uniSynK: a ratio-optimized bicistronic chemically induced dimerization tool for robust induction of dendritic spine enlargement

Synaptic plasticity is widely implicated in learning and brain-state regulation. Although correlations between synaptic modifications and diverse brain functions have been extensively reported, establishing causality requires tools that can directly and selectively perturb synapses in vivo. Recently, the chemogenetic actuator SYNCit-K (SynK) enabled causal induction of dendritic spine enlargement and synaptic potentiation. However, SynK relies on the co-expression of two independent components delivered by separate viral vectors, making it difficult to reliably achieve appropriate expression levels of both components within the same neuron. Here, using neocortical dissociated cultures from mouse embryos of undetermined sex, we sought to develop a unified, single-vector version of SynK (uniSynK) that enables robust and safe induction of dendritic spine enlargement. We first found that each SynK component induces distinct, concentration-dependent abnormalities in neuronal morphology when overexpressed, underscoring the need for precise control of their expression ratio. To address this challenge, we engineered a single construct in which the two components are linked by internal ribosome entry site (IRES) variants with graded translational efficiencies. Systematic screening of IRES variants identified an optimized configuration that preferentially drives each component within its respective concentration range that preserves normal spine morphology. The optimized uniSynK efficiently induces dendritic spine enlargement, while exhibiting fewer off-target morphological abnormalities than observed in the multi-vector system when expression levels were not properly controlled. By simplifying experimental design, this single-vector, ratio-optimized synaptic chemogenetic tool provides a practical and broadly applicable platform that should facilitate causal interrogation of synaptic plasticity across neural circuits and behavior. Significance Statement Synaptic plasticity is thought to shape learning, brain states, and neuropsychiatric disease, but testing its causal roles requires tools that can directly manipulate synapses with minimal side effects. Here, we developed uniSynK, a single-vector chemogenetic system that induces dendritic spine enlargement by coordinating the expression of two functional components. By characterizing the expression-level-dependent effects of each component and using IRES variants to optimize their expression ratio, uniSynK achieves robust spine enlargement while reducing unintended morphological abnormalities associated with imbalanced expression. This work provides both a practical tool for synaptic manipulation and a general strategy for optimizing multi-component biological systems.