Dorsal horn neurons release extracellular ATP in a VNUT-dependent manner that underlies neuropathic pain

Abstract

Activation of purinergic receptors in the spinal cord by extracellular ATP is essential for neuropathic hypersensitivity after peripheral nerve injury (PNI). However, the cell type responsible for releasing ATP within the spinal cord after PNI is unknown. Here we show that PNI increases expression of vesicular nucleotide transporter (VNUT) in the spinal cord. Extracellular ATP content ([ATP]e) within the spinal cord was increased after PNI, and this increase was suppressed by exocytotic inhibitors. Mice lacking VNUT did not show PNI-induced increase in [ATP]e and had attenuated hypersensitivity. These phenotypes were recapitulated in mice with specific deletion of VNUT in spinal dorsal horn (SDH) neurons, but not in mice lacking VNUT in primary sensory neurons, microglia or astrocytes. Conversely, ectopic VNUT expression in SDH neurons of VNUT-deficient mice restored PNI-induced increase in [ATP]e and pain. Thus, VNUT is necessary for exocytotic ATP release from SDH neurons which contributes to neuropathic pain.

Introduction

ATP is the well-known energy currency found in all living cells1. ATP also plays a distinct role in extracellular spaces, where it acts as a signalling molecule that controls cellular physiology, connectivity and dynamics through the activation of purinergic receptors1,2. Recently, extracellular ATP has emerged as a key player for a variety of diseases, including chronic inflammatory diseases3, and several neurological4,5and psychiatric disorders6,7. Thus, characterizing the nature of ATP-mediated physiological phenomena is important for better understanding their aetiology, as well as identifying potential therapeutic targets of the diseases.

Neuropathic pain is one of the most debilitating chronic pain syndromes. It occurs concomitantly with neuronal damage as a consequence of multiple sclerosis, diabetes mellitus, cancer and traumatic injury8,9. Accumulating evidence has indicated the crucial roles of microglia, the immune-related glial cells of the central nervous system (CNS)10,11, in the spinal cord in the development of pain hypersensitivity following peripheral nerve injury (PNI). In response to PNI, spinal microglia transform into a reactive state through a sequence of cellular and molecular changes. These changes include morphological hypertrophy, proliferation and alteration in gene expression12,13 including genes encoding purinergic receptors14, such as ionotropic P2X4 receptor (P2X4R)9,15,16 and metabotropic P2Y12 receptor (P2Y12R)17 which are markedly upregulated after PNI15. In response to stimulation of these ATP receptors, microglia release several bioactive factors18,19, which cause abnormal neurotransmission in the dorsal horn nociceptive network18,20. These pathological alterations result in converting innocuous inputs to nociceptive signals9,10,21. The fact that the PNI-induced pain hypersensitivity is reversed rapidly by pharmacological blockade of spinal P2X4Rs15 or P2Y12Rs17 suggests that such pain behaviours require ongoing activation of these receptors by extracellular ATP in the spinal cord. These studies have led to two major questions: which type of cells release ATP, and what is the mechanism by which ATP is released from those cells, that causes pain hypersensitivity after nerve injury within the spinal cord9,22.

In the present study, we investigated the mechanisms and cell types that are involved in supplying extracellular ATP in the spinal cord. We also examined whether increased extracellular ATP release occurs within the spinal cord after PNI, and if it does, whether such ATP enhancement contributes to neuropathic pain. We identify vesicular nucleotide transporter (VNUT; also known as Slc17a9), a secretory vesicle protein responsible for the storage and release of ATP23,24, expressed in spinal dorsal horn (SDH) neurons as being crucial for exocytotic ATP release within the spinal cord and for neuropathic pain after PNI. Our findings may provide a novel target for treating neuropathic pain.

Results

Increase in spinal extracellular ATP after PNI requires VNUT

To date it is unknown if extracellular ATP ([ATP]e) level within the spinal cord is increased after PNI. Therefore, we first determined the [ATP]econtent within the spinal cord of wild-type (WT) mice subjected to PNI (Fig. 1a). Seven days after PNI, the concentration of [ATP]e in the artificial cerebral spinal fluid (ACSF) incubated with spinal cord slices of the ipsilateral side was significantly higher than that of the contralateral side (P<0.05, Fig. 1b). A slight increase in [ATP]e content was observed 3 days, but not 28 days, post-PNI (Supplementary Fig. 1). These results indicate that the level of [ATP]e within the ipsilateral spinal cord is increased after PNI.

Figure 1: PNI-induced increase in extracellular ATP within the spinal cord is dependent on VNUT.

(a) Schematic diagram of the experimental protocol for spinal ATP detection (Ipsi, ipsilateral; Contra, contralateral). (b) Measurement of extracellular ATP ([ATP]e) content in the ACSF media of ipsilateral and contralateral spinal cord slices taken from wild-type (WT) and VNUT-deficient (Slc17a9−/−) mice before (naive) and 7 days after PNI (naive: n=12, WT and Slc17a9−/−: n=16; *P<0.05, **P<0.01, one-way ANOVA with post hoc Tukey Multiple Comparison test). (c,d) Measurement of [ATP]e content in the ACSF media of ipsilateral and contralateral spinal cord slices taken from WT mice with or without (w/o) (c) tetanus toxin (TeNT) or (d) thapsigargin (TG) 7 days after PNI (c: n=6, d: n=11; *P<0.05, ***P<0.001, one-way ANOVA with post hoc Tukey Multiple Comparison test). Values are means±s.e.m.