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.
Introduction
Activity-dependent long-term potentiation (LTP) and long-term depression (LTD) are enduring changes in synaptic strength in the CNS and are generally viewed as the cellular representation of learning and memory. Increased activity of primary afferent nerves is critically involved in the initiation of pain, and a major feature of chronic pain is persistent synaptic plasticity in the spinal cord (i.e., central sensitization; Zhou et al., 2012; Chen et al., 2013, 2014b). Conditioning tetanic stimulation, typically at 100 Hz, is commonly applied to primary afferent nerves to induce LTP in spinal cord slices (recorded at room temperature) from young animals (Randić et al., 1993; Ikeda et al., 2003; Lee et al., 2010). However, single-unit recordings in vivo indicate that primary afferent nerves display rhythmic bursting discharge activity at the theta frequency (4–12 Hz) under many painful conditions, including tissue ischemia, traumatic nerve injury, and neuropathy associated with diabetes and chemotherapy (Pan et al., 1997, 1999; Amir et al., 2002; Khan et al., 2002; Xiao and Bennett, 2008). Thus, tetanic high-frequency stimulation does not accurately mimic naturally occurring discharge patterns of primary afferents in many painful conditions. Theta-burst stimulation (TBS), consisting of short bursts and typically repeated at 5 Hz, resembles the physiological theta frequency and firing patterns of neurons and is widely used to study LTP in the brain (DeCoteau et al., 2007; Tort et al., 2008; Larson and Munkácsy, 2015). Although the repetitive bursting firing pattern of primary afferents during painful conditions has long been documented, there is no information about how TBS of primary afferent nerves impacts synaptic plasticity in the spinal cord. Also, the functional significance of TBS-driven synaptic plasticity in the development of chronic pain remains largely unknown.
The glutamate NMDA receptors (NMDARs) in the spinal cord play a pivotal role in LTP induced by primary afferent stimulation and in the development of chronic neuropathic pain (Randić et al., 1993; Ikeda et al., 2000; Zhou et al., 2012; Chen et al., 2014b). Tetanic stimulation of primary afferents induces NMDAR-dependent LTP and LTD in dorsal horn neurons (Randić et al., 1993; Kim et al., 2015). NMDARs at the spinal cord level are normally inactive, and intrathecal injection of NMDAR antagonists has no effect on nociception under physiological conditions (Yamamoto and Yaksh, 1992; Zhao et al., 2012; Zhou et al., 2012). Although the functional significance of NMDARs in LTP induction and chronic pain is well recognized, it is uncertain how brief conditioning stimulation of primary afferents leads to long-lasting activation of NMDARs at the spinal cord level. α2δ-1 (encoded by the Cacna2d1 gene) is the main target of gabapentinoids (Gee et al., 1996; Fuller-Bicer et al., 2009) that are used clinically for treating chronic neuropathic pain. In neuropathic pain caused by traumatic nerve injury and chemotherapy, α2δ-1 is upregulated in the dorsal root ganglion and spinal dorsal horn (Li et al., 2004; Chen et al., 2019). Recent studies revealed that α2δ-1 physically interacts with NMDARs independent of its conventional role as a Ca2+ channel subunit and is essential for augmented synaptic activity and trafficking of NMDARs in the spinal dorsal horn in neuropathic pain (Chen et al., 2018, 2019; Zhang et al., 2021). However, little is known as to whether α2δ-1 and its interaction with NMDARs are involved in the induction of spinal cord LTP by primary afferent stimulation.
In this study, we determined how TBS, by mimicking bursting discharges of primary afferents at the theta frequency, affects synaptic plasticity in the spinal dorsal horn and nociception. We tested the hypothesis that TBS of primary afferents elicits LTP preferentially in excitatory neurons in the spinal dorsal horn and pain hypersensitivity via α2δ-1-bound NMDARs. We demonstrated for the first time that TBS induces persistent pain and LTP predominantly in primary afferent synapses with dorsal horn excitatory interneurons. Furthermore, our study revealed that α2δ-1-dependent NMDAR activation is essential for TBS-induced LTP in the spinal cord and for persistent pain.
Materials and Methods
Animals.
All surgical procedures and experimental protocols were approved by the Institutional Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center and were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male and female adult mice (weight, 25–32 g; age range, 8–12 weeks) were used in this study, and they were housed at 24°C on a 12 h light/dark cycle with food and water available ad libitum. Cacna2d1 knock-out (KO) mice were obtained by crossing Cacna2d1 heterozygous mice (stock #6900, Medical Research Council, UK). VGluT2-ires-Cre knock-in mice (stock #028863), VGAT-ires-Cre knock-in mice (stock #028862), and tdTomatoflox/flox mice (stock #007909) were purchased from The Jackson Laboratory. VGluT2Cre:tdTomatoflox/flox mice were obtained by mating male VGluT2-ires-Cre mice with female tdTomatoflox/flox mice as described previously (Wang et al., 2018). Similarly, male VGAT-ires-Cre knock-in mice and female tdTomatoflox/flox mice were crossed to generate VGATCre/+:tdTomatoflox/flox mice.
