Extended Data Fig. 1: Nodal and not terminal GABA_A receptors in mice and rats.

a-b, In the sacrocaudal spinal cord of mice we examined the distribution of GABA_A receptor subunits on nodes and terminals of sensory axons, including extrasynaptic α5 subunits, synaptic α1 and α2 subunits, and ubiquitous γ2 subunits (for example forming the common α1βγ2 or α5βγ2 receptors, though less common extrasynaptic α1βδ have been reported)^{106,107,108,109}. We genetically labelled primary sensory axons by their expression of the vesicular glutamate transporter VGLUT1 with a reporter in VGLUT1^Cre/+; R26^LSL-tdTom mice (tdTom reporter displayed as green, for consistency with Fig. 1). VGLUT1 is mainly only expressed in sensory axons⁶⁰, especially ventral proprioceptive afferents, as other afferents do not reach the ventral horn¹⁵. Axons were reconstructed in 3D as detailed in Fig. 1. 3D reconstructed nodes of Ranvier on myelinated 2^nd order ventral branches are shown identified by paranodal Capsr immunolabelling (a, bottom), along with raw confocal image stacks (maximum projection from z-stack) prior to 3D reconstruction (a, top; receptors red). As in rats, nodes were often near branch points (green arrow). Terminal boutons from the ventral horn are likewise shown with raw and 3D rendered images (b). GABA receptors colocalized with the axon are labelled yellow in the 3D reconstructions. Receptor clusters specifically in the plasma membrane are indicated by yellow arrows in (a). Similar to rats, the α5, α1, and γ2 GABA_A receptor subunits were found on large axon branches (1^st and 2^nd order) in the dorsal, intermediate, and ventral cord, near nodes (a), but not on ventral horn terminal boutons (b, 3^rd order). As also seen in rats (Fig. 1e), synaptic GABA_A receptors were usually in single large membrane bound clusters at nodes, whereas extrasynaptic α5 receptors were often broken up into multiple clusters, with the largest clusters in the membrane (yellow arrows), and smaller cytoplasmic clusters several µm from the edge of the node under the paranodal Caspr (grey arrows; Fig. 1f). Cytoplasmic α5 receptors have been reported previously⁹⁵. Many receptors were in neighboring neurons (red arrows), and in our previous publication¹⁵ these and cytoplasmic axon receptors may have been mistaken for nodal receptors in the axon membrane, though this is corrected here with evaluation of higher resolution confocal images. The presence of these α5, α1, and γ2 GABA_A subunits is consistent with their mRNA previously reported in the dorsal root ganglion¹¹⁰. Also, the finding of α1 subunits on these axons is consistent with the recent observation that α1 is only on myelinated sensory axons, rather than unmyelinated C fibres¹¹¹. c, Synaptic α2 GABA_A receptor labelling in mouse and rat axons (rat axon labelled as in Fig. 1) with nodes labelled with both antibodies to sodium channels Na_V and Caspr, to confirm the relation of receptors to the node and paranodal region. Receptors are often at the transition between the node and paranodal Caspr, as here. Putative GABAergic synaptic contact labelled with VGAT. d, Ventral terminal boutons in VGLUT1^Cre/+; R26^LSL-tdTom mouse (with reporter labelling the complete axon, green) immunolabelled with VGLUT1 to verify that these are afferent terminals, which have vesicular VGLUT1⁺ protein expression. Immunolabelling for γ2 GABA_A receptors again showed that terminals lacked this ubiquitous subunit that makes up most GABA_A receptors. e, Immunolabelling for GABA_B receptors on 3D reconstructed sensory axons, with same format and mice of (a-b). GABA_B receptors were generally absent from nodes identified by paranodal Caspr, but present on ventral terminal boutons, as in rats (Fig. 1). Similar results (a- e) were obtained from n = 5 mice. f, Synaptic α1 GABA_A receptor labelling at a rat Ia afferent node labelled with both antibodies to sodium channels Na_V and Caspr, to confirm the relation of receptors to the node and paranodal region in rat, like in mouse (c, same node as in left of Fig. 1c, but Na_V and raw images included). Putative GABAergic synaptic contact labelled with VGAT, where left red arrow shows GABAA receptor contacting VGAT at edge of node. VGAT is near, but does not contact Caspr (in 3D view), but may well contact the paranodal myelin loops, since oligodendrocytes express GABA receptors³⁸. Node is at dorsal 2^nd order axon branch point. (a-f) representative of 5 mice. g-h, Box plots of the proportion of ventral terminal boutons with GABA receptors in both mice (g,VGLUT1⁺) and rats (h), again showing that few boutons contain GABA_A receptors, whereas many contain GABA_B receptors. * significantly more GABA_B than GABA_A receptors, ANOVA with Bonferroni correction, P < 0.05, n = 5 animals per condition, with 70–120 terminals examined per animal and receptor. Box plots show the interquartile range (box), median (thin line), mean (thick line), 10 and 90 percentiles (whiskers), and extremes (dots).