Electrical stimulation of low-threshold afferent fibers induces a prolonged synaptic depression in lamina II dorsal horn neurons to high-threshold afferent inputs in mice

Abstract

Electrical stimulation of low-threshold Aβ-fibers (Aβ-ES) is used clinically to treat neuropathic pain conditions that are refractory to pharmacotherapy. However, it is unclear how Aβ-ES modulates synaptic responses to high-threshold afferent inputs (C-, Aδ-fibers) in superficial dorsal horn. Substantia gelatinosa (SG) (lamina II) neurons are important for relaying and modulating converging spinal nociceptive inputs. We recorded C-fiber-evoked excitatory postsynaptic currents (eEPSCs) in spinal cord slices in response to paired-pulse test stimulation (500 μA, 0.1 millisecond, 400 milliseconds apart). We showed that 50-Hz and 1000-Hz, but not 4-Hz, Aβ-ES (10 μA, 0.1 millisecond, 5 minutes) induced prolonged inhibition of C-fiber eEPSCs in SG neurons in naive mice. Furthermore, 50-Hz Aβ-ES inhibited both monosynaptic and polysynaptic forms of C-fiber eEPSC in naive mice and mice that had undergone spinal nerve ligation (SNL). The paired-pulse ratio (amplitude second eEPSC/first eEPSC) increased only in naive mice after 50-Hz Aβ-ES, suggesting that Aβ-ES may inhibit SG neurons by different mechanisms under naive and nerve-injured conditions. Finally, 50-Hz Aβ-ES inhibited both glutamatergic excitatory and GABAergic inhibitory interneurons, which were identified by fluorescence in vGlut2-Td and glutamic acid decarboxylase-green fluorescent protein transgenic mice after SNL. These findings show that activities in Aβ-fibers lead to frequency-dependent depression of synaptic transmission in SG neurons in response to peripheral noxious inputs. However, 50-Hz Aβ-ES failed to induce cell-type-selective inhibition in SG neurons. The physiologic implication of this novel form of synaptic depression for pain modulation by Aβ-ES warrants further investigation.

Conflict of interest statement

Disclosures: The authors declare no conflict of interest. None of the authors has a commercial interest in the material presented in this paper. There are no other relationships that might lead to a conflict of interest in the current study.

Figures

Fig. 1. Calibrating the intensity of dorsal root electrical stimulation

(A) Left: Configuration for extracellular recording of compound action potentials (APs) produced by graded dorsal root electrical stimulation. In a transverse spinal cord slice, the stimulating electrode was placed distally on the dorsal root, and the compound AP recording electrode was placed close to the dorsal root entry zone. Right: Examples of increasing stimulus intensities of dorsal root stimulation that activated Aβ- and Aδ-fiber components of compound APs. DRG, dorsal root ganglion. (B) The stimulus response curves were established by plotting the amplitude of the compound APs, shown as a fraction of the maximum amplitude (MM, inset), against the increasing stimulus intensity (0–1.0 mA, 0.1 ms). The Aβ-compound AP amplitude plateaued first, followed by the Aδ-component (left), and then, at much higher intensities (>100 μA), the C-component (right). Data are expressed as mean ± SEM in B.

Fig. 2. Electrical stimulation of Aβ-fibers in the dorsal root inhibits evoked postsynaptic currents in substantia gelatinosa neurons

(A) Configuration for whole-cell patch-clamp recording in a substantia gelatinosa (SG) neuron during dorsal root stimulation in a spinal cord slice. First, a baseline recording was obtained (5 min). Then electrical stimulation of Aβ-fibers (Aβ-ES, 50 Hz, 10 μA, 0.1 ms, 1 min) was administered to the ipsilateral dorsal root. Finally, post-stimulation tests were obtained for 20 minutes after stimulation. (B) Representative traces of postsynaptic currents in an SG neuron evoked in response to paired-pulse test stimulation (500 μA, 0.1 ms, 400 ms apart) before (black) and after (red) Aβ-ES. The traces of 1st evoked postsynaptic currents in response to high-threshold afferent (i.e., C-fiber) inputs are also shown on a smaller timescale (inset). (C) Time course of amplitudes of the 1st C-fiber–evoked postsynaptic currents in SG neurons of naïve mice before and after Aβ-ES. The 50 Hz Aβ-ES induced progressive inhibition of C-fiber–evoked postsynaptic currents in the presence (n=6) and absence of GABA-A receptor (bicuculline, 20 μM) and glycine receptor (strychnine, 2 μM) blockers (bath application, n=11). (D) The amplitudes of the 1st C-fiber–evoked postsynaptic currents during each 5-minute period were averaged for analysis (one-way repeated measures ANOVA). (E) Time course of paired-pulse ratio (2nd amplitude / 1st amplitude) before and after Aβ-ES. (F) The averaged paired-pulse ratios were increased at 15–20 minutes after Aβ-ES in both groups (one-way repeated measures ANOVA). Data are expressed as mean ± SEM (C-F). *P<0.05 as compared with the baseline.