Grin1flox/flox mice (stock #005246) were purchased from The Jackson Laboratory, and AdvillinCre/+ mice (da Silva et al., 2011) were provided by Fan Wang (Duke University, Durham, NC). Female Grin1flox/flox mice were first crossed with male AdvillinCre/+ mice to obtain male AdvillinCre/+:Grin1flox/+ mice, which were crossed again with female Grin1flox/flox mice to generate AdvillinCre/+:Grin1flox/flox mice, referred to as Grin1-conditional KO (cKO) mice. Mice were earmarked 3 weeks after birth, and tail biopsies were used for PCR genotyping. Grin1-cKO in the dorsal root ganglion has been validated using a specific anti-GluN1 antibody in our previous study (Huang et al., 2020). Cre-negative littermates were used as wild-type (WT) control mice, and all the mice had a C57BL/6J genetic background and were housed at no more than five per cage. Final data were pooled from males and females because there was no sex difference in behavioral data and slice recording results.
Spared nerve injury.
Spared nerve injury (SNI) was conducted in mice as previously described (Laedermann et al., 2014). In brief, mice were anesthetized with 2% isoflurane, and the left sciatic nerve and its three branches (the sural, common peroneal, and tibial nerves) were exposed under a surgical microscope. The common peroneal and tibial nerves were tightly ligated and sectioned (leaving the sural nerve intact) under the microscope. Sham mice were subjected to the same procedure but without the nerve injury. Mice were allowed to recover in a warm cage before being returned to the housing facility. Spinal cord slice recordings were performed 2–3 weeks after surgery.
Electrophysiological recordings in spinal cord slices.
Mice were anesthetized under 3% isoflurane, and the lumbar spinal cord at L5 and L6 levels was quickly removed through laminectomy. The spinal cords were immediately placed and immerged in ice-cold sucrose artificial CSF containing the following (in mm): 234 sucrose, 26 NaHCO3, 3.6 KCl, 1.2 NaH2PO4, 2.5 CaCl2, 1.2 MgCl2, and 25 glucose, presaturated with 95% O2 and 5% CO2. Transverse slices (400 μm thick) were cut using a vibratome (Leica) and then transferred to 95% O2 and 5% CO2 oxygenated Krebs solution consisting of the following (in mm): 117 NaCl, 25 NaHCO3, 3.6 KCl, 1.2 NaH2PO4, 2.5 CaCl2, 1.2 MgCl2, and 11 glucose. After incubation for at least 1 h, the spinal cord slices were transferred into a glass-bottomed recording chamber and perfused continuously by oxygenated Krebs solution at 34°C at a speed of 3 ml/min.
Neurons in spinal lamina II were selected for recording because they predominantly receive nociceptive information from primary afferents (Pan and Pan, 2004; Santos et al., 2007; Chen et al., 2014a). Lamina II neurons were visualized under an upright microscope (Olympus) with a 60× water-immersion objective with a combination of differential interference contrast and infrared illumination. Glutamate AMPA receptor-mediated EPSCs were recorded via whole-cell voltage-clamp mode using a glass pipette (6–9 MΩ) filled with a solution containing the following (in mm): 135 K-gluconate, 5 KCl, 5 HEPES, 5 EGTA, 2 MgCl2, 0.5 CaCl2, 5 Mg-ATP, 0.5 Na2-GTP, and 10 lidocaine N-ethyl bromide (QX314), pH 7.3, 280–300 mOsm, at a holding potential of –60 mV. EPSCs were elicited by electrical stimulation (0.6 mA, 0.5 ms) of the ipsilateral dorsal root using a tungsten bipolar electrode. Evoked monosynaptic EPSCs were identified if the latency was constant, and no conduction failure was caused by a 20 Hz stimulation (Li et al., 2002; Zhou et al., 2010). Signals were digitized at 10 kHz using a MultiClamp 700B Amplifier (Molecular Devices), filtered at 1–2 kHz, and stored in a computer for offline analysis.
TBS and tetanic stimulation of primary afferents in spinal cord slices.
A stable baseline of evoked EPSCs was recorded for at least 10 min before TBS or tetanic stimulation was applied. The TBS protocol consisted of 10 trains of stimuli spaced at 10 s intervals, with each train containing bursts of four pulses at 100 Hz, repeated 10 times at 5 Hz (Fig. 1A), as previously described (Hawes et al., 2013; Zhou et al., 2018). In some spinal cord slices, a standard tetanic stimulation protocol (100 Hz pulses for 1 s, three times at 10 s intervals; Fig. 1B) was applied as previously reported (Randić et al., 1993; Ikeda et al., 2003). After induction with TBS or tetanic stimulation, EPSCs were evoked with a single stimulus pulse at 0.033 Hz and recorded for at least 40 min.
