Extrasynaptic α5GABAA receptors on proprioceptive afferents produce a tonic depolarization that modulates sodium channel function in the rat spinal cord

Abstract

Activation of GABA_A receptors on sensory axons produces a primary afferent depolarization (PAD) that modulates sensory transmission in the spinal cord. While axoaxonic synaptic contacts of GABAergic interneurons onto afferent terminals have been extensively studied, less is known about the function of extrasynaptic GABA receptors on afferents. Thus, we examined extrasynaptic α₅GABA_A receptors on low-threshold proprioceptive (group Ia) and cutaneous afferents. Afferents were impaled with intracellular electrodes and filled with neurobiotin in the sacrocaudal spinal cord of rats. Confocal microscopy was used to reconstruct the afferents and locate immunolabelled α₅GABA_A receptors. In all afferents α₅GABA_A receptors were found throughout the extensive central axon arbors. They were most densely located at branch points near sodium channel nodes, including in the dorsal horn. Unexpectedly, proprioceptive afferent terminals on motoneurons had a relative lack of α₅GABA_A receptors. When recording intracellularly from these afferents, blocking α₅GABA_A receptors (with L655708, gabazine, or bicuculline) hyperpolarized the afferents, as did blocking neuronal activity with tetrodotoxin, indicating a tonic GABA tone and tonic PAD. This tonic PAD was increased by repeatedly stimulating the dorsal root at low rates and remained elevated for many seconds after the stimulation. It is puzzling that tonic PAD arises from α₅GABA_A receptors located far from the afferent terminal where they can have relatively little effect on terminal presynaptic inhibition. However, consistent with the nodal location of α₅GABA_A receptors, we find tonic PAD helps produce sodium spikes that propagate antidromically out the dorsal roots, and we suggest that it may well be involved in assisting spike transmission in general. NEW & NOTEWORTHY GABAergic neurons are well known to form synaptic contacts on proprioceptive afferent terminals innervating motoneurons and to cause presynaptic inhibition. However, the particular GABA receptors involved are unknown. Here, we examined the distribution of extrasynaptic α₅GABA_A receptors on proprioceptive Ia afferents. Unexpectedly, these receptors were found preferentially near nodal sodium channels throughout the afferent and were largely absent from afferent terminals. These receptors produced a tonic afferent depolarization that modulated sodium spikes, consistent with their location.

Keywords: GABA; antidromic action potential; branch point failure; dorsal root; dorsal root reflex; extrasynaptic; intracellular recording; primary afferent depolarization; spinal cord.

Fig. 1.

Localization of GABA receptors on 3D reconstructed afferents. A, left: neurobiotin-filled afferent (green) and immunolabelled α₅GABA_A receptors (red) imaged sequentially at 0.1-µm increments in depth with a laser-scanning confocal microscope and displayed as maximum intensity projection across the image stack. Right: same image with the afferent and receptors reconstructed in 3D, and any receptor within the afferent volume was labeled yellow rather than green, whereas receptors not on the afferent remained red. Amplified images are shown from insets in the middle panels. Arrows show a large cluster of α₅GABA_A receptors on the afferent. The lower panel shows the afferent from a side view or slightly rotated lateral view, again showing receptor clusters within the afferent in yellow. B, top: images stacks from the same afferent, with NF200 immunolabelling shown additionally to provide a background stain of other axons in the spinal cord, with presynaptic contacts approaching the afferent in the zone where the receptors are located. Bottom: sequential images from the same stack as in A, showing the receptor cluster on the afferent at the arrow.

Fig. 2.

