Abstract
Endogenous neuropeptide Y (NPY) exerts long-lasting spinal inhibitory control of neuropathic pain, but its mechanism of action is complicated by the expression of its receptors at multiple sites in the dorsal horn: NPY Y1 receptors (Y1Rs) on post-synaptic neurons and both Y1Rs and Y2Rs at the central terminals of primary afferents. We found that Y1R-expressing spinal neurons contain multiple markers of excitatory but not inhibitory interneurons in the rat superficial dorsal horn. To test the relevance of this spinal population to the development and/or maintenance of acute and neuropathic pain, we selectively ablated Y1R-expressing interneurons with intrathecal administration of an NPY-conjugated saporin ribosomal neurotoxin that spares the central terminals of primary afferents. NPY-saporin decreased spinal Y1R immunoreactivity but did not change the primary afferent terminal markers isolectin B4 or calcitonin-gene-related peptide immunoreactivity. In the spared nerve injury (SNI) model of neuropathic pain, NPY-saporin decreased mechanical and cold hypersensitivity, but disrupted neither normal mechanical or thermal thresholds, motor coordination, nor locomotor activity. We conclude that Y1R-expressing excitatory dorsal horn interneurons facilitate neuropathic pain hypersensitivity. Furthermore, this neuronal population remains sensitive to intrathecal NPY after nerve injury. This neuroanatomical and behavioral characterization of Y1R-expressing excitatory interneurons provides compelling evidence for the development of spinally-directed Y1R agonists to reduce chronic neuropathic pain.
Highlights
Peripheral nerve damage can lead to a debilitating neuropathic pain syndrome that persists for years[1]
Y1 receptors (Y1Rs) immunoreactivity (Y1R-ir) in the dorsal horn presents as a dense plexus of axons and dendrites that complicates analysis of co-labeling. We reduced this problem by pretreating animals with two intrathecal injections of neuropeptide Y (NPY) (30 μg) separated by 1 hour so as to promote receptor internalization, thereby concentrating Y1R from more distal dendritic locations to within the cell soma
We found Y1R-ir to co-localize with multiple markers of excitatory neurons in superficial lamina: calbindin (Figs 1A,B and 2A), calretinin (Figs 1C,D and 2B), and somatostatin (Fig. 1I), but neither PKCγ-ir (Figs 1E,F and 2D) nor Pax2-ir (Figs 1G,H and 2C)
Summary
Peripheral nerve damage can lead to a debilitating neuropathic pain syndrome that persists for years[1]. Distinct populations of excitatory interneurons have been identified within the spinal dorsal horn microcircuitry that are required for the behavioral expression of neuropathic pain[3,4,5,6,7,8] These subpopulations can be defined by the expression of either a small molecule neurotransmitter (e.g. gamma-aminobutyric acid, GABA), a neuropeptide transmitter (e.g. somatostatin)[5], transporter protein (e.g. vesicular glutamate transporter 3, VGlut3)[7], or opioid receptor (e.g. DOR)[8]; none of these neural population have been found to be readily druggable targets for the development of pharmacological agents directed at non-opioid neurotransmitter receptors. Our approach, which uses NPY-saporin, readily discriminates between targets on cell bodies and axon terminals[21], and avoids pitfalls associated with the use of Y1R-Cre transgenic mice that include germline recombination, transient expression, and aberrant expression at non-targeted sites[25]
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