Distinct Modes of Presynaptic Inhibition of Cutaneous Afferents and Their Functions in Behavior

Abstract

Presynaptic inhibition (PSI) of primary sensory neurons is implicated in controlling gain and acuity in sensory systems. Here, we define circuit mechanisms and functions of PSI of cutaneous somatosensory neuron inputs to the spinal cord. We observed that PSI can be evoked by different sensory neuron populations and mediated through at least two distinct dorsal horn circuit mechanisms. Low-threshold cutaneous afferents evoke a GABA_A-receptor-dependent form of PSI that inhibits similar afferent subtypes, whereas small-diameter afferents predominantly evoke an NMDA-receptor-dependent form of PSI that inhibits large-diameter fibers. Behaviorally, loss of either GABA_A receptors (GABA_ARs) or NMDA receptors (NMDARs) in primary afferents leads to tactile hypersensitivity across skin types, and loss of GABA_ARs, but not NMDARs, leads to impaired texture discrimination. Post-weaning age loss of either GABA_ARs or NMDARs in somatosensory neurons causes systemic behavioral abnormalities, revealing critical roles of two distinct modes of PSI of somatosensory afferents in adolescence and throughout adulthood.

Figure 1.. There are at least two mechanistically distinct forms of sensory neuron-evoked primary afferent depolarization (PAD).

A. Recording configuration for dorsal root (DR) or optogenetic stimulation. B. Effect of GABA_AR antagonist gabazine (GZ, 10μM) on low threshold (≤3T) and high intensity (500μA, 500μs) DR evoked DRPs in control and Avil^Cre; Gabrb3^f/f mice. Note the loss of low-threshold evoked DRPs in Avil^Cre; Gabrb3^f/f mice. C. Recording configuration for dorsal cutaneous nerve stimulation. D. Similar to DR stimulation, GZ blocks 3T dorsal cutaneous nerve evoked DRP but enhances the high intensity electrical stimulation evoked DRP. E. Example traces of optogenetically evoked DRPs at baseline and in the presence of GABA_AR antagonists GZ (5–10μM) or bicuculline (BIC, 10–20μM). Blue and yellow bars denote light pulse timing. The following crosses were used: Small diameter nociceptors (Nav1.8^Cre; Avil^Flpo; Rosa26^{LSL-FSF-ReaChR}), CGRP nociceptors (Avil^Cre; CGRP^Flpe; Rosa26^{LSL-FSF-ReaChR}), Mrgprd⁺ polymodal nociceptors (Mrgprd^Cre; Rosa26^LSL-ChR2), Aδ-LTMRs (TrkBCreER; Rosa26LSL-ChR2), and Aβ RA-LTMRs (RetCreER; AvilFlpo; Rosa26LSL-FSF-ReaChR). F and G. Summary of the effects of GABA_AR antagonists on electrically (F) and optogenetically evoked DRPs (G). All bar graphs in figures are represented as mean ± SEM. Asterisks above bar graphs denote statistical significance (**** p<.0001, *** p< .001, ** p<.01, * p<.05). Scale bars represent 0.05mV, 100ms.

Figure 2.. Presynaptic NMDARs contribute to high threshold PAD.