Distribution of α₅GABA_A receptors on proprioceptive group Ia afferent. A, left: low-power image of a transverse section of the spinal cord with neurobiotin-filled group Ia afferent (green) lying in the plane of the section, with many collateral branches arising from the afferent in the deep dorsal horn (DH) above the central canal (CC) and in the ventral horn (VH). Right, same section with immunolabelling for α₅GABA_A receptors (red). B: sagittal section through the spinal cord showing fragments of the afferent as it traverses from the dorsal columns to the ventral horn. Neurofilament labeling counterstain (purple). Right, fully reconstructed primary axon collateral branching off from the dorsal columns and descending to the ventral horn, reconstructed from sequential sagittal sections. Boxes (i, ii, iii, and iv) indicate regions expanded below. The axon was penetrated at the boundary of the dorsal columns (DC) and the gray matter (injection). C: high-power image of the dorsal columns axon of the afferent forming a descending primary branch (1°; primary branch point) reconstructed in 3D. α₅GABA_A receptors colocalized with afferent are labeled yellow, and others labeled red (using methods of Fig. 1). Neurofilament is labeled purple. Note GABA receptor cluster near branch point (arrow). D: secondary branch point (from 1° to 2° collaterals), again with GABA receptors nearby (yellow). E and F: terminal branches and boutons in the dorsal horn, with some GABA receptor labeling near the branch points (yellow arrows). Terminal boutons sometimes lacked GABA receptors (green arrow). Inset in E expanded in F. G: long descending collateral from the central canal to the ventral horn, with GABA receptors at branch points, as well as nodes in long unbranched sections (yellow). Insets on right show expanded regions at branch points. Inset on left shows peculiar branch from small secondary to much larger ventral axon (ventral columns), again with GABA receptors nearby. H: afferent terminal branches (3°) and boutons on motoneurons lacking GABA receptors (green arrows), but larger secondary branch points containing receptor clusters (yellow arrows). Motoneurons (white arrows) contained large α₅GABA_A receptor clusters (red, pink arrow).

Fig. 3.

Distribution of α₅GABA_A receptors on putative low-threshold cutaneous afferent. A: low-power image of a low-threshold afferent traveling in dorsal columns (DC) with short primary collaterals descending into the superficial dorsal horn (DH) and forming compact terminal clusters (putative cutaneous afferent; neurobiotin labeled green). CC, central canal; VH, ventral horn. B: branch point from the DC axon to primary collateral (1°), with α₅GABA_A receptors nearby (yellow, arrows), reconstructed in 3D. Receptors outside afferent are red. Neurofilament NF200 is purple. C: secondary (2°) collaterals and tertiary terminal branches (3°) densely covered with α₅GABA_A receptors (yellow arrows), including near branch points and on boutons (arrows). Green arrows show terminals lacking GABA receptors. Inset on left expanded at right. D: secondary to terminal branch point with multiple GABA receptor clusters (yellow at yellow arrows), and GABA receptors on some but not all terminals. Lower panel, same collateral, but with neurofilament labeling and many GABA receptors not in axon shown (red).

Fig. 4.

Distribution of sodium channels on afferents, relative to α₅GABA_A receptors. A: large dorsal column branch of a proprioceptive Ia afferent (horizontal, top) branching to primary collateral (1°) that descended into the gray matter and formed a secondary branch (2°). Afferent labeled with neurobiotin (green). The sodium channels labeled with a pan-sodium antibody and colocalized on the afferent are indicated in white (by white arrows, channels outside afferent not shown). Sodium channel immunolabelling occurred in clusters at branch point nodes and at other nodes on long unbranched sections of the afferent. Sodium α₅GABA_A receptor clusters on the afferent (yellow arrows) were expressed near the sodium channel nodes (receptors outside node red). Nodes expanded in box insets. NF200, purple. B: branch points in primary and secondary collateral with sodium channel clusters in cutaneous afferent. Terminal branches (3°) and boutons typically lacked sodium channels, with a few exceptions in long or complex terminal branches (white arrows). Terminals lacking GABA receptors are marked with green arrows.

Fig. 5.

Distribution of primary afferent depolarization (PAD) in the spinal cord. A: extracellular recording (EC; black) of the group Ia afferent volley (field) in lamina I near the dorsal columns (DC) in response to S4 dorsal root (DR) stimulation (1.2 × threshold (T), for volley), expanded 10× in gray. Intracellular (IC) spike (red) in response to the same DR stimulation after penetrating a nearby proprioceptive group Ia afferent (S4), demonstrating that extracellular fields are negative when there is a nearby positive intracellular event. Afferent resting at −72 mV. B: same cell as in A, but on longer time scale and stimulation set lower (1.1 × T), subthreshold to direct orthodromic spike in the same afferent. Intracellular recording (red) shows a PAD in response to the DR stimulation. Extracellular recording (black) shows the afferent volley field, followed by a second field corresponding to the synaptic excitation of local interneurons in the dorsal horn, and finally a slow long-lasting negative field corresponding to PAD (expanded in gray, 7×; truncated at vertical line). Subtraction of the EC from the IC recordings gives the actual transmembrane potential (green). C: representative intracellular (red) and extracellular (black) recordings from different laminae in the spinal cord during dorsal root stimulation (~2.0 × T), with peak PAD observed in the deep dorsal horn (positive red, IC and negative black, EC), including both an early, fast phasic PAD and later tonic PAD (n = 22). In the ventral horn, only a transient 20 ms long PAD was seen in the ventral afferent terminals recorded intracellularly (Lamina IX, red, n = 4), and extracellularly there was no negative PAD field (n = 20/20). Scale 0.5 mV for dorsal EC fields, 0.1 mV for ventral EC fields and 2 mV for all IC recordings.

