α1A-adrenaline receptors in dorsal horn inhibitory neurons have an inhibitory role in the regulation of chloroquine-induced itch in mice

Our previous study showed the intrinsic ability of descending noradrenergic neurons projecting from the locus coeruleus to the spinal dorsal horn (SDH) to suppress itch-related behaviors. Noradrenaline and α1A-adrenaline receptor (α1A-AR) agonist increase inhibitory synaptic inputs onto SDH interneurons expressing gastrin-releasing peptide receptors, which are essential for itch transmission. However, the contribution of α1A-ARs expressed in SDH inhibitory interneurons to itch-related behavior remains to be determined. In this study, RNAscope in situ hybridization revealed that Adra1a mRNA is expressed in SDH inhibitory interneurons that are positive for Slc32a1 mRNA (known as vesicular GABA transporter). Mice with conditional knock-out of α1A-ARs in inhibitory interneurons (Vgat-Cre;Adra1aflox/flox mice) exhibited an increase in scratching behavior when induced by an intradermal injection of chloroquine, but not compound 48/80, which are known as models of histamine-independent and dependent itch, respectively. Furthermore, knockout of inhibitory neuronal α1A-ARs in the SDH using the CRISPR–Cas9 system also increased the scratching behavior elicited by chloroquine but not compound 48/80. Our findings demonstrated for the first time that α1A-ARs in SDH inhibitory interneurons contribute to the regulation of itch signaling with preference for histamine-independent itch.

Itch is defined as an unpleasant sensation that provokes a desire to scratch to transiently attenuate such sensations. Sensory itch information from the cutaneous nerve endings of primary afferent fibers is conveyed to the spinal dorsal horn (SDH). Recent studies have made great progress in furthering our understanding of the neuronal circuits for itch transmission in the SDH [1]. Specifically, gastrin-releasing peptide receptor (GRPR)-expressing (GRPR + ) neurons in the SDH have been identified as an essential component of spinal itch transmission [2]. Furthermore, it has also been shown that GRPR + neurons are controlled not only locally by subsets of excitatory and inhibitory interneurons in the SDH [3] but also remotely by descending monoaminergic neurons from the brainstem [4,5]. A recent study has shown that the activation of descending serotonergic (5-HTergic) neurons facilitates GRPR signaling and itch transmission in the SDH via activation of 5-HT 1A receptors [4]. As for noradrenaline (NA), we recently demonstrated that NAergic neurons descending from the locus coeruleus Open Access *Correspondence: tsuda@phar.kyushu-u.ac.jp † Yuto Shiraishi and Keisuke Koga contributed equally to this work 3 Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan Full list of author information is available at the end of the article (LC) to the SDH negatively control itch-related scratching behaviors. Furthermore, application of either NA or an α 1A -adrenaline receptor (α 1A -AR) agonist to spinal cord slices facilitates the transmission of inhibitory synaptic inputs into GRPR + SDH neurons [5]. Thus, it is possible that α 1A -ARs in inhibitory SDH interneurons may play a role in controlling itch-related behavior, although this is entirely unknown. The aim of this study was to determine the role of α 1A -ARs expressed in inhibitory SDH interneurons using genetic tools that enable cell-and region-specific gene manipulations.
By generating two lines of genetically manipulated mice in a cell type-and region-specific manner, we demonstrate for the first time that α 1A -ARs in SDH inhibitory interneurons play an inhibitory role in the regulation of itch-related behavioral responses. The present data reinforce and extend our previous findings indicating an intrinsic inhibitory control for spinal itch transmission by SDH-projecting LC-NAergic neurons [5]. An unexpected and interesting finding of the present study was that the knockout of α 1A -ARs in SDH inhibitory interneurons enhanced scratching behavior elicited by chloroquine but not by compound 48/80, whereas silencing SDHprojecting LC-NAergic neurons is known to enhance scratching induced by both pruritogens [5]. Given that chloroquine and compound 48/80 cause histamineindependent and -dependent itch, respectively [2,8], it is possible that α 1A -ARs in SDH inhibitory interneurons preferentially contribute to the regulation of histamineindependent itch. This study showed that Adra1a was expressed in SDH inhibitory interneurons, and activation of spinal α 1A -ARs has been shown to facilitate the transmission of inhibitory synaptic inputs onto GRPR + SDH neurons [5]. It is thus conceivable that inhibitory signals acting on GRPR + neurons via α 1A -AR-expressing SDH inhibitory interneurons suppress histamine-independent itch (Additional file 2). Other ARs, such as α 2 -ARs in GRPR + neurons [10] and at primary afferent terminals [11], may be involved in histamine-dependent itch.
However, scratching behaviors evoked by chloroquine and compound 48/80 have been shown to be dramatically suppressed in mice with ablated GRPR + SDH neurons [2]. Recent single-cell transcriptome analysis in SDH neurons revealed the presence of several putative subsets expressing Grpr mRNA [10]. Thus, it is speculated that histamine-independent itch transmission may be relayed through a subset of GRPR + neurons that is regulated by α 1A -AR-expressing SDH inhibitory interneurons (Additional file 2). Alternatively, considering previous findings that showed that GRPR-knockout mice exhibit clearer suppression of scratching behaviors evoked by chloroquine than by compound 48/80 [12], and that these mice fail to suppress histamine-dependent itch [2], interactions between signals from GRPRs and GABA (and glycine) receptors in GRPR + SDH neurons may also occur.
Considering the data from single-cell transcriptome of SDH neurons, it appears that there are some subsets of Adra1a-expressing inhibitory interneurons including ones that express Npy mRNA [10]. However, SDH neurons expressing Cre under the control of the Npy promoter (NPY::Cre) have been reported to selectively contribute to mechanical (but not chemical) itch [13]. Furthermore, intrathecal administration of an Y1 receptor agonist has no effect on scratching caused by chloroquine [14]. Thus, it is hypothesized that a subset of α 1A -AR-expressing inhibitory SDH interneurons that are negative to NPY expression could be involved in the regulation of histamine-independent itch transmission.
Although these are important points to be elucidated by further investigations, our findings reveal a selective role of α 1A -ARs in SDH inhibitory interneurons in histamine-independent itch and provide a clue to understanding the neuronal circuits of spinal itch transmission.

Additional file 1: Materials and methods.
Additional file 2: Schematic illustration of possible neuronal circuits regulated by α 1A -ARs expressed in Vgat-Cre + inhibitory interneurons in the SDH.

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