Laminar-selective spinal astrocyte population capable of converting tactile information into nociceptive in rats.

We previously identified a spinal astrocyte population that expresses hairy and enhancer of split 5 (Hes5) and is selectively present in superficial laminae in mice. However, it was unclear whether such astrocyte heterogeneity is commonly observed across species. Using adeno-associated viral (AAV) vectors incorporating a rat Hes5 promotor (AAV-Hes5P), we found that AAV-Hes5P-captured astrocytes were selectively located in the superficial laminae in rats. Furthermore, activation of AAV-Hes5P+ astrocytes elicited allodynia-like behavior and increased c-FOS+ cells in the superficial laminae. Thus, laminar-selective Hes5+ astrocytes are conserved beyond species and have the capability to convert tactile information to nociceptive.

Identification of Spinal Inhibitory Interneurons Required for Attenuating Effect of Duloxetine on Neuropathic Allodynia-like Signs in Rats.

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 Aß 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.

Selective Involvement of a Subset of Spinal Dorsal Horn Neurons Operated by a Prodynorphin Promoter in Aß Fiber-Mediated Neuropathic Allodynia-Like Behavioral Responses in Rats.

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 Aß 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, Aß fiber stimulation did not excite AAV-PdynP+ SDH neurons under normal conditions, but after PNI, this induced excitation, possibly due to enhanced Aß 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 Aß 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 Aß fiber stimulation, which selectively contributes to the conversion of Aß 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.

Improvement of the affinity of an anti-rat P2X4 receptor antibody by introducing electrostatic interactions.

We have recently developed a mouse monoclonal antibody (12-10H) binding to the head domain region in rat P2X4 receptor (rP2X4R, which is crucial for the pathogenesis of neuropathic pain) expressed on the cell with the highest binding affinity (KD = 20 nM). However, the 12-10H antibody failed to detect endogenously expressed P2X4Rs in microglia isolated from the spinal cord of rats whose spinal nerves were injured. Then, we prepared R5 mutant, in which five arginine residues were introduced into variable regions except for the "hot spot" in the 12-10H antibody to increase electrostatic interactions with the head domain, an anionic region, in rP2X4R. The mutation resulted in an increase of 50-fold in the affinity of the R5 mutant for the head domain with respect to the intact 12-10H antibody. As a result, detection of P2X4Rs endogenously expressed on primary cultured microglial cells originated from the neonatal rat brain and spinal cord microglia isolated from a rat model of neuropathic pain was achieved. These findings suggest a strategy to improve the affinity of a monoclonal antibody for an anionic antigen by the introduction of several arginine residues into variable regions other than the "hot spot" in the paratope.

A subset of spinal dorsal horn interneurons crucial for gating touch-evoked pain-like behavior.

A cardinal, intractable symptom of neuropathic pain is mechanical allodynia, pain caused by innocuous stimuli via low-threshold mechanoreceptors such as Aß fibers. However, the mechanism by which Aß fiber-derived signals are converted to pain remains incompletely understood. Here we identify a subset of inhibitory interneurons in the spinal dorsal horn (SDH) operated by adeno-associated viral vectors incorporating a neuropeptide Y promoter (AAV-NpyP+) and show that specific ablation or silencing of AAV-NpyP+ SDH interneurons converted touch-sensing Aß fiber-derived signals to morphine-resistant pain-like behavioral responses. AAV-NpyP+ neurons received excitatory inputs from Aß fibers and transmitted inhibitory GABA signals to lamina I neurons projecting to the brain. In a model of neuropathic pain developed by peripheral nerve injury, AAV-NpyP+ neurons exhibited deeper resting membrane potentials, and their excitation by Aß fibers was impaired. Conversely, chemogenetic activation of AAV-NpyP+ neurons in nerve-injured rats reversed Aß fiber-derived neuropathic pain-like behavior that was shown to be morphine-resistant and reduced pathological neuronal activation of superficial SDH including lamina I. These findings suggest that identified inhibitory SDH interneurons that act as a critical brake on conversion of touch-sensing Aß fiber signals into pain-like behavioral responses. Thus, enhancing activity of these neurons may offer a novel strategy for treating neuropathic allodynia.