Fig. 6.

Phasic primary afferent depolarization (PAD) mediated by GABA_A receptor chloride currents. A: intracellular recording of group I afferent in the dorsal horn (DH) from S4 dorsal root (DR). VH, ventral horn. B: PAD evoked by stimulation of adjacent Ca1 dorsal root. Inset: onset of PAD, at black arrow. Afferent volley at red arrow. Resting potential at dotted line, −67 mV. C: PAD amplitude variation with initial holding potential, with reversal potential near −15 mV. D: reversal potential increased by blocking NKCC1 chloride pump with bumetanide (50 µM). Reversal potential unchanged by varying intracellular chloride, without injecting current, proving that the ultrasharp electrode tips (50 nm opening) do not allow mixing of the electrode and cellular contents; n = 5 per group. E–F: high-dose bicuculline (50 µM) or gabazine (30 µM) blocked most of PAD (n = 18 and 4, respectively), leaving only a small and very slow component. G: PAD in a group Ia afferent held at different potentials with a bias current [iCa1 afferent, with contralateral Ca1 DR stimulation at 2 × threshold (T)]. Fast phasic PAD and sustained tonic PAD indicated. Variability shown with standard deviation (SD) error bars. *Significantly changed, P < 0.05.

Fig. 7.

Tonic primary afferent depolarization (PAD) is mediated by α₅GABA_A receptors. A: intracellular recording from group I afferent (S4). Blocking all GABA_A with high-dose bicuculline (50 µM) eliminated a spontaneous tonic PAD, hyperpolarizing the afferent. Resting potential at dotted line, −74 mV. B: selectively blocking α₅GABA_A receptors with L655708 (0.1–0.2 µM), likewise, hyperpolarized group I afferents (different afferent), and increased input resistance (R_m measured with −0.1 nA pulse; loss of shunt). C: on average, bicuculline decreased both spontaneous tonic PAD (n = 5) and dorsal root-evoked phasic PAD (n = 22), with a larger reduction in tonic PAD, indicating that spontaneous tonic PAD is large in relation to phasic PAD. In contrast, L655708 only blocked tonic PAD and not phasic PAD (n = 5). Error bars SD; *significantly different, P < 0.05.

Fig. 8.

Extrasynaptic α₅GABA_A receptors contribute to a tonic primary afferent depolarization (PAD), assist antidromic spikes, and are partly activated by a TTX-resistant GABA leak. A: extracellular recording from cut central end of an S4 dorsal root (DR) in grease very close to the cord, to observe the compound potentials from many afferents. B: dorsal root stimulation [Ca DR; 2 × threshold (T)] evoked a dorsal root potential (DRP) corresponding to phasic PAD in Fig. 5. Sometimes this PAD appeared alone (top), but more often it appeared with a compound action potential riding on the DRP (bottom), which correspond to spikes evoked by PAD (dorsal root reflex; DRR). C–D: blocking α₅GABA_A receptors with L655708 (0.1 µM) did not change phasic PAD (DRP) but hyperpolarized the afferents (reduced tonic DRP; tonic PAD) and reduced the DRR (n = 7). E: blocking spike-mediated synaptic activity with high-dose tetrodotoxin (TTX; 2 µM; n = 9) also hyperpolarized the afferents. F: overall, blocking GABA receptors with L655708, bicuculline (50 µM, n = 5) or gabazine (30 µM, n = 3) reduced tonic PAD computed from the reduction in DRP, as did TTX. Bicuculline and gabazine data combined (and abbreviated Bicuc). Both drugs also eliminated the phasic PAD (DRP). After TTX application, subsequent application of L655708, bicuculline or glutamate receptor blockers (CNQX and APV; 10 µM and 50 µM; abbreviated CNQX) further reduced tonic PAD in the presence of TTX (n = 9, 5 and 5, respectively). Error bars SD; *significantly different, P < 0.05.

Fig. 9.

