Gucy2d selectively marks inhibitory dynorphin neurons in the spinal dorsal horn but is dispensable for pain and itch sensitivity

Abstract

Introduction: Inhibitory neurons in the spinal dorsal horn can be classified based on expression of neurochemical marker genes. However, these marker genes are often expressed throughout the central nervous system, which poses challenges for manipulating genetically identified spinal neurons without undesired off-target effects.

Objectives: We investigated whether Gucy2d, previously identified as a highly selective marker of dynorphin-lineage neurons in the dorsal horn, is expressed in other locations within the adult mouse spinal cord, dorsal root ganglia (DRG), or brain. In addition, we sought to molecularly characterize Gucy2d-expressing dorsal horn neurons and investigate whether the disruption of Gucy2d gene expression affects sensitivity to itch or pain.

Methods: In situ hybridization experiments assessed Gucy2d mRNA expression in the adult mouse spinal cord, DRG, and brain, and its colocalization with Pax2, Bhlhb5, and Pde2a in dorsal horn neurons. Knockout mice lacking Gucy2d expression were compared with littermate controls to assess sensitivity to chloroquine-induced itch and dry skin-mediated chronic itch, as well as heat, cold, or mechanical stimuli.

Results: Gucy2d is selectively expressed in dynorphin-lineage neurons in lamina I-III of the adult mouse spinal cord but not in the brain or DRG. Spinal Gucy2d-expressing neurons are inhibitory neurons that also express the transcription factor Bhlhb5 and the cGMP-dependent phosphodiesterase Pde2a. Gucy2d knockout mice did not exhibit altered responses to itch or pain.

Conclusions: The selective expression of Gucy2d within a subpopulation of inhibitory dorsal horn neurons may yield a means to selectively manipulate inhibitory signaling at the level of the spinal cord without effects on the brain.

Keywords: Dynorphin; Guanylate cyclase; Gucy2d; Itch; Pain; Spinal cord.

Figure 1.

Gucy2d is expressed in the spinal dorsal horn but not in the dorsal root ganglia or brain. (A) Gucy2d mRNA (white) is strongly expressed in the superficial dorsal horn (SDH) but not the deeper dorsal horn laminae or the ventral horn. Scale bar = 500 µm. (B) Gucy2d mRNA expression detected by in situ hybridization is reduced in mice heterozygous for Gucy2d expression and (C) abolished in homozygous Gucy2d knockout mice. Scale bars in B-C = 50 µm. (D) Gucy2d mRNA (magenta) was not detected in dorsal root ganglia. Scale bar = 100 µm, inset scale bar = 20 µm. Nuclei are stained with DAPI, 4′,6-diamidino-2-phenylindole (blue). (E) A representative parasagittal section of the brain shows a lack of Gucy2d mRNA (white). Although fluorescent signal was detected in the glomerular layer of the rostral olfactory bulb (asterisk), it was determined not to be specific for Gucy2d mRNA (see supplemental Fig. S1, available at http://links.lww.com/PR9/A125). Scale bar = 1 mm.

Figure 2.

Gucy2d is not expressed in the brain. In situ hybridization experiments reveal the absence of Gucy2d mRNA (white) throughout the brain. Selected brain regions are shown: (A) hindbrain, (B) lateral hippocampus, (C) thalamus and anterior hippocampus, (D) amygdala and lateral hypothalamus, (E) primary sensory cortex and primary/secondary motor cortex, and (F) cerebellum. Distances from bregma (A–E) or from midline (F) are approximated based on Paxinos & Franklin's The Mouse Brain in Stereotaxic Coordinates fourth Ed. Scale bars = 200 µm. 4 V, fourth ventricle; DPGi, dorsal paragigantocellular nucleus; Gi, gigantocellular reticular nucleus; CA1, field CA1 of the hippocampus; CA3, field CA3 of the hippocampus; DG, dentate gyrus of the hippocampus; LV, lateral ventricle; AH, anterior hippocampus; Th, lateral thalamic nuclei; D3V, dorsal third ventricle; LH, lateral hypothalamus; AA, amygdaloid area; S1, primary sensory cortex of the hind limb; M1, primary motor cortex; M2, secondary motor cortex; CC, corpus callosum; Int, interposed cerebellar nucleus; Icp, inferior cerebellar peduncle; Crus2, crus2 of the ansiform lobule; PM, paramedian lobule.

