The Neurokinin-1 Receptor Is Expressed with Gastrin-Releasing
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Research Articles: Cellular/Molecular The neurokinin-1 receptor is expressed with gastrin-releasing peptide receptor in spinal interneurons and modulates itch https://doi.org/10.1523/JNEUROSCI.1832-20.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.1832-20.2020 Received: 15 July 2020 Revised: 25 August 2020 Accepted: 21 September 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 the authors 1 Title: The neurokinin-1 receptor is expressed with gastrin-releasing peptide receptor in spinal 2 interneurons and modulates itch 3 4 Abbreviated Title: Neurokinin-1 receptor spinal neurons mediate itch 5 6 7 Authors: Tayler D. Sheahan1, Charles A. Warwick1, Louis G. Fanien1, Sarah E. Ross1 8 9 10 1Pittsburgh Center for Pain Research and Department of Neurobiology, University of Pittsburgh, 11 Pennsylvania 12 13 Corresponding author: Sarah E. Ross, Department of Neurobiology, University of Pittsburgh, 14 200 Lothrop Street, Pittsburgh, PA, e-mail: [email protected] 15 The authors have no conflicts of interest to declare. 16 17 18 Number of text pages: 19 19 20 21 Number of figures and tables: 5 figures 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 1 50 Abstract 51 52 The neurokinin-1 receptor (NK1R, encoded by Tacr1) is expressed in spinal dorsal horn 53 neurons and has been suggested to mediate itch in rodents. However, previous studies relied 54 heavily on neurotoxic ablation of NK1R spinal neurons, which limited further dissection of their 55 function in spinal itch circuitry. To address this limitation, we leveraged a newly developed 56 Tacr1CreER mouse line to characterize the role of NK1R spinal neurons in itch. We show that 57 pharmacological activation of spinal NK1R and chemogenetic activation of Tacr1CreER spinal 58 neurons increases itch behavior in male and female mice, whereas pharmacological inhibition of 59 spinal NK1R suppresses itch behavior. We use fluorescence in situ hybridization to characterize 60 the endogenous expression of Tacr1 throughout the superficial and deeper dorsal horn, as well 61 as the lateral spinal nucleus, of mouse and human spinal cord. Retrograde labeling studies in 62 mice from the parabrachial nucleus show that less than 20% of superficial Tacr1CreER dorsal 63 horn neurons are spinal projection neurons, and thus the majority of Tacr1CreER are local 64 interneurons. We then use a combination of in situ hybridization and ex vivo two-photon Ca2+ 65 imaging of the mouse spinal cord to establish that NK1R and the gastrin-releasing peptide 66 receptor (GRPR) are coexpressed within a subpopulation of excitatory superficial dorsal horn 67 neurons. These findings are the first to suggest a role for NK1R interneurons in itch and extend 68 our understanding of the complexities of spinal itch circuitry. 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 2 101 Significance Statement 102 103 The spinal cord is a critical hub for processing somatosensory input, yet which spinal neurons 104 process itch input and how itch signals are encoded within the spinal cord is not fully 105 understood. We demonstrate neurokinin-1 receptor (NK1R) spinal neurons mediate itch 106 behavior in mice and that the majority of NK1R spinal neurons are local interneurons. These 107 NK1R neurons comprise a subset of gastrin-releasing peptide receptor interneurons and are 108 thus positioned at the center of spinal itch transmission. We show NK1R mRNA expression in 109 human spinal cord, underscoring the translational relevance of our findings in mice. This work is 110 the first to suggest a role for NK1R interneurons in itch and extends our understanding of the 111 complexities of spinal itch circuitry. 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 3 152 Introduction 153 154 Acute itch, much like pain, serves as a protective warning signal to the body, indicating there is 155 an irritant on the skin that needs to be removed. Itchy stimuli are first detected by primary 156 afferent fibers innervating the skin, which then relay inputs to neurons within the spinal cord 157 dorsal horn including local interneurons, as well as ascending spinal projection neurons that 158 target brainstem, midbrain, and thalamic structures. However, how these spinal neurons 159 process and encode itch as well as other somatosensory stimuli is not fully understood. 