Tweety-homolog 1 protein overexpression alters dendritic complexity and dendritic spine morphology of CA1 and CA3 pyramidal neurons in organotypic slices in vitro

Tweety-homolog 1 protein (Ttyh1) is a presumed volume-regulated chloride channel that is widely expressed in neurons in vitro and in vivo. It was previously implicated in regulating dendrite morphology in dissociated hippocampal neurons in vitro, indicating its possible role in structural neuronal plasticity. This study tested the hypotheses that (i) Ttyh1 influences dendritic tree formation in rat organotypic hippocampal slice cultures in an in vitro model with preserved cytoarchitecture and synaptic circuits, and (ii) Ttyh1 influences dendritic spine morphology in the same experimental model. Neurons were transfected via a Helios gene gun with a plasmid that carried Ttyh1-EGFP under control of the synapsin promoter (or green fluorescent protein as a control) and a plasmid that encoded red fluorescent protein under a β-actin promoter. To evaluate dendritic tree morphology, transfected neurons were reconstructed using Neuromantic software and morphometrically analyzed using the Sholl method and L-measure software. To quantify dendritic spine density and the distributions of thin, mushroom, and stubby spines on basilar dendrites and proximal and distal apical dendrites, we used SpineMagick! software. The results indicated that Ttyh1 impacted dendritic spine morphology and density on apical and basilar dendrites of CA1 and CA3 pyramidal neurons, and moderately regulated the complexity of their apical and basilar dendritic arbors. Our findings support the hypothesis that Ttyh1 participates in shaping neuronal morphology.