Extended Data Fig. 4: GABAergic innervation of nodes and nearby boutons in viral vector labelled proprioceptive afferents.

a, Sensory afferents labelled in the spinal cord and DR of adult GAD2//ChR2-EYFP mouse (GAD2-EYFP) by a peripheral adeno-associated virus (AAV9-tdTom); confocal image of a transverse spinal cord section. Large myelinated proprioceptive Ia afferents were identified by characteristic extensive ventral horn branching and innervation of motor neurons (as in Fig. 1a). These Ia afferents left the dorsal columns in bundles that were readily traced to the ventral horn in serial sections, with an S-shaped projection path from the dorsal columns to the motor neurons (as in Fig. 3j)¹¹². The relatively sparce afferent labelling helped facilitate this tracing of afferent innervation. The peripheral AAV9-tdTom injection occasionally also labelled one motor neuron in a transverse (lower white arrow), though this only occurred a couple times per spinal cord segment, making them possible to distinguished from afferents elsewhere. No other central neurons were labelled, indicated that the viral vector only affected peripheral sensory and motor axons, as we previously found⁴³. EYFP⁺ cells (GABA_axo neurons) were only in the dorsal and intermediate laminae (GAD2-EYFP⁺), with projections into the ventral horn and dorsal columns along the entire path of the Ia afferent bundles, as detailed in Fig. 3. The approximate regions where images in (b-h) were taken are indicated in (a), but in different transverse sections, oriented similarly. b, GABA_axo neurons formed a dense plexus that wound around Ia afferents as they branched out of the dorsal columns into the grey matter (at dashed line). c, Main 1^st order myelinated branch of a Ia afferent in intermediate laminae with three nodes at branch points labelled with axonal caspr, approximate myelin regions marked with white lines, and GABA_axo neurons seen wrapping the axon (top images). Node 1 had a GAD2-EYFP + contact that had presynaptic GAD2 labelled with an antibody (GAD2-Ab, cyan; yellow marks contact of afferent, GAD2-EYFP and GAD2-Ab computed in 3D), whereas Nodes 2 and 3 lacked contacts, though the short branch arising off of Node 3 had a nearby unmyelinated terminal branch with a GAD2⁺ contact. GAD2 (GAD65) is known to be closely associated with GABAergic terminals and vesicular GABA and highly enriched in terminals compared to elsewhere in neurons^5,7,113,114, and thus the GAD2-Ab provides a presynaptic contact label, consistent with the intense GAD2-Ab labelling we observed in GAD2-EYFP boutons (also bassoon⁺), and similar to the GAD65-intense boutons on afferents previously detailed⁷. Expanded view of Node 1 shown at bottom left, where all caspr labelling is displayed, including in overlapping nearby axons, whereas in other images only the axonal caspr in the 3D volume of the afferent is shown for clarity, together with raw images from other antibodies. Expanded view of Node 1 contact shown on bottom right, where all GAD2-ab labelling is shown, whereas in other images only GAD2-Ab in the GAD2-EYFP neuron volume is shown. Not all GAD2-Ab was in GAD2-EYFP⁺ neurons, likely due to variability in tamoxifen induced cre or transport of EYFP in GAD2//Ch2R-EYFP mice. d, Nodal contact in the ventral horn, shown with similar format to (c), with again a GABA_axo neuron wrapping around the axon and specifically making a contact at the node (yellow; EYFP + , GAD2-Ab⁺). White lines: estimated myelin regions. 1^st order afferent. e, Two nodes on two 1^st order Ia afferent branches in the dorsal horn plexus of (b), again delineated by paranodal caspr, one of which had a GABA_axo neuron contact (GAD2-EYFP, marked yellow again) with presynaptic GAD2-Ab (cyan, shown in 3D volume of GABA_axo neuron) and the other did not. f-g, Nodes from 1^st order Ia afferent branches, identified by paranodal Caspr, in control wild-type mice, and so lacking EYFP, but inhibitory innervation examined with VGAT or GAD2 antibodies. In (f) the node had a direct VGAT⁺ contact and nearby contacts on the small unmyelinated branch arising from the node (contacts computed in 3D labelled yellow). In (g) the node lacked a direct GABAergic contact, but had a nearby GAD2-Ab⁺ contact on a bouton of a short unmyelinated branch arising from the node. h, Complex node in intermediate laminae with enlarged inter-myelin region and two unmyelinated branches, one with a GAD2-Ab⁺ contact on the neck of the branch, and the other with contacts on nearby boutons (above top of image). Same format as (d-e). Similar to enlarged nodal boutons of Walmsley²⁵. (a-h) representative of n = 3 mice. i, Quantification of the fraction of total nodes with direct synaptic GABAergic contacts (nodal contacts, GAD2-Ab⁺, ~25%), nearby contacts at a node or unmyelinated terminal branch/bouton on the same axon (within λ_S = 90 µm; 98–77%; as in c) and putative extrasynaptic innervation (within 5 µm, ~95%) from GABA_axo neurons, with presynaptic GABA inferred from GAD2-Ab immunolabelling, in n = 3 mice from n = 43–53 nodes, each on 1^st and 2^nd order branches of Ia afferents. VGAT⁺ contacts on or near nodes occurred with similar incidence (28% of nodes had synaptic contacts, from n = 50 nodes in n = 3 mice). Similar incidence of contacts occurred in rat afferents labelled with neurobiotin (n = 5 rats). Synaptic contacts occurred at unbranched or branched nodes, as did extrasynaptic innervation, with about half the nodes branched overall. Synaptic contacts that occurred on short unmyelinated afferent terminal branches arising from the node usually occurred at a bouton and are labelled as: Nearby bouton. Synaptic GAD2⁺ contacts occurred most frequently at or near complex branch points where a parent branch split into large daughter branches (d) or the daughter branches had large boutons (g-h), which theoretically increased the local conductance and probability of spike failure downstream to the branch point (as in Extended Data Fig. 5).