160 161 A major unresolved question regarding the spinal circuitry of itch and pain is how to distinguish 162 spinal projection neurons from interneurons. Proposed distinguishing criteria include 163 morphology or genetic markers (Al Ghamdi et al., 2009; Todd, 2010). For instance, the 164 neurokinin-1 receptor (NK1R), an excitatory G-protein coupled receptor that is expressed 165 throughout the nervous system and activated by the neuropeptide substance P (SP), is often 166 highlighted as a selective marker for spinal projection neurons (Todd, 2010; Zhao et al., 2014; 167 Cameron et al., 2015; Häring et al., 2018; Huang et al., 2018; Bardoni et al., 2019). Yet Tacr1, 168 which encodes NK1R, appears to be expressed within multiple excitatory dorsal horn neuron 169 populations recently identified by single cell RNA-sequencing (Häring et al., 2018; Zeisel et al., 170 2018). Thus, whether NK1R is a true marker of spinal projection neurons requires further 171 investigation. 172 173 NK1R has been studied extensively as a putative target for pain relief in preclinical rodent 174 models (Mantyh et al., 1997; Nichols et al., 1999; Vierck et al., 2003; Wiley et al., 2007), but 175 demonstrated negligible clinical success and was quickly dropped as a therapeutic target (Hill, 176 2000; Boyce and Hill, 2004). Intriguingly, NK1R expressed on spinal neurons has recently 177 emerged as a potential therapeutic target for chronic itch. Neurotoxic ablation studies show that 178 loss of NK1R spinal neurons reduces scratching in rodents (Carstens et al., 2010; Akiyama et 179 al., 2015; Acton et al., 2019). However, the effect of modulation of NK1R spinal neurons in an 180 intact spinal circuit remains unclear. Furthermore, little is known about which NK1R neurons are 181 involved in itch processing or where they fit within the current model of itch transmission. 182 183 The current model of itch spinal circuitry positions neurons that express the gastrin-releasing 184 peptide receptor (GRPR) as a gate for itch input (Sun and Chen, 2007; Sun et al., 2009; Liu et 185 al., 2019; Pagani et al., 2019; Lay and Dong, 2020). We explored the possibility that NK1R and 186 GRPR neurons might represent an overlapping dorsal horn neuron subpopulation that 187 modulates itch. 188 189 In this study, we demonstrate that activation of NK1R spinal neurons potentiates itch, while 190 inhibition of NK1R attenuates itch. We highlight that, despite being widely used as a selective 191 marker of spinal projection neurons, NK1R is expressed predominately in interneurons within 192 the dorsal horn. Moreover, we reveal a likely role for NK1R interneurons in spinal itch 193 transmission, and that these NK1R neurons comprise a subpopulation of excitatory GRPR 194 interneurons within the superficial dorsal horn. 195 196 197 198 199 200 201 202 4 203 Materials and Methods 204 205 Donors 206 Human spinal cord tissue was obtained from the University of Pittsburgh NIH NeuroBioBank 207 Brain and Tissue Repository in compliance with the University of Pittsburgh’s Committee for the 208 Oversight of Research involving the Dead and Institutional Review Board for Biomedical 209 Research. Spinal cord tissue from the C1 spinal segment was collected from adult human organ 210 donors (donor 1: white 45-year-old male, donor 2: white 44-year-old female), flash frozen, and 211 stored at -80qC until use. 212 213 Animals 214 All animals were cared for in compliance with the National Institutes of Health guidelines and 215 approved by the University of Pittsburgh Institutional Animal Care and Use Committee. 216 Experiments were conducted on both male and female mice, with littermates randomly assigned 217 to experimental groups. Adult, C57BL/6 mice were obtained from Charles River (strain 027) and 218 allowed to acclimate to the University of Pittsburgh’s vivarium for at least one week prior to 219 beginning experimentation. Previously generated Tacr1CreER mice (also referred to as NK1R- 220 CreER mice (Huang et al., 2016)) were bred in house and maintained on a C57BL/6 221 background. For histology and in situ hybridization studies, Tacr1CreER mice were crossed with 222 Cre-dependent RosatdT reporter mice (Jackson Laboratory, stock no. 007909). For two-photon 223 Ca2+ imaging studies, Vglut2Cre mice (Jackson Laboratory, stock no. 016963) were crossed with 224 Cre-dependent RosaGCaMP6s mice (Jackson Laboratory, strain no. 028866). 225 226 Mice were housed in cages of 4 (males) or 5 (females) in the animal facility under a 12 hr 227 light/dark cycle (7AM – 7PM) and provided food and water ad libitum. Cages were lined with 228 woodchip bedding. Behavioral studies on and tissue harvesting from C57BL/6 and Tacr1CreER 229 mice began when animals were 6-8 week old. Spinal cord Ca2+ imaging studies were similarly 230 performed on 6-8 week old Vglut2Cre::RosaGCaMP6s mice.