Cell type specific targeting of nicotinic receptor activity with tethered ligand viral vectors

Heteromeric β2-containing nicotinic acetylcholine receptors (nAChRs) regulate diverse neural processes, yet their study in native circuits has been limited by the inability to manipulate receptor function with cell-type specificity, without transgenics, and across species. Existing tethered-ligand approaches, including photoswitchable and knock-in-based systems, provide valuable control of engineered receptors but do not readily integrate with Cre-driver lines and cannot be used in rats, where many nicotine-related behaviors are more robust. To address these limitations, we developed Cre-dependent adeno-associated viral (AAV) vectors expressing the β2 E61C nAChR subunit, a cysteine-bearing variant that enables covalent antagonism by the maleimide-PEG4-choline tethered ligand MPEG₄Ch. We generated AAVs encoding either β2 E61C or hypersensitive β2Leu9'Ser E61C subunits and validated their functional incorporation into native nAChR pentamers in mouse and rat ventral tegmental area (VTA) neurons. Patch-clamp recordings demonstrated that MPEG₄Ch produced strong, long-lasting, and E61C-dependent suppression of ACh-evoked currents, with near-complete blockade following prolonged ligand exposure. The system performed robustly in both DAT-Cre mice and rats receiving co-infused AAV-Cre, establishing effective cross-species use. From our results, we infer that any heteromeric receptor containing ≥1 engineered β2 subunit becomes ligand-sensitive, and that viral expression efficiently converts the endogenous β2 receptor pool without increasing total receptor abundance. This AAV-based, Cre-dependent E61C system provides a practical, optics-free, and species-flexible platform for cell-type-specific silencing of β2-containing nAChRs. Its compatibility with widely used Cre lines and suitability for rat behavioral paradigms-including nicotine self-administration and sustained-attention tasks-positions it as a versatile tool for dissecting nicotinic signaling in intact neural circuits.