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

Abstract

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-HT1A receptors [4]. As for noradrenaline (NA), we recently demonstrated that NAergic neurons descending from the locus coeruleus (LC) to the SDH negatively control itch-related scratching behaviors. Furthermore, application of either NA or an α1A-adrenaline receptor (α1A-AR) agonist to spinal cord slices facilitates the transmission of inhibitory synaptic inputs into GRPR+ SDH neurons [5]. Thus, it is possible that α1A-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 α1A-ARs expressed in inhibitory SDH interneurons using genetic tools that enable cell- and region-specific gene manipulations.

We generated mice in which α1A-ARs are conditionally knocked out in inhibitory neurons by crossing Vgat-Cre mice (in which Cre is expressed in inhibitory neurons under the control of the promoter of vesicular GABA transporter [6]) with the Adra1aflox/flox mice that we recently developed [7] (Additional file 1). Scratching behavior was induced by injecting pruritogens (compound 48/80 and chloroquine, models of histamine-dependent and -independent itch, respectively [2, 8]) intradermally into the nape of the neck of Adra1aflox/flox and Vgat-Cre;Adra1aflox/flox mice. The scratching responses induced by compound 48/80 were indistinguishable between Adra1aflox/flox and Vgat-Cre;Adra1aflox/flox mice; however, the chloroquine-induced scratching responses were significantly enhanced in Vgat-Cre;Adra1aflox/flox mice compared to Adra1aflox/flox mice (Fig. 1a). In addition, nociceptive behavior (wiping responses to the capsaicin-injected cheek) was not different between the two genotypes (Adra1aflox/flox mice, 46.0 ± 9.6, n = 5; Vgat-Cre;Adra1aflox/flox mice, 40.2 ± 9.7, n = 6; P = 0.623, unpaired t-test). Our previous study showed that the application of α1A-AR agonist to spinal cord slices facilitates the transmission of inhibitory synaptic inputs into GRPR+ SDH neurons, raising the possibility that α1A-ARs in inhibitory SDH interneurons have a role in the itch response [5]. We examined the expression of α1A-ARs in the SDH using RNAscope in situ hybridization and found that Adra1a mRNA was clearly detected in inhibitory interneurons positive for Slc32a1 mRNA (also known as Vgat) in the SDH of control Adra1aflox/flox mice (Fig. 1b, c). Quantitative analysis revealed that 18.3% of Slc32a1+ SDH interneurons were positive for Adra1a mRNA (Fig. 1c). SDH cells expressing both Adra1a and Slc32a1 mRNA were almost absent in Vgat-Cre;Adra1aflox/flox mice (Fig. 1b, c), confirming the specificity of the probe to detect Adra1a mRNA and suggesting that α1A-ARs are expressed in inhibitory SDH interneurons. Because Vgat-Cre;Adra1aflox/flox mice might lack α1A-AR expression in inhibitory neurons in the brain, we employed the CRISPR–Cas9 system using adeno-associated virus (AAV) vectors [9] to knock out inhibitory neuronal α1A-ARs specifically in the SDH. Cre-dependent Staphylococcus aureus Cas9 (SaCas9)-expressing vectors (AAV-FLEX-SaCas9) and single guide RNA-expression vectors (AAV-FLEX-mCherry-U6-sgAdra1a or -sgRosa [as control]) were injected into the bilateral cervical SDH of Vgat-Cre mice (Fig. 1d). Vgat-Cre mice injected with AAV-FLEX-SaCas9 and AAV-FLEX-mCherry-U6-sgAdra1a did not show changes in scratching behavior as elicited by compound 48/80; however, they showed a significant increase in scratching when itch was induced by chloroquine (Fig. 1e).