Gabapentin (#1287303) was purchased from Millipore Sigma. MK-801 (catalog #0924) was obtained from Tocris Bioscience. d(–)-2-amino-5-phosphonopentanoic acid (AP5; catalog #HB0251) was purchased from Hello Bio. α2δ-1 C-terminus peptide (VSGLNPSLWSIFGLQFILLWLVSGSRHYLW) and scrambled control peptide (FGLGWQPWSLSFYLVWSGLILSVLHLIRSN), both fused to the Tat domain (YGRKKRRQRRR), were synthesized by Synpeptide and validated using liquid chromatography and mass spectrometry. All agents were freshly dissolved in artificial CSF before the recording and were perfused via syringe pumps, except that MK-801 was included in the recording internal solution.
TBS of the sciatic nerve in vivo.
Mice were anesthetized with 2% isoflurane, and the left sciatic nerve was exposed at the mid-thigh under the surgical microscope and immersed in mineral oil. The isolated sciatic nerve was electrically stimulated (10 V, 0.5 ms) using a bipolar hook electrode connected to a stimulator (model S48, Grass Instruments; Pan et al., 1993, 1996). The TBS protocol was the same as used in the spinal cord slice recordings and comprised 10 trains of stimuli at 10 s intervals (four pulses at 100 Hz, 10 times at 5 Hz), repeated three times at 5 min intervals. The sham control mice were subjected to the same procedure without electrical stimulation. Mice were allowed to fully recover in a warm cage before being returned to the housing facility.
Behavioral assessment of nociception.
Tactile allodynia was assessed using a calibrated series of von Frey filaments (Stoelting). Mice were placed in individual chambers suspended on a mesh floor to adapt for 30 min before testing. The filaments were applied vertically to the hindpaw plantar surface with sufficient force to bend the filament for 6 s, and paw withdrawal or flinching was regarded as a positive response (Chaplan et al., 1994). If a response occurred, a filament of the next lower force was applied. In the absence of a response, a filament of the next greater force was applied. The 50% paw withdrawal threshold was calculated using the “up-down” method (Chaplan et al., 1994).
Mechanical nociception was measured using a Randall-Selitto analgesiometer (IITC Life Science) as described previously (Chen et al., 2014a,b). In brief, the mouse hindpaw was placed on the device, and a pressure applicator was activated to generate a constantly increasing force. The force was immediately released when the animal withdrew the paw or vocalized, and the value was recorded as the pressure withdrawal threshold.
A thermal testing apparatus (IITC Life Science) was used to quantify the heat sensitivity as previously described (Chen et al., 2014a; Huang et al., 2020). Mice were individually placed in a transparent chamber on the glass surface maintained at 30°C and allowed to acclimate for 30 min. A radiant heat source was positioned under the glass floor targeting the hindpaw. The paw withdrawal latency was recorded by an electronic stopwatch that switched off an infrared beam when the animal withdrew its paw. Memantine (catalog #M9292) was purchased from Millipore Sigma. All drugs were freshly prepared in saline before intraperitoneal or intrathecal injection.
Conditioned place preference test.
We performed the conditioned place preference (CPP) test to assess whether there was “ongoing” pain in mice subjected to TBS of the sciatic nerve, as described previously (Juarez-Salinas et al., 2019). The CPP apparatus consisted of two chambers of identical size separated by a removable door, with a grid-rod style floor in the white compartment and a mesh style floor in the black compartment. Animal movement was tracked by infrared photobeam detectors inside the chamber and automatically recorded using the MED-PC software (MED Associates). Mice were subjected to TBS of the sciatic nerve or a sham procedure before starting habituation and conditioning. To habituate the mice and minimize stress, on postsurgery days 3–4 (habituation days 1–2), we placed the mice individually into the CPP chamber and with the door open, allowing free access to both chambers for 30 min each day. Baseline chamber preference was determined on habituation day 2 by recording of the time spent by each mouse in the CPP apparatus for the last 15 min (900 s). To ensure that there was no chamber preference bias, animals spending <20% or >80% of the total time in either chamber were excluded from further testing.
Mice were then randomly assigned to the saline chamber or the gabapentin chamber. In the morning of postsurgery days 5–7 (conditioning days 1–3), each mouse received intraperitoneal administration of saline 20 min before the mouse was placed in the assigned chamber for 30 min with the door closed. In the afternoon, each mouse received intraperitoneal injection of 60 mg/kg gabapentin and was placed 20 min later in the opposite chamber for 30 min with the door closed. During this session, no activity was recorded. After the drug-conditioning period, mice received no drug treatment and were tested for chamber preference (place preference test) on postsurgery day 8. Each mouse was placed in the CPP apparatus with access to both chambers and was allowed to explore freely for 15 min. CPP scores were calculated as the time spent in each chamber during the place preference test minus the time spent in the same chamber at baseline (Juarez-Salinas et al., 2019).
Synaptosome preparation, immunoblotting, and coimmunoprecipitation.