A. Optogenetic activation of small diameter afferents (Nav1.8^Cre; Avil^Flpo; Rosa26^{LSL-FSF-ReaChR}) and high intensity electrical stimulation of the dorsal root evoked DRPs in baseline conditions and with the sequential addition of an NMDAR antagonist (CPP, 20μM), kainate receptor antagonist (UPB301, 10μM), metabotropic glutamate receptor antagonists (MCPG, 50μM and LY341495, 50μM), and AMPA and kainate receptor antagonist (NBQX, 10μM). Only the combination of ionotropic glutamate receptor inhibitors blocks both types of DRP. B. Summary of the effects of CPP (20μM) on low threshold electrical, high intensity electrical and small diameter optogenetically evoked DRPs. Only the latter two types of evoked DRPs are inhibited by CPP (one-way ANOVA, F(2,13)=24.75, p<.0001). Asterisks denote Tukey’s post hoc test. C. Example of the effect of CPP on DRPs in control mice and Avil^Cre, Grin1^f/f mutants. D. Summary of CPP-evoked changes on 500μA, 500μs evoked DRPs. Asterisks below bar graph denote statistical significance from baseline, asterisk above denotes two-tailed student’s t-test, p=.0003. E. Control and GZ subtracted traces of 500μA, 500μs dorsal root stimulation in control, Avil^Cre; Grin1^f/f and Avil^Cre; Gabrb3^f/f mutants. ^ denotes gabazine-sensitive peak, asterisk denotes gabazine-enhanced trough. F. Normalized GZ-sensitive peak and trough amplitude. Two-way ANOVA, genotype F(2,26)=30.61, p<.0001; component F(1,26)=206.6, p<.0001. Dunnett’s multiple comparison post hoc test comparing each mutant to control mice, denoted by asterisks. G. Immunohistochemistry of lamina III-IV for NR1 and myelinated afferent terminals marked by VGlut1. White arrows denote presynaptic NR1 puncta and yellow arrows denote postsynaptic NR1 puncta in control mice and Avil^Cre; Grin1^f/f mutants. H. Average number of NR1 puncta per VGlut1 afferent terminal. Unpaired t-test, p=0.0005. (**** p<.0001, *** p< .001, ** p<.01, * p<.05)

Figure 3.. GABA_AR PAD and NMDA-R PAD are engaged by different populations of dorsal horn excitatory interneurons.

A. Recording configuration denoting light stimulation of the dorsal root entry zone. B. CCK^iCre-labeled interneuron induced DRPs in neonatal or juvenile mice are completely or partially blocked by GABA_AR antagonists, respectively. Shaded region demarcates area under the DRP calculated in C. C. Composite data, integrating the area under the filtered DRP up to its peak in both neonatal and juvenile mice. Asterisks denote statistical significance from no effect. D. Transient VGlut3⁺ interneuron (labeled using VGlut3^Cre, Lbx1^Flpo mice) activated DRPs depend on ionotropic glutamate receptors, particularly NMDARs, and are enhanced by GABA_AR antagonists. E. Composite data, integrating area under the full filtered DRP for each antagonist. F. Application of tetrodotoxin (TTX, 1uM) greatly diminishes the transient VGlut3⁺ interneuron optogenetically evoked DRP, which is partially recovered by the addition of 600 μM 4-AP. Electrically evoked DRPs were completely blocked by TTX and could not be recovered with the addition of 4-AP (400μM-1mM). G. Composite data of the effect of TTX (1 μM) and 4-AP (400–600μM) on optogenetically evoked DRPs. One-way ANOVA, F(1.241,2.482)=307.2, p=.0011. Asterisks denote Tukey’s multiple comparison post hoc test. Physiology scale bars denote 0.05mV, 100ms. H. Immunohistochemistry of transient VGlut3⁺ interneurons and CCK^iCre labeled interneurons. Upper: Sagittal sections from CCK^iCre; Rosa26^LSL-ChR2 mouse counterstained for IB4 and VGlut1; Lower: Sagittal sections from VGlut3^Cre; Lbx1^Flpo; Rosa26^{LSL-FSF-Synaptohpysin-GFP} mouse counterstained for VGlut1 and IB4. Inset is high magnification of close proximity of transient VGlut3 interneuron terminals to VGlut1 presumptive afferent terminals. (*** p< .001, ** p<.01, * p<.05).

Figure 4.. GABA_ARs and NMDA-Rs are both required in adult somatosensory afferents for normal tactile sensitivity.

