Theta-Burst Stimulation of Primary Afferents Drives Long-Term Potentiation in the Spinal Cord and Persistent Pain via α2δ-1-Bound NMDA Receptors

Abstract

Long-term potentiation (LTP) and long-term depression (LTD) in the spinal dorsal horn reflect activity-dependent synaptic plasticity and central sensitization in chronic pain. Tetanic high-frequency stimulation is commonly used to induce LTP in the spinal cord. However, primary afferent nerves often display low-frequency, rhythmic bursting discharges in painful conditions. Here, we determined how theta-burst stimulation (TBS) of primary afferents impacts spinal cord synaptic plasticity and nociception in male and female mice. We found that TBS induced more LTP, whereas tetanic stimulation induced more LTD, in mouse spinal lamina II neurons. TBS triggered LTP, but not LTD, in 50% of excitatory neurons expressing vesicular glutamate transporter-2 (VGluT2). By contrast, TBS induced LTD and LTP in 12-16% of vesicular GABA transporter (VGAT)-expressing inhibitory neurons. Nerve injury significantly increased the prevalence of TBS-induced LTP in VGluT2-expressing, but not VGAT-expressing, lamina II neurons. Blocking NMDARs, inhibiting α2δ-1 with gabapentin, or α2δ-1 knockout abolished TBS-induced LTP in lamina II neurons. Also, disrupting the α2δ-1-NMDAR interaction with α2δ-1Tat peptide prevented TBS-induced LTP in VGluT2-expressing neurons. Furthermore, TBS of the sciatic nerve induced long-lasting allodynia and hyperalgesia in wild-type, but not α2δ-1 knockout, mice. TBS significantly increased the α2δ-1-NMDAR interaction and synaptic trafficking in the spinal cord. In addition, treatment with NMDAR antagonists, gabapentin, or α2δ-1Tat peptide reversed TBS-induced pain hypersensitivity. Therefore, TBS-induced primary afferent input causes a neuropathic pain-like phenotype and LTP predominantly in excitatory dorsal horn neurons via α2δ-1-dependent NMDAR activation. α2δ-1-bound NMDARs may be targeted for reducing chronic pain development at the onset of tissue/nerve injury.SIGNIFICANCE STATEMENT Spinal dorsal horn synaptic plasticity is a hallmark of chronic pain. Although sensory nerves display rhythmic bursting discharges at theta frequencies during painful conditions, the significance of this naturally occurring firing activity in the induction of spinal synaptic plasticity is largely unknown. In this study, we found that theta-burst stimulation (TBS) of sensory nerves induced LTP mainly in excitatory dorsal horn neurons and that the prevalence of TBS-induced LTP was potentiated by nerve injury. This TBS-driven synaptic plasticity required α2δ-1 and its interaction with NMDARs. Furthermore, TBS of sensory nerves induced persistent pain, which was maintained by α2δ-1-bound NMDARs. Thus, TBS-induced LTP at primary afferent-dorsal horn neuron synapses is an appropriate cellular model for studying mechanisms of chronic pain.

Keywords: Cacna2d1; dorsal root ganglion; gabapentinoid; interneuron; nociceptor; synaptic plasticity.

Copyright © 2022 the authors.

Figures

Figure 1.

TBS and tetanic stimulation of primary afferents differentially induce LTP and LTD in spinal dorsal horn neurons. A, Schematic of the TBS protocol, representative original recording traces, and time course of TBS-induced differential changes (percentage of baseline) in the amplitude of evoked EPSCs from a total of 40 spinal lamina II neurons recorded from seven mice. The time course of evoked EPSC amplitude in spinal lamina II neurons without TBS was included as a control (n = 17 neurons from 4 mice). B, Schematic of the tetanic stimulation protocol, representative current traces, and time course of tetanic stimulation-induced differential changes in the amplitude of evoked EPSCs in a total of 38 spinal lamina II neurons recorded from eight mice. Pie charts in A and B show the proportion of lamina II neurons displaying LTP and LTD in response to TBS or tetanic stimulation. Data are the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, compared with respective baselines (one-way ANOVA followed by Dunnett's post hoc test).

Figure 2.

Nerve injury increases the prevalence of TBS-induced LTP in excitatory dorsal horn neurons. A, Representative recording traces and time course of TBS-induced changes (% of baseline) in the amplitude of evoked EPSCs in VGluT2-expressing lamina II neurons from mice 2 weeks after sham surgery (n = 26 neurons from seven mice). B, Original traces and time course of TBS-induced changes in the amplitude of evoked EPSCs from VGluT2-expressing lamina II neurons from mice 2 weeks after SNI (n = 24 neurons from eight mice). Pie charts in A and B show the proportion of VGluT2-expressing lamina II neurons displaying LTP in mice subjected to SNI and sham surgery. Data are the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, compared with respective baselines (one-way ANOVA followed by Dunnett's post hoc test).

Figure 3.

Nerve injury does not affect the occurrence of TBS-induced LTP or LTD in inhibitory dorsal horn neurons. A, Original recording traces and time course of TBS-induced changes (percentage of baseline) in the amplitude of evoked EPSCs in VGAT-expressing lamina II neurons from sham control mice (n = 25 neurons from five mice). B, Recording traces and time course of TBS-induced changes in the amplitude of evoked EPSCs in VGAT-expressing lamina II neurons from mice 2 weeks after SNI (n = 27 neurons from five mice). Pie charts in A and B show the proportion of VGAT-expressing lamina II neurons displaying LTP in mice subjected to SNI and sham surgery. Data are the mean ± SEM. *p < 0.05, **p < 0.01, compared with respective baselines (Kruskal–Wallis one-way ANOVA followed by Dunn's post hoc test).