We isolated synaptosomes from the dorsal half of mouse lumbar spinal cord tissues as described previously (Huang et al., 2020; Zhang et al., 2021). Tissues were dissected and homogenized using ice-cold Syn-PER Synaptic Protein Extraction Reagent (catalog #87793, Thermo Fisher Scientific) in the presence of a protease inhibitor cocktail (catalog #P8340, Millipore Sigma). The homogenate was centrifuged at 1200 × g for 10 min at 4°C to discard the nuclei and large debris. The supernatant was then centrifuged at 15,000 × g for 20 min at 4°C to obtain the pellets. The pellets were resuspended in RIPA Lysis and Extraction Buffer (catalog #89901, Thermo Fisher Scientific) with the protease inhibitor cocktail for 1 h on ice and centrifuged again at 13,000 rpm for 15 min at 4°C to obtain crude synaptosomal fractions for immunoblotting analysis. Samples were subjected to 4–15% Tris-HCl sodium dodecyl sulfate–polyacrylamide gel electrophoresis (catalog #NP0336BOX, Thermo Fisher Scientific) and then transferred to a polyvinylidene difluoride membrane (catalog #IPVH00010, Millipore Sigma). After 1 h of incubation in blocking buffer (catalog #1706404, BIO-RAD), the membrane was incubated with mouse anti-α2δ-1 (1:1000; catalog #sc-271697, Santa Cruz Biotechnology), rabbit anti-GluN1 (1:1000; catalog #G-8913, Millipore Sigma), or mouse anti-PSD-95 (catalog #75–348, NeuroMab) antibody. Horseradish peroxidase-conjugated anti-mouse IgG (1:7000; catalog #7076S, Cell Signaling Technology) or anti-rabbit IgG (1:7000; #7074S, Cell Signaling Technology) was used as the secondary antibody. We used a SuperSignal West Pico PLUS Chemiluminescent Substrate (catalog #34580, Thermo Fisher Scientific) to detect the protein band, which was visualized and quantified with the Odyssey Fc Imager (LI-COR Bioscience) and normalized to the PSD-95 protein band on the same gel.
For coimmunoprecipitation (co-IP) assays, dorsal spinal cord tissues were homogenized in ice-cold immunoprecipitation lysis buffer (catalog #87788, Thermo Fisher Scientific) containing a cocktail of protease and phosphatase inhibitors (catalog #PPC2020, Millipore Sigma) and centrifuged at 13,000 rpm for 15 min at 4°C to obtain the supernatant. Equal amounts of protein samples were incubated with Protein G Agarose Beads (catalog #16–266, Millipore Sigma) prebound to the rabbit anti-GluN1 antibody (catalog #G-8913, Millipore Sigma) or rabbit IgG beads (catalog #A2909, Millipore Sigma) at 4°C overnight, then washed and extracted using 10% SDS. For immunoblotting, the above mouse anti-α2δ-1 and rabbit anti-GluN1 primary antibodies were used. Horseradish peroxidase-conjugated anti-mouse IgG (1:7000; catalog #7076S, Cell Signaling Technology) or True-Blot anti-rabbit IgG (1:7000; catalog #18–8816-31, Rockland) was used as the secondary antibody. Protein bands were normalized to the GluN1 band on the same blot.
Experimental design and statistical analysis.
All data were presented as the mean ± SEM. Data collection was randomized, and the investigators were blinded to the mouse genotypes and experimental treatments. For spinal cord slice recordings, at least four mice were used in each recording protocol, and only one neuron was recorded from each slice. The cell capacitance, input resistance, and series resistance were continuously monitored during recording. The recording was abandoned if any of these parameters changed by >15%. The amplitude of evoked EPSCs was analyzed by averaging two consecutive EPSCs using Clampfit 11.0 software (Molecular Devices) and normalized to the baseline averaged over 10 min. Neurons with LTP or LTD were defined as those with the amplitude of EPSCs changed at least 20% from the baseline and lasting for >15 min after TBS or tetanic stimulation. A two-tailed Student's t test was used to compare two groups. One-way or two-way ANOVA followed by the Dunnett's or Tukey's post hoc test was used to compare more than two groups. Kruskal–Wallis one-way ANOVA followed by Dunn's post hoc test was used to compare differences between more than two groups, when the data were not normally distributed. A χ2 test was used to determine the difference between two groups in the proportion of neurons displaying LTP and LTD. All statistical analyses were performed using Prism software (version 8; GraphPad Software). p < 0.05 was considered statistically significant.
Results
TBS of primary afferents induces LTP in spinal dorsal horn neurons more consistently than does tetanic stimulation
To determine the effect of TBS of primary afferents on synaptic plasticity in the spinal cord, we recorded EPSCs of spinal lamina II neurons monosynaptically evoked by dorsal root stimulation in spinal cord slices from adult mice. We first confirmed that in the absence of TBS or tetanic stimulation, the amplitude of EPSCs evoked by a single pulse of stimulation in lamina II neurons was stable during the 50 min recording period (n = 17 neurons; Fig. 1A). In 45% of lamina II neurons (18 of 40 neurons), TBS of the dorsal root caused a large and persistent increase in the amplitude of EPSCs (i.e., LTP); the increase occurred ∼10 min after TBS and lasted for at least 30 min (Fig. 1A). Only 5% of lamina II neurons (2 of 40 neurons) exhibited a long-lasting reduction in the amplitude of evoked EPSCs (i.e., LTD) after TBS (Fig. 1A). The remaining 50% of lamina II neurons (20 of 40 neurons) showed no significant change in the amplitude of evoked EPSCs.