A. qPCR of mRNA knockdown in both Avil^CreER conditional knockout models. Unpaired two-tailed student’s t-test with Welch’s correction, p=0.0003 for Grin1 and p=.0018 for Gabrb3. B. tPPI, 50ms ISI, Avil^CreER; Grin1^f/f. Two-way ANOVA: interaction F(1,79)=7.437, p=.0079; sex F(1,79)=10.39, p=.0018; genotype F(1, 79)=6.146, p=.0153. Tukey’s multiple comparisons test denoted by asterisks. C. tPPI, 50ms ISI, Avil^CreER; Gabrb3^f/f. Two-way ANOVA, asterisks denote main effect of genotype (F(1,64)=8.618, p=.0046), no main effect of sex or interaction. D and E. Von Frey thresholds and response rates for Avil^CreER; Grin1^f/f (D) and Avil^CreER; Gabrb3^f/f (E). Two-way ANOVA of threshold, main effect of genotype denoted by asterisks. Avil^CreER; Grin1^f/f: F(1,37)=16.05, p=0.0003; Avil^CreER; Gabrb3^f/f: F(1,40)=6.937, p=0.0119. For response rates, a two-way repeated measures ANOVA was performed, main effect of genotype noted by the p-value in the upper left and asterisks denoting Sidak multiple comparisons post hoc test. No main effect of sex or interaction for either genotype. F-H. Textured NORT (F), shape NORT (G), and rough floor aversion tests (H) for both Grin1^f/f and Gabrb3^f/f crosses. Asterisks above bar graphs denote preference significantly different than zero. Unpaired two-tailed student’s t-test per cross, p=.0122 for Avil^CreER; Gabrb3^f/f mice textured NORT. (**** p<.0001, *** p< .001, ** p<.01, * p<.05)

Figure 5.. GABA_ARs mediate PSI of both large diameter and small diameter sensory neurons, whereas NMDARs mediate PSI predominantly in large diameter neurons.

A. Sample traces of same-day-recorded littermate control and Nav1.8^Cre; Gabrb3^f/f mutant DRPs. B. Composite data of DRP integrals, two-way ANOVA: interaction of stimulus intensity and genotype, F(1,20)=6.461, p=0.0194. Asterisks denote Sidak’s multiple comparisons test, compared within each stimulus intensity. C. Example traces from Nav1.8^Cre; Gabrb3^f/f mutant DRPs evoked by stimulating an adjacent dorsal root at stimulus intensity noted, and with the addition of 10μM gabazine (GZ). D. Composite data of GZ sensitivity. Control and Nav1.8^Cre; Gabrb3^f/f mutant mice have bidirectional GZ modulation of evoked DRPs (two-way ANOVA, no main effect of genotype or interaction with stimulus intensity). E. Example traces from Nav1.8^Cre, Grin1^f/f mutant DRPs evoked by dorsal root stimulation using the stimulation intensities noted, and with the addition of CPP. Note marked CPP-induced reduction of the high intensity DRP remains. F. Composite data comparing control, Nav1.8^Cre; Grin1^f/f, and Avil^Cre; Grin1^f/f mice (repeated from Figure 2). One-way ANOVA: F(2,12)=5.228, p=.0233. Asterisks below bars denote Dunnett’s multiple comparison post hoc test. All scalebars denote 0.05mV, 100ms. G. Diagrams of three afferent activity-dependent modes of PSI. Left most spinal cord dorsal horn represents GABA_AR dependent PSI of large diameter cutaneous afferents. Activity in Aβ-LTMRs can lead to activation of a subset of CCK^iCre-labeled excitatory interneurons, which in turn activate GABAergic interneurons and induce GABA_AR-dependent PAD in predominantly other Aβ subtypes. Middle spinal cord represents GABA_AR-dependent PSI of small diameter afferents, induced by activity in small diameter afferents. Currently unidentified interneuron(s) are colored grey. The right spinal cord dorsal horn represents NMDAR-dependent PSI, whereby activity in C and Aδ afferents predominantly inhibits Aβ LTMRs through transient VGlut3⁺ excitatory interneurons. The anatomical substrate of this form of PSI is unknown but does not require additional synapses.