Figure 4.

TBS induces LTP in excitatory dorsal horn neurons through NMDARs. A, Representative current traces and time course data show that bath application of 50 μm AP5 blocked TBS-induced LTP in VGluT2-expressing lamina II neurons (n = 19 neurons from six mice). B, Original recording traces and time course of TBS-induced changes (percentage of baseline) in the amplitude of evoked EPSCs in VGluT2-expressing lamina II neurons recorded with intracellular solution containing 1 mm MK-801 (n = 27 neurons from six mice). C, Original recording traces and time course of TBS-induced changes (percentage of baseline) in the amplitude of evoked EPSCs in lamina II neurons from Grin1-cKO mice (n = 25 neurons from five mice). Data are the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, compared with respective baselines (one-way ANOVA followed by Dunnett's post hoc test).

Figure 5.

TBS induces LTP in spinal dorsal horn neurons via α2δ-1-bound NMDARs. A, Original recording traces and time course data show that pretreatment of spinal cord slices with 100 μm gabapentin for 30 min abolished TBS-induced LTP in VGluT2-expressing lamina II neurons (n = 19 neurons from six mice). B, Representative current traces and time course of TBS-induced changes (percentage of baseline) in the amplitude of evoked EPSCs in spinal lamina II neurons from Cacna2d1 KO mice (n = 22 neurons from four mice). C, D, Original recording traces and time course data show the effect of pretreatment of spinal cord slices with 1 μm α2δ-1Tat peptide (n = 18 neurons from four mice; C) or 1 μm control peptide (n = 20 neurons from six mice; D) for 30 min on TBS-induced changes in the amplitude of evoked EPSCs in VGluT2-expressing lamina II neurons. Data are the mean ± SEM. *p < 0.05, **p < 0.01, compared with respective baselines (one-way ANOVA followed by Dunnett's post hoc test).

Figure 6.

TBS induces long-lasting pain hypersensitivity through α2δ-1. A, Time course of changes in hindpaw withdrawal thresholds in response to von Frey filaments, pressure, and noxious heat in WT (n = 8 mice) and Cacna2d1 KO mice (n = 7 mice) after TBS of the sciatic nerve (at time 0). WT mice subjected to sham surgery and untreated Cacna2d1 KO mice were used as controls. B, Time course data show the effect of intraperitoneal injection of 60 mg/kg gabapentin on withdrawal thresholds measured with von Frey filaments, pressure, and noxious heat in WT mice 9–12 d after TBS of the sciatic nerve or sham surgery (n = 10 mice). Data are the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the baseline (time 0). #p < 0.05, ##p < 0.01, ###p < 0.001, compared with WT-TBS group at the same time point (two-way ANOVA followed by Dunnett's or Tukey's post hoc test in A and B). C, D, CPP data show the time spent in saline-paired and gabapentin-paired chambers (C) and the CPP score (D) of saline-treated and gabapentin-treated (60 mg/kg) mice subjected to TBS of the sciatic nerve. Data are the mean ± SEM. **p < 0.01 compared between groups as indicated (one-way ANOVA followed by Tukey's post hoc test in C; two-tailed Student's t test in D).

Figure 7.

TBS-induced pain hypersensitivity is sustained via NMDARs. A, Effect of intraperitoneal injection of 10 mg/kg memantine on the paw withdrawal thresholds in response to von Frey filaments, pressure, and noxious heat of WT mice 9–12 d after TBS of the sciatic nerve or sham surgery (n = 10 mice/group). B, Effect of intrathecal injection of 5 μg of AP5 on the withdrawal thresholds of WT mice 9–12 d after TBS of the sciatic nerve or sham surgery (n = 10 mice/group). Data are the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the respective baseline (time 0, one-way ANOVA followed by Dunnett's post hoc test).

Figure 8.

TBS of the sciatic nerve increases the α2δ-1–NMDAR interaction and synaptic trafficking of α2δ-1-bound NMDARs in the spinal cord. A, Original blotting images and mean data show the interaction between α2δ-1 and GluN1 in the tissue extracts of dorsal spinal cords from WT mice 10 d after TBS of the sciatic nerve or sham surgery (n = 8 mice/group). Proteins were initially IP with a rabbit anti-GluN1 antibody or IgG. Immunoblotting was then performed using mouse anti-α2δ-1 or anti-GluN1 antibodies. IgG and input (tissue lysates only, without immunoprecipitation) were used as negative and positive controls, respectively. Values were normalized to GluN1 protein bands in the same gel. B, Representative gel images (two pairs of samples) and quantification of the protein levels of GluN1, α2δ-1, and PSD-95 (a synaptic marker) in synaptosomes isolated from dorsal spinal cord tissues of WT mice 10 d after TBS of the sciatic nerve or sham surgery (n = 8 mice/group). PSD-95 was used as the internal control for normalizing the protein level of GluN1 and α2δ-1 on the same gel. Data are the mean ± SEM. *p < 0.05, ***p < 0.001, compared with the sham group (two-tailed Student's t test).

Figure 9.

TBS-induced pain hypersensitivity is maintained via α2δ-1-bound NMDARs. A, B, Effect of intrathecal injection of 1 μg of α2δ-1Tat peptide (A) or 1 μg of control peptide (B) on the hindpaw withdrawal thresholds in response to von Frey filaments, pressure, and noxious heat in mice 9–12 d after TBS of the sciatic nerve or sham surgery (n = 10 mice/group). Data are the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the respective baseline (time 0; one-way ANOVA followed by Dunnett's post hoc test).