Distinct contribution of spinal neuropeptide Y and NPY1R neurons to morphine analgesia

Opioids produce potent antinociception by activating μ-opioid receptors (MOR) in the spinal dorsal horn (SDH), where MOR is broadly expressed in both inhibitory and excitatory neurons. However, the neuron subtype-specific actions and mechanisms of spinal MOR in opioid analgesia remain elusive. We employed chemogenetic approaches and conditional gene knockout strategies across multiple pain models to assess the roles of the NPY-NPY1R pathway and MOR signaling in spinal morphine analgesia. In addition, in situ hybridization combined with electrophysiological recordings from spinal cord slices was used to further investigate the underlying cellular and functional mechanisms. We demonstrate that MOR signaling in spinal neuropeptide Y (NPY)+ and NPY1 receptor (NPY1R)+ neurons exerts distinct effects on morphine analgesia under acute and inflammatory pain conditions in mice. Intrathecal injection of NPY synergistically enhanced morphine analgesia across pain models, an effect abolished by NPY1R antagonism or in Npy1r-/- mice. Chemogenetic activation of NPY+ interneurons or inhibition of NPY1R+ interneurons significantly reduced acute and persistent inflammatory pain. In situ hybridization further revealed that the MOR gene Oprm1 was co-expressed in 34% of NPY⁺ and 21% of NPY1R⁺ interneurons in the SDH. Notably, morphine analgesia is enhanced in mice with a specific Oprm1 deletion in NPY+ neurons (NpyCre;Oprm1fl/fl). However, loss of Oprm1 in NPY1R+ interneurons (Oprm1fl/fl/AAV-Npy1r-Cre-EGFP) impairs morphine analgesia. Furthermore, co-treatment with NPY or knockout of MOR in NPY+ neurons also prevented opioid-induced hyperalgesia and tolerance. Whole-cell recordings of spinal slices revealed that inflammation-induced hyperexcitability in both NPY+ and NPY1R+ neurons was suppressed by morphine perfusion, as evidenced by increased rheobase, hyperpolarized resting membrane potential (RMP), and reduced action potential firing. These findings indicate that in inflammatory pain, morphine suppresses nociception by activating MOR expressed on NPY1R⁺ neurons, but undermines its antinociceptive efficacy by suppressing NPY⁺ neurons and disinhibiting the downstream nociception circuit in the spinal cord. Our data provides mechanistic insights into opioid analgesia at the spinal level and highlights the pharmacotherapeutic potential of the NPY-NPY1R pathway in pain management.