Mechanical allodynia (pain produced by innocuous stimuli such as touch) is the main symptom of neuropathic pain. Its underlying mechanism remains to be elucidated, but peripheral nerve injury (PNI)-induced malfunction of neuronal circuits in the central nervous system, including the spinal dorsal horn (SDH), is thought to be involved in touch-pain conversion. Here, we found that intra-SDH injection of adeno-associated viral vectors including a prodynorphin promoter (AAV-PdynP) captured a subset of neurons that were mainly located in the superficial laminae, including lamina I, and exhibited mostly inhibitory characteristics. Using transgenic rats that enable optogenetic stimulation of touch-sensing Aβ fibers, we found that the light-evoked paw withdrawal behavior and aversive responses after PNI were attenuated by selective ablation of AAV-PdynP-captured SDH neurons. Notably, the ablation had no effect on withdrawal behavior from von Frey filaments. Furthermore, Aβ fiber stimulation did not excite AAV-PdynP+ SDH neurons under normal conditions, but after PNI, this induced excitation, possibly due to enhanced Aβ fiber-evoked excitatory synaptic inputs and elevated resting membrane potentials of these neurons. Moreover, the chemogenetic silencing of AAV-PdynP+ neurons of PNI rats attenuated the Aβ fiber-evoked paw withdrawal behavior and c-FOS expression in superficial SDH neurons. Our findings suggest that PNI renders AAV-PdynP-captured neurons excitable to Aβ fiber stimulation, which selectively contributes to the conversion of Aβ fiber-mediated touch signal to nociceptive. Thus, reducing the excitability of AAV-PdynP-captured neurons may be a new option for the treatment of neuropathic allodynia.

Neuropathic pain is a chronic pain condition that occurs after nerve damage; allodynia, which refers to pain caused by generally innocuous stimuli, is a hallmark symptom. Although allodynia is often resistant to analgesics, the antidepressant duloxetine has been used as an effective therapeutic option. Duloxetine increases spinal noradrenaline (NA) levels by inhibiting its transporter at NAergic terminals in the spinal dorsal horn (SDH), which has been proposed to contribute to its pain-relieving effect. However, the mechanism through which duloxetine suppresses neuropathic allodynia remains unclear. Here, we identified an SDH inhibitory interneuron subset (captured by adeno-associated viral (AAV) vectors incorporating a rat neuropeptide Y promoter; AAV-NpyP+ neurons) that is mostly depolarized by NA. Furthermore, this excitatory effect was suppressed by pharmacological blockade or genetic knockdown of α1B-adrenoceptors (ARs) in AAV-NpyP+ SDH neurons. We found that duloxetine suppressed Aβ fiber-mediated allodynia-like behavioral responses after nerve injury and that this effect was not observed in AAV-NpyP+ SDH neuron-selective α1B-AR-knockdown. These results indicate that α1B-AR and AAV-NpyP+ neurons are critical targets for spinal NA and are necessary for the therapeutic effect of duloxetine on neuropathic pain, which can support the development of novel analgesics.

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Pain information transmission/processing in the spinal dorsal horn (SDH) is strongly controlled by descending neurons from the brain. One of the major neurotransmitters of descending pathways is noradrenaline (NA). Descending NAergic neurons from the locus coeruleus (LC) is known to produce analgesic effects via activation of inhibitory interneurons in the SDH. However, the identity of the inhibitory interneuron subset in the SDH is poorly understood. Recently, we have found a subset of the SDH inhibitory interneurons captured by adeno-associated viral (AAV) vectors incorporating a neuropeptide Y promoter (AAV-NpyP + ) that is crucial for neuropathic allodynia. Here, we showed that this neuronal subset is a major target of spinal NA to inhibit pain information transmission/processing. Whole-cell patch-clamp recordings using spinal cord slices revealed that NA predominantly depolarizes AAV-NpyP + neurons. This effect was suppressed by a pharmacological blockade and genetic knockdown of α 1B -adrenoceptor (AR) in AAV-NpyP + neurons in the SDH. Furthermore, we found that the analgesic effect of duloxetine on neuropathic pain which is associated with an increase in the spinal NA level by inhibiting NA reuptake into presynaptic terminals is reduced by AAV-NpyP + neuron-selective knockdown of α 1B -ARs. These results indicate that α 1B -ARs expressed in AAV-NpyP + neurons would be a target of spinal NA presumably from descending LC neurons and contribute to the analgesic effect of duloxetine. Thus, spinal α 1B -ARs could be a new therapeutic target.