Fig. 1
figure1

Knockout of α1A-ARs in inhibitory interneurons enhances scratching behavior evoked by chloroquine. a Scratching behaviors induced by intradermal injection of compound 48/80 (50 μg/50 μl) or chloroquine (200 μg/50 μl) in Adra1aflox/flox mice or Vgat-Cre;Adra1aflox/flox mice. *P < 0.05. b RNAscope in situ hybridization for mRNAs of Adra1a (red) and Slc32a1 (green) in the SDH of Adra1aflox/flox mice or Vgat-Cre;Adra1aflox/flox mice. 4,6-diamidino-2-phenylindole (DAPI) to mark nuclei is blue. Arrowheads and arrows indicate Adra1a+ Slc32a1+ cells and Adra1a Slc32a1+ cells, respectively. Scale bar, 20 μm. c Percentage of Adra1a+ Slc32a1+ cells per total Slc32a1+ cells tested in the SDH of Adra1aflox/flox mice or Vgat-Cre;Adra1aflox/flox mice. **P < 0.005. d Schematic illustration of transduction strategy of SaCas9 and guide RNA (sgAdra1a and sgRosa (control)) expression in SDH inhibitory neurons. e Effect of genome editing on scratching behaviors induced by intradermal injection of compound 48/80 (50 μg/50 μl) or chloroquine (200 μg/50 μl). *P < 0.05. Data show the mean ± SEM

By generating two lines of genetically manipulated mice in a cell type- and region-specific manner, we demonstrate for the first time that α1A-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 α1A-ARs in SDH inhibitory interneurons enhanced scratching behavior elicited by chloroquine but not by compound 48/80, whereas silencing SDH-projecting LC-NAergic neurons is known to enhance scratching induced by both pruritogens [5]. Given that chloroquine and compound 48/80 cause histamine-independent and -dependent itch, respectively [2, 8], it is possible that α1A-ARs in SDH inhibitory interneurons preferentially contribute to the regulation of histamine-independent itch. This study showed that Adra1a was expressed in SDH inhibitory interneurons, and activation of spinal α1A-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 α1A-AR-expressing SDH inhibitory interneurons suppress histamine-independent itch (Additional file 2). Other ARs, such as α2-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 α1A-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 α1A-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 α1A-ARs in SDH inhibitory interneurons in histamine-independent itch and provide a clue to understanding the neuronal circuits of spinal itch transmission.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its Additional file.

Abbreviations

SDH:

Spinal dorsal horn

GRPR:

Gastrin-releasing peptide receptor

GRPR+ neurons:

Gastrin-releasing peptide receptor-expressing neurons

5-HTergic neurons:

Serotonergic neurons

NA:

Noradrenaline

LC:

Locus coeruleus

α1A-AR:

α1A adrenaline receptor

AAV:

Adeno-associated virus

SaCas9:

Staphylococcus aureus Cas9

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Acknowledgements

We thank the University of Pennsylvania vector core for providing pZac2.1, pAAV2/9, and pAd DeltaF6 plasmids. We would like to thank Editage (www.editage.com) for English language editing.

Funding

This work was supported by JSPS KAKENHI Grant Numbers JP19K22500, JP19H05658, JP20H05900 (M.T.), by the Core Research for Evolutional Science and Technology (CREST) program from AMED under Grant Number JP20gm0910006 (M.T.), and by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP20am0101091 (M.T.).

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YS designed experiments, performed behavioral experiments, analyzed the data and wrote the manuscript. KK designed experiments, performed plasmid construction and viral injection. RY performed viral injection, behavioral experiments and analyzed the data. IH provided Adra1aflox/flox mice. MSH advised on behavioral experiments. MT conceived this project, supervised the overall project, designed experiments and wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Makoto Tsuda.

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Supplementary Information

Additional file 1:

Materials and methods.

Additional file 2:

Schematic illustration of possible neuronal circuits regulated by α1A-ARs expressed in Vgat-Cre+ inhibitory interneurons in the SDH.

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Shiraishi, Y., Koga, K., Yamagata, R. et al. α1A-adrenaline receptors in dorsal horn inhibitory neurons have an inhibitory role in the regulation of chloroquine-induced itch in mice. Mol Brain 14, 55 (2021). https://0-doi-org.brum.beds.ac.uk/10.1186/s13041-021-00768-9

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Keywords

  • α1A-Adrenaline receptor
  • Spinal dorsal horn
  • Inhibitory interneurons
  • Itch
  • Mouse