Fig. 3. Electrical stimulation of Aβ-fibers induces frequency-dependent inhibition of evoked excitatory postsynaptic currents (eEPSCs) produced by high-threshold afferent fiber inputs in substantia gelatinosa (SG) neurons

(A) Representative traces of eEPSCs in SG neurons. The eEPSCs were produced by paired-pulse test stimulation (500 μA, 0.1 ms, 400 ms apart) before (black) and after (red) sham stimulation (control) and 50 Hz electrical stimulation of the dorsal root at Aβ-intensity (Aβ-ES, 10 μA, 0.1 ms, 5 min). (B) Upper panel: Example of an SG neuron that showed only Aβ-fiber eEPSCs in response to a 10 μA test stimulus but Aβ-, Aδ-, and C-fiber eEPSCs in response to a high-intensity (500 μA) test stimulus. Lower panel: eEPSCs evoked by a high-intensity test stimulus were completely blocked by glutamate receptor antagonists (APV, 50 μM; CNQX, 50 μM). (C) Time-dependent changes in the amplitudes of the 1st C-fiber eEPSCs after Aβ-ES in naïve mice (4 Hz: n=10, 50 Hz: n=12, 1000 Hz: n=10). Data from sham stimulation (control) in naïve and nerve-injured mice were combined for analysis (n=12). (D) The amplitudes of the 1st C-fiber eEPSC during each 5-minute period were averaged for analysis (one-way repeated measures ANOVA). (E) The paired-pulse ratio (2nd amplitude / 1st amplitude) was significantly increased from baseline at 15–20 minutes after 50 Hz Aβ-ES (paired t-test). Data are expressed as mean ± SEM (C-E). *P<0.05, **P<0.01, ***P<0.001 as compared with the baseline.

Fig. 4. Electrical stimulation of Aβ-fibers inhibits evoked excitatory postsynaptic currents (eEPSCs) produced by high-threshold afferent fiber inputs in both naive and nerve-injured mice

(A) Time-dependent changes in the amplitudes of the 1st C-fiber eEPSCs after Aβ-ES in naïve mice (n=12) and mice that underwent spinal nerve ligation (SNL, n=11). Data from sham stimulation (control) in naïve and SNL mice were combined for analysis (n=12). (B) The amplitudes of the 1st C-fiber eEPSC during each 5-minute period were averaged for analysis in each group (one-way repeated measures ANOVA). (C) The paired-pulse ratio (2nd amplitude / 1st amplitude) at 15–20 minutes after Aβ-ES was significantly increased from baseline (paired t-test) in naïve mice, but not in SNL mice. Data are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 as compared with the baseline.

Fig. 5. Dorsal root stimulation inhibits both monosynaptic and polysynaptic forms of evoked excitatory postsynaptic currents (eEPSCs) produced by high-threshold afferent fiber inputs

(A) Examples of monosynaptic (left) and polysynaptic (right) eEPSCs produced by high-threshold afferent fiber (i.e., C-fiber) inputs in substantia gelatinosa (SG) neurons. The C-fiber eEPSCs were judged to be monosynaptic if no failures occurred during 20 test pulses (500 μA, 0.1 ms) applied to the dorsal root at 1 Hz, despite variability of C-fiber eEPSC amplitude (left). The eEPSCs of the 20 traces are superimposed. (B) In naïve mice, 50 Hz Aβ-ES (10 μA, 0.1 ms, 5 min) at the dorsal root reduced the 1st C-fiber eEPSC in SG neurons that showed monosynaptic (n=7) and polysynaptic (n=5) components of C-fiber eEPSCs. (C) The amplitudes of the 1st C-fiber eEPSCs during each 5-minute period in naïve mice were averaged for analysis (one-way repeated measures ANOVA). (D) Changes in the 1st C-fiber eEPSCs in SNL mice after Aβ-ES in SG neurons that showed monosynaptic (n=6) and polysynaptic components (n=5). (E) The amplitudes of the 1st C-fiber eEPSCs during each 5-minute period in SNL rats were averaged for analysis (one-way repeated measures ANOVA). Data are expressed as mean ± SEM (B–E). *P<0.05, **P<0.01, ***P<0.001 as compared with the baseline.

Fig. 6. Effects of 50 Hz electrical stimulation of Aβ-fibers on inhibitory and excitatory dorsal horn neurons

(A) Confocal images of spinal cord slices from GAD-GFP and vGlut2-Td mice. GABAergic inhibitory interneurons can be identified by green fluorescence in slices from GAD-GFP mice, and glutamatergic excitatory neurons can be identified by red fluorescence in slices from vGlut2-Td mice. (B) Aβ-ES (50 Hz, 10 μA, 0.1 ms, 5 min) induces prolonged inhibition of C-fiber eEPSCs in both GAD-GFP–positive (+, n=11) and vGlut2-Td–positive (n=10) neurons in mice with spinal nerve ligation (one-way repeated measures ANOVA). Data are expressed as mean ± SEM. *P <0.05, **P <0.01 as compared with the baseline.