, MAP2 to highlight pyramidal neuron dendrites, fluorescent secondary antibodies, and DAPI to reveal cell nuclei. After imaging the slices, we quantified the intensity of fluorescence across dorsal hippocampal layers from the alveus to stratum moleculare to measure the dendritic expression gradient in CA1 hippocampal neurons using ImageJ.  Results: We found that WT CA1 pyramidal neurons exhibit a robust dendritic gradient of HCN1 channel expression, with higher expression in the distal dendrites. Our experiments indicate that this gradient may be abnormal in Scn1b KO neurons. Previous studies have shown that dendrites are shorter in some types of Scn1b KO pyramidal neurons. Thus, our findings could be influenced by a decrease in distal dendritic length. We are making further measurements of dendritic length and HCN1 expression in order to account for these structural changes. The mechanisms by which loss of β1 causes disruption of the HCN1 dendritic gradient are not clear. HCN1 channels exhibit activity-dependent changes in surface expression, so this phenotype may be induced by changes in network activity (i.e., seizures). β1 is involved in the trafficking of multiple different types of ion channels, so a more direct and cell autonomous effect on HCN1 distribution is also possible.  Conclusions: The HCN1 channel dendritic gradient is vital for controlling excitability in pyramidal neurons. Thus, abnormal HCN1 expression in Scn1b KO neurons could play a role in the etiology of both the seizures and cognitive deficits of DS. Understanding how loss of β1 disrupts the subcellular localization and expression levels of ion channels, and cell physiology, is key to developing novel therapies and interventions for DS. Funding: This work is supported by the Neuroscience Studies Foundation (SAK) and an American Epilepsy Society Young Investigator Award (MAH).