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).
We also determined the effect of conventional tetanic high-frequency stimulation on LTP and LTD in lamina II neurons in spinal cord slices from adult mice. Tetanic stimulation of the dorsal root-induced LTP in 37% of lamina II neurons (14 of 38 neurons), which was a significantly lower proportion than that induced by TBS (p = 0.0326, χ2 [2, n = 78] = 6.845; Fig. 1B). Furthermore, tetanic stimulation-induced LTD in more lamina II neurons (26%; 10 of 38 neurons) than TBS did (p = 0.0326, χ2 [2, n = 78] = 6.845; Fig. 1B). In the remaining 37% of lamina II neurons (14 of 38 neurons) tested, tetanic stimulation had no significant effect on the amplitude of evoked EPSCs. These results suggest that compared with tetanic stimulation, TBS of primary afferents induces LTP in more spinal dorsal horn neurons.
Nerve injury increases the prevalence of TBS-induced LTP in VGluT2-expressing excitatory neurons
The spinal lamina II is composed of excitatory and inhibitory interneurons (Santos et al., 2007; Yasaka et al., 2010). Vesicular glutamate transporter-2 (VGluT2)-expressing excitatory neurons in the spinal dorsal horn are critically involved in nociceptive transmission (Wang et al., 2018). To determine the effect of TBS of primary afferents on synaptic plasticity of excitatory dorsal horn neurons, we recorded EPSCs monosynaptically evoked by dorsal root stimulation in tdTomato-labeled VGluT2-expressing neurons in the lamina II of spinal cord slices from VGluT2Cre/+:tdTomatoflox/flox mice subjected to sham surgery. After induction with TBS, 50% of labeled lamina II neurons (13 of 26 neurons) displayed LTP, and the other half showed no significant change in the amplitude of evoked EPSCs (Fig. 2A). TBS did not induce LTD in any of the tdTomato-labeled neurons tested.
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).
Augmented glutamatergic input from primary afferents to spinal dorsal horn neurons is a hallmark of neuropathic pain (Chen et al., 2014b; Xie et al., 2017). Thus, we next determined whether peripheral nerve injury alters TBS-induced LTP in VGluT2-expressing dorsal horn neurons. We recorded EPSCs monosynaptically evoked by dorsal root stimulation in tdTomato-labeled lamina II neurons from VGluT2Cre/+:tdTomatoflox/flox mice 2 weeks after SNI. Remarkably, 79% of tdTomato-labeled neurons (19 of 24 neurons) exhibited LTP after TBS; this proportion was significantly higher than that recorded from VGluT2Cre/+:tdTomatoflox/flox mice subjected to the sham procedure (p = 0.0318, χ2 [1, n = 50] = 4.608; Fig. 2B). These data indicate that peripheral nerve injury increases the occurrence of LTP in VGluT2-expressing excitatory neurons induced by TBS of primary afferents.
Nerve injury has no effect on the prevalence of TBS-induced LTP in VGAT-expressing inhibitory neurons
GABAergic/glycinergic inhibitory neurons expressing vesicular GABA transporter (VGAT; also termed as vesicular inhibitory amino acid transporter, VIAAT) are involved in the modulation of nociceptive transmission in the spinal dorsal horn (Koga et al., 2017). Tetanic stimulation seems to induce mainly LTD in spinal inhibitory neurons, labeled with GAD67, in juvenile (2- to 3-week old) mice (Kim et al., 2015). To determine whether TBS of primary afferents induces LTP and/or LTD in VGAT-expressing inhibitory neurons, we recorded EPSCs monosynaptically evoked by dorsal root stimulation in tdTomato-labeled neurons in the lamina II of spinal cord slices from adult VGATCre/+:tdTomatoflox/flox mice subjected to sham surgery. Among 25 neurons tested, TBS-induced LTP in four neurons (16%) and LTD in three neurons (12%). However, most tdTomato-labeled lamina II neurons (18 of 25 neurons; 72%) showed no significant change in the amplitude of evoked EPSCs after TBS induction (Fig. 3A).
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).
We then determined whether peripheral nerve injury alters the ratio of TBS-induced LTP and LTD in spinal inhibitory dorsal horn neurons. We recorded evoked EPSCs in tdTomato-labeled neurons in the lamina II of spinal cord slices from VGATCre/+:tdTomatoflox/flox mice 2 weeks after SNI. Among a total of 27 neurons tested, 81% of neurons (22 of 27 neurons) showed no significant change in the amplitude of evoked EPSCs after TBS of the dorsal root. Furthermore, TBS-induced LTP in 11% of neurons (3 of 27 neurons) and LTD in 7% of neurons (2 of 27 neurons; Fig. 3B). The proportion of tdTomato-labeled neurons displaying LTP and LTD induced by TBS did not differ significantly between the sham and SNI groups (Fig. 3B). These results indicate that nerve injury has no effect on TBS-induced synaptic plasticity in VGAT-expressing inhibitory neurons in the spinal dorsal horn.
