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.