Figure 3.

Gucy2d is selectively expressed within spinal inhibitory neurons located in lamina I to III. (A) Gucy2d mRNA (white) was detected in cells within laminae I-III of the spinal dorsal horn. Scale bar = 50 µm. (B) The majority of Gucy2d-expressing cells reside in lamina II (76.44% ± 1.68%), although smaller percentages are located in lamina I (18.79% ± 1.61%) or III (4.19% ± 0.84%). N = 23 sections from 4 mice. (C) UMAP plot showing normalized Gucy2d expression in single prodynorphin-lineage spinal nuclei. (D) UMAP plot of prodynorphin-lineage spinal nuclei identifying inhibitory (red) and excitatory (blue) neuron clusters. Nonneuronal or indeterminate clusters (gray) were not classified. (E) A comparison of Gucy2d expression levels in neurons in designated inhibitory clusters (red) and designated excitatory clusters (blue) indicates higher levels of expression in inhibitory neurons. Violin plot in E depicts scaled and log-transformed normalized expression (gene UMIs/total cell UMIs). Single-nucleus RNA sequencing data shown in panels C-E obtained in previously published study. UMAP, Uniform Manifold Approximation Projection.

Figure 4.

Spinal Gucy2d-expressing neurons in laminae I-III coexpress Pax2, Bhlhb5, and Pde2a. (A) Neurons expressing Gucy2d mRNA (magenta) coexpress Pax2 mRNA (green). (B) Bhlhb5 mRNA (green) and (C) Pde2a mRNA (green), encoding cGMP-stimulated phosphodiesterase 2, were also detected in Gucy2d+ neurons. Scale bars in low-magnification (20X) panels = 50 µm. Individual fluorescent channels are shown at high magnification (40X) in panels 1 to 3; scale bars = 20 µm. Nuclei are stained with DAPI, 4′,6-diamidino-2-phenylindole (blue).

Figure 5.

Global Gucy2d knockout mice do not exhibit altered response to pruriceptive stimuli. (A) Quantification of time spent scratching (in seconds) over a 30-minute period after the intradermal injection of chloroquine. No effect of Gucy2d genotype was observed (one-way ANOVA; P = 0.44, F_{(2, 35)} = 0.84). (B) Quantification of time spent scratching (in seconds) over a 1-hour period after induction of dry skin by repeated application of acetone: ether and water (AEW). No effect of Gucy2d genotype was observed (Kruskal–Wallis test; P = 0.23, Kruskal–Wallis statistic = 2.92). ANOVA, analysis of variance.

Figure 6.

Global Gucy2d knockout mice do not exhibit altered response to nociceptive stimuli. (A) No effect of Gucy2d genotype was observed on the mechanical paw withdrawal threshold (PWT; in gram Force) of naive adult mice (Kruskal–Wallis test; P = 0.92, Kruskal–Wallis statistic = 0.18). (B) Latency to withdraw (in seconds) from a radiant noxious heat stimulus was unaffected by Gucy2d genotype (Kruskal–Wallis test; P = 0.36, Kruskal–Wallis statistic = 2.07). Each data point represents the mean latency of 3 stimulus presentations per animal. (C) Latency to withdraw (in seconds) from a noxious cold stimulus was unaffected by Gucy2d genotype (one-way ANOVA; P = 0.24, F_{(2, 36)} = 1.50). Each data point represents the mean latency of 4 stimulus presentations per animal. ANOVA, analysis of variance.