TBS of primary afferents induces LTP in spinal dorsal horn neurons through NMDARs
NMDARs play a key role in tetanic stimulation-induced LTP in the spinal cord (Randić et al., 1993; Kim et al., 2015) and TBS-induced LTP in the brain (Park et al., 2014; Zhou et al., 2018). We determined whether NMDARs are also required for TBS-induced LTP in spinal dorsal horn neurons. Because TBS of primary afferents consistently induced LTP, but not LTD, in VGluT2-expressing dorsal horn neurons, we recorded EPSCs monosynaptically evoked by dorsal root stimulation in tdTomato-labeled lamina II neurons of spinal cord slices from VGluT2Cre/+:tdTomatoflox/flox mice. AP5 (50 μm), a specific NMDAR antagonist, was bath applied for 6 min immediately before TBS induction. In the presence of AP5, TBS of the dorsal root failed to induce LTP in any of 19 tdTomato-labeled neurons tested (Fig. 4A).
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).
To determine the role of postsynaptic NMDARs in TBS-induced LTP, we included 1 mm MK-801, an NMDAR open-channel blocker, in the pipette recording solution to selectively block postsynaptic NMDARs via intracellular dialysis (Zhou et al., 2010). Among a total of 27 tdTomato-labeled lamina II neurons recorded with MK-801-containing solution, TBS of the dorsal root had no significant effect on the amplitude of evoked EPSCs in 22 neurons (81%). TBS-induced LTP in five neurons (19%) only (Fig. 4B), which was significantly less than the proportion of neurons displaying TBS-induced LTP among VGluT2-expressing neurons recorded with a solution that contained no MK-801 (50% of neurons; p = 0.0156, χ2 [1, n = 53] = 5.853).
Presynaptic NMDARs are expressed at primary afferent central terminals and mediate increased glutamatergic input to spinal dorsal horn neurons in neuropathic pain (Chen et al., 2018, 2019; Zhang et al., 2021). We thus determined the role of NMDARs expressed at primary afferent central terminals in TBS-induced LTP. We recorded EPSCs monosynaptically evoked by dorsal root stimulation in lamina II neurons in spinal cord slices from Grin1-cKO mice in which GluN1, the obligatory subunit of NMDARs, was genetically ablated in primary sensory neurons (Huang et al., 2020). Among a total of 25 lamina II neurons tested, TBS of the dorsal root stimulation-induced LTP in only five neurons (20%) and LTD in two neurons (8%). TBS had no significant effect on the amplitude of evoked EPSCs in 18 neurons (72%) from Grin1-cKO mice (Fig. 4C). Together, these results indicate that TBS-driven LTP in the spinal dorsal horn requires both presynaptic and postsynaptic NMDARs.
α2δ-1 is essential for TBS-induced LTP at primary afferent–dorsal horn neuron synapses
Recent studies indicate that α2δ-1 is essential for increased synaptic NMDAR activity in the spinal cord in neuropathic pain (Chen et al., 2018, 2019; Zhang et al., 2021). To study the potential role of α2δ-1 in TBS-induced LTP in spinal excitatory dorsal horn neurons, we incubated spinal cord slices from VGluT2Cre/+:tdTomatoflox/flox mice with 100 μm gabapentin, an α2δ-1 inhibitory ligand, for 30 min immediately before recording. Strikingly, TBS of the dorsal root failed to induce LTP in any of 19 tdTomato-labeled lamina II neurons in gabapentin-pretreated spinal cord slices (Fig. 5A).
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).
To further validate the role of α2δ-1 in TBS-induced LTP, we recorded evoked EPSCs of spinal lamina II neurons in spinal cord slices from Cacna2d1 KO mice (Fuller-Bicer et al., 2009). As expected, TBS induced no LTP in any of 22 lamina II neurons tested (Fig. 5B). Notably, TBS-induced LTD in two lamina II neurons from Cacna2d1 KO mice, which may have been because of the recording of inhibitory neurons in the lamina II.
In addition, we determined whether the α2δ-1–NMDAR interaction is required for TBS-induced LTP in spinal excitatory dorsal horn neurons. We used a Tat-fused 30 aa peptide mimicking the C-terminal domain of α2δ-1 (α2δ-1Tat peptide), which effectively disrupts the α2δ-1–NMDAR interaction (Chen et al., 2018). We incubated spinal cord slices from VGluT2Cre/+:tdTomatoflox/flox mice with 1 μm α2δ-1Tat peptide or 1 μm control peptide for 30 min immediately before recording. TBS of the dorsal root induced LTP in 50% of tdTomato-labeled lamina II neurons (10 of 20 neurons) treated with control peptide. In contrast, in spinal cord slices pretreated with α2δ-1Tat peptide, TBS failed to induce LTP in any of 18 tdTomato-labeled lamina II neurons tested (Fig. 5C,D). Collectively, these findings indicate that α2δ-1-bound NMDARs are essential for TBS-induced LTP at primary afferent–dorsal horn neuron synapses.