Antidromic spikes are facilitated by depolarization, including tonic primary afferent depolarization (PAD). A: intracellular recording from Ia afferent with (red; Ca1 afferent) and without an antidromic spike triggered by phasic PAD evoked by S4 dorsal root stimulation [2 × threshold (T)]. This spike is insecurely propagated, often failing spontaneously, and leaving only a small failed spike (failure potential). Resting potential (dotted line, −70 mV). Spike peaked at +15 mV overshoot but is truncated. B: two separate phasic PADs evoked in an Ia afferent (Ca1 root afferent) by stimulating an adjacent dorsal root [(DR); S4, black; 2 × T] or a contralateral dorsal root (Ca1, green; 2 × T). Neither stimuli alone evoked an antidromic spike, but the extra combined potential from the two stimuli together evoked an antidromic spike (red). C: spontaneous depolarization of the afferent facilitates an antidromic spike evoked by PAD in an axon without an antidromic spike initially (phasic PAD evoked by S4 DR stimulation, 2 × T). D–E: antidromic spikes (dorsal root reflex; DRR) recorded on dorsal root (black and gray) are reduced by blocking tonic PAD with L655708 (0.1 µM). F–H: Another example of antidromic spikes reduced by L655708 and then further reduced by bicuculline (10 µM).

Fig. 10.

Spatial and temporal summation of tonic primary afferent depolarization (PAD), including facilitation by cutaneous afferents. A: intracellular recording from proprioceptive group I afferent (Ca1 afferent), resting at −83 mV (dotted line). Stimulating the adjacent S4 dorsal root (DR) at group I intensity [1.5 × T (T, afferent volley threshold, blue)] evoked a phasic PAD with a small tonic PAD. Increasing the DR stimulation to additionally recruit low- (2.5 × T, Aβ; red) and high-threshold (6 × T, black) cutaneous afferents progressively increased the tonic PAD (starting 20 ms later, relative to group-I-evoked PAD, shown in light blue for reference), without increasing the early portion of phasic PAD. Each plot is an average of 10 responses with DR stimulation delivered at 1-s intervals. B: repeated stimulation of the DR at 2.5 × T (in same afferent as in A) facilitated tonic PAD shown on a longer time scale but decreases phasic PAD that rides on top of tonic PAD (first few phasic PADs expanded in insets, black). Temporal facilitation of tonic PAD was maximal at the 2 Hz DR stimulation rate and decreased with rate, mostly gone by 10 s interstimulus intervals. Similar facilitation also occurred for smaller and larger DR stimuli [1.5 × T and 6 × T], being larger with larger stimuli (not shown). C: PAD recorded from the dorsal root (DRP) in response to stimulating an adjacent DR at low-threshold cutaneous intensity (2.5 × T, red). The tonic PAD component is increased by turning the stimulation up to C fiber intensity (100 × T; 5 sweeps at 10-s intervals averaged). Repeated C fiber stimulation to compute averages displayed increased the dorsal root reflex (DRR) by tonically depolarizing the root (tonic PAD; not shown). D: Another proprioceptive group I axon recorded intracellularly, showing the same slow buildup of tonic PAD with repeated 1 Hz DR stimulation at group I and C fiber intensity, lasting a minute poststimuli for the latter. E: tonic PAD facilitated by repeated DR stimulation was consistently blocked by L655708 (0.1 µM; n = 6/6).

Fig. 11.

Local GABAergic microcircuits in the dorsal horn contribute to tonic primary afferent depolarization (PAD). A: in vitro rat spinal cord in low-dose tetrodotoxin (TTX) to block all spike transmission, except in TTX-resistant C fibers. B: prior to TTX, dorsal roots stimulation at both low and high intensity [2.5 × and 100 × threshold (T)] evoked a dorsal root reflex (DRP; PAD related) and PAD-evoked spikes [dorsal root reflex (DRR)], with both early phasic and late tonic PAD components. C: bath application of TTX (100 nM) blocked the PAD evoked by low-threshold stimulation, but there remained a delayed slow C fiber-mediated tonic PAD evoked by high-intensity stimulation (100 × T). PAD evoked spikes (DRR) were blocked by TTX, confirming the TTX block of spike transmission in low-threshold afferents (n = 6/6). D: application of the α₅GABA_A receptor antagonist L655708 consistently inhibited the C fiber-mediated tonic PAD (n = 6/6). Top trace: amplified from part C.