Figure 6.. NMDAR-mediated PAD of Aα/Aβ afferents underlies hairy skin hypersensitivity, while GABA_AR-mediated PAD of both C-/Aδ-afferents and Aα/Aβ afferents underlies glabrous skin hypersensitivity.

A. Diagram of viral strategy to conditionally delete receptors only in DCN projecting A-fibers. B. Characterization of viral strategy of AAV2/9-fDIO-CreGFP in Avil^FlpO; Rosa26^LSL-tdTomato mice and control (Rosa26^LSL-tdTomato) mice in DRG and spinal cord dorsal horn (right most panels). GFP expression and Cre driven tdTomato expression was observed only in NF200⁺ neurons in the DRG of Avil^FlpO mice. Scalebars denote 20μm for DRG and 50μm for spinal cord. C. RNAscope for Cre and Grin1 expression in Avil^FlpO; Grin1^f/f mice and immunohistochemistry for GFP expression. White arrowhead denotes Cre⁺/GFP⁺ soma, yellow arrowheads note Cre⁺/GFP^- cells. D. Labeling efficiency of virus injected mice previously run on behavioral assays. E. 50% withdrawal threshold for Von Frey test for Gabrb3^f/f crosses. Two-way ANOVA was run separately on each cross; asterisks denote main effect of genotype. Nav1.8^Cre; Gabrb3^f/f main effect of genotype: F(1,26)=6.136, p=.0201; Avil^FlpO; Gabrb3^/f main effect of genotype: F(1,36)=8.506, p=.0061. F. tPPI with 50ms ISI for all three Gabrb3^f/f crosses, M labels male data, F labels female data. No significant effects of sex or interactions between sex and genotype for any cross. G. Von Frey thresholds for Grin1^f/f crosses, asterisks denote main effect of genotype. H. tPPI with 50ms ISI for all three Grin1^f/f crosses. Two-way ANOVA interaction between sex and genotype for Avil^FlpO; Grin1^f/f mutants, F(1,61)=6.244, p=.0132. For each cross, Sidak’s multiple comparison post hoc test was performed within each sex and denoted with asterisks. Avil^CreER; Gabrb3^f/f and Avil^CreER; Grin1^f/f data are replotted from data in Figure 4. (*** p< .001, ** p<.01, * p<.05)

Figure 7.. Loss of GABA_ARs and NMDARs in somatosensory neurons leads to anxiety-like and exploratory behavioral alterations.

A. Sample open field traces of Avil^CreER; Grin1^f/f and Avil^CreER; Gabrb3^f/f female mice and their littermate controls. B-F. Composite data of behavioral paradigms, asterisks and statistics denote two-way ANOVA main effect of genotype, data shown are pooled across sex. B. Distance traveled during open field; Avil^CreER; Grin1^f/f: F(1,54)=8.943, p=.0042; and Avil^CreER; Gabrb3^f/f: F(1,54)=10.36, p=.0022. C. Time in center of open field, Avil^CreER; Grin1^f/f: F(1,54)=42.34, p<0.0001; Avil^CreER; Gabrb3^f/f: F(1,54)=13.49, p=0.0006. D. Elevated plus maze, Avil^CreER; Grin1^f/f F(1,50)=20.53, p<0.0001; Avil^CreER; Gabrb3^f/f: F(1,51)=11.14, p=0.0016. E. Acoustic startle reflex, Avil^CreER; Grin1^f/f: F(1,74)=27.85, p<0.0001; and Avil^CreER; Gabrb3^f/f: F(1,64)=13.15, p=0.0006. F. Short term habituation to acoustic startle, asterisks above bars denote statistically significant habituation. Two-way ANOVA main effect of genotype of Avil^CreER; Grin1^f/f: F(1,55)=4.366, p=.0413; Avil^CreER; Gabrb3^f/f: F(1,60)=4.856, p=0.0314.