TBS of the sciatic nerve induces persistent pain hypersensitivity in mice via α2δ-1
To explore the functional significance of TBS-induced LTP in vivo, we determined whether TBS of primary afferents induces pain hypersensitivity. We performed TBS of the sciatic nerve or sham surgery in WT mice. TBS caused a large reduction in the withdrawal thresholds in response to tactile stimuli, noxious pressure, and heat stimuli applied to the ipsilateral hindpaw, and this reduction persisted for >3 weeks (n = 8 mice; Fig. 6A). In contrast, WT sham-operated mice displayed a transient reduction in the hindpaw withdrawal thresholds within 3 d after surgery and then quickly returned to the baseline levels (n = 8 mice; Fig. 6A).
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).
α2δ-1 is critically involved in TBS-induced LTP in the spinal cord, and gabapentin, which targets α2δ-1, is effective in reducing neuropathic pain in animal models and in patients (Rowbotham et al., 1998; Pan et al., 1999; Chen et al., 2018, 2019). We thus determined whether α2δ-1 plays a role in persistent pain induced by TBS of the sciatic nerve. We tested the effect of gabapentin in WT mice 9–12 d after TBS or sham surgery. Intraperitoneal injection of 60 mg/kg gabapentin fully reversed the reduction in the withdrawal thresholds in TBS-treated mice but had no such effects in sham-operated mice (n = 10 mice/group; Fig. 6B). Furthermore, we determined the role of α2δ-1 in the development of pain hypersensitivity caused by TBS of the sciatic nerve using Cacna2d1 KO mice. TBS of the sciatic nerve induced a smaller reduction in the withdrawal thresholds in Cacna2d1 KO mice (n = 7 mice) than in WT mice 3 d after surgery. Remarkably, the withdrawal thresholds returned to the baseline levels 6 d after TBS induction in Cacna2d1 KO mice (Fig. 6A).
Next, we used gabapentin and an operant CPP test to determine whether TBS of the sciatic nerve causes “ongoing” pain in mice 8 d after TBS. After recovery and habituation to the CPP apparatus, mice were subjected to 3 d of conditioning, where each mouse was paired with either saline or gabapentin, before the final CPP test. These TBS-treated mice exhibited a significant preference for the gabapentin-paired chamber, and the CPP score was significantly higher in gabapentin-paired mice than in saline-paired mice (p = 0.0019, t(7) = 4.826, n = 8 mice/group; Fig. 6C,D). Together, these data strongly suggest that TBS of primary afferents induces persistent pain hypersensitivity, which is dependent on α2δ-1.
TBS of the sciatic nerve induces pain hypersensitivity through NMDARs
NMDARs at the spinal cord level play a critical role in the development of chronic neuropathic pain (Yamamoto and Yaksh, 1992; Zhou et al., 2012; Chen et al., 2014b, 2019). To determine the role of NMDARs in pain hypersensitivity induced by TBS of primary afferents, we tested the effect of systemically administered memantine, a clinically used NMDAR antagonist, and intrathecal injection of the specific NMDAR antagonist AP5 in WT mice 9–12 d after TBS surgery. A single intraperitoneal injection of 10 mg/kg memantine rapidly increased the withdrawal thresholds in response to von Frey filaments, pressure, and heat stimuli (n = 10 mice; Fig. 7A). Similarly, intrathecal injection of 5 μg of AP5 fully reversed TBS-induced reduction in withdrawal thresholds (n = 10 mice; Fig. 7B). These findings indicate that TBS-induced pain hypersensitivity is maintained by NMDARs at the spinal cord level. Because inhibiting α2δ-1 and NMDARs effectively diminished TBS-induced pain hypersensitivity, it seems that TBS of primary afferents mainly causes a neuropathic pain-like phenotype.
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).
TBS of primary afferents potentiates the α2δ-1 interaction with NMDARs and synaptic NMDAR trafficking in the spinal cord
Because both α2δ-1 and NMDARs are critically involved in TBS-induced pain hypersensitivity and LTP in the spinal cord, we used co-IP assays to determine whether TBS of the sciatic nerve affects the α2δ-1 interaction with NMDARs in the spinal cord. Dorsal spinal cord tissues were obtained from WT mice 10 d after TBS of the sciatic nerve or the sham operation. Co-IP showed that the anti-GluN1 antibody precipitated more α2δ-1 proteins in tissues from TBS-treated mice than in those of sham control mice (p = 0.013, t(14) = 2.843, n = 8 mice/group; Fig. 8A).
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).
We then determined whether TBS of the sciatic nerve increases synaptic expression of NMDARs in the dorsal spinal cord. We isolated synaptosomes from dorsal spinal cord tissues of mice 10 d after TBS of the sciatic nerve or the sham operation. Immunoblotting of spinal cord synaptosomes showed that the protein levels of GluN1 (p = 0.0263, t(14) = 2.482) and α2δ-1 (p = 0.0008, t(14) = 4.256) were significantly greater in TBS-treated mice than in sham control mice (n = 8 mice/group; Fig. 8B). These results suggest that TBS of primary afferents potentiates the α2δ-1–NMDAR interaction and synaptic trafficking of α2δ-1-bound NMDARs in the spinal cord.
TBS-induced pain hypersensitivity is maintained via α2δ-1-bound NMDARs
Finally, we determined whether α2δ-1-bound NMDARs at the spinal cord level mediate TBS-induced pain hypersensitivity. α2δ-1 interacts with voltage-gated Ca2+ channels and thrombospondin via its von Willebrand factor type A domain on the α2 (Cantí et al., 2005; Eroglu et al., 2009), whereas the α2δ-1 interaction with NMDARs mainly involves its C terminus on the δ-1 (Chen et al., 2018). We have shown that the α2δ-1Tat peptide has no effect on the α2δ-1 interaction with voltage-gated Ca2+ channels (Chen et al., 2018) and that the C terminus of α2δ-1 is essential for α2δ-1 overexpression-induced synaptic plasticity and neuropathic pain (Chen et al., 2018, 2019; Li et al., 2021). We intrathecally injected 1 μg of α2δ-1Tat peptide or 1 μg of control peptide in WT mice 9–12 d after TBS of the sciatic nerve or sham surgery. Treatment with α2δ-1Tat peptide largely reversed the reduced withdrawal thresholds in response to von Frey filaments, noxious pressure, and heat stimuli (n = 10 mice; Fig. 9A). In contrast, intrathecal injection of control peptide produced no significant effect on the withdrawal thresholds reduced by TBS (n = 10 mice; Fig. 9B). Treatment with α2δ-1Tat peptide or control peptide had no effect on the withdrawal thresholds in sham control mice (n = 10 mice/group; Fig. 9A,B). These data suggest that persistent pain hypersensitivity induced by TBS of primary afferents is maintained by α2δ-1-bound NMDARs at the spinal cord level.
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).
Discussion
Our study provides substantial new evidence about the significance of theta rhythmic bursting discharges of primary afferents in LTP induction and nociceptive processing in the spinal dorsal horn. TBS mimics the theta frequency of bursting firing patterns of brain neurons during various cognitive tasks (DeCoteau et al., 2007; Tort et al., 2008; Hoy et al., 2016). Transcranial magnetic TBS has been used in humans to improve learning and memory (Bonnì et al., 2020; Hermiller et al., 2020). Compared with tetanic stimulation, TBS is more efficient in evoking LTP in the hippocampus (Larson and Munkácsy, 2015) and striatum (Hawes et al., 2013). TBS can cause prolonged axonal Ca2+ elevation in the corticostriatal synapses under normal Mg2+ levels (Park et al., 2014). An initial burst can “prime” the circuit so that succeeding bursts consistently induce LTP. The depolarization by the initial priming burst not only triggers a massive amount of glutamate release from presynaptic terminals but also allows NMDARs to be sufficiently activated at the same time (Larson and Lynch, 1988). In the present study, using spinal cord slices from adult mice, we showed that by mimicking bursting discharge patterns of primary afferent nerves (Pan et al., 1999; Chen et al., 2001; Khan et al., 2002), TBS induced a robust form of LTP in more dorsal horn neurons than did conventional tetanic stimulation. Thus, TBS is relevant to the bursting firing pattern recorded from nociceptive primary afferents and is an appropriate induction paradigm for studying synaptic plasticity in the spinal cord initiated by nociceptive primary afferent input and the underlying signaling mechanisms. Nevertheless, the total number of stimuli applied to primary afferents is different between the two stimulation protocols. Although we recorded a large number of neurons in a blind fashion, we cannot ensure that the same population of dorsal horn neurons was sampled for each stimulation protocol.
We found in the present study that although the NMDAR antagonist AP5 abolished TBS-induced LTP in spinal cord slices, removing presynaptic NMDARs expressed at primary afferent central terminals or blocking postsynaptic NMDARs via intracellular dialysis of MK-801 alone only partially reduced the occurrence of LTP in dorsal horn neurons. These findings suggest that both presynaptic and postsynaptic NMDARs are likely required for the full manifestation of LTP in the spinal cord induced by TBS of primary afferents. NMDARs are essential for both LTP and LTD in dorsal horn neurons elicited by tetanic stimulation of primary afferents (Randić et al., 1993; Kim et al., 2015). Spatial summation of neuronal depolarization produced by TBS governs spike timing-dependent plasticity and is important for optimal activation of presynaptic and postsynaptic NMDARs (Dan and Poo, 2006; Feldman, 2012; Park et al., 2014). For TBS-induced primary afferent activity, the later-arriving bursts could maintain depolarization activated by the first burst to sustain NMDAR channel activity at the synapses formed by primary afferents and dorsal horn neurons. Thus, bursting activity repeated at the theta frequency may allow sufficient presynaptic and postsynaptic depolarization to activate NMDARs in the spinal dorsal horn.
An important finding of our study is that α2δ-1, via its direct interaction with NMDARs, is critically involved in TBS-driven LTP in primary afferent synapses with spinal dorsal horn neurons. α2δ-1 plays a crucial role in TBS-induced LTP in corticostriatal synapses (Zhou et al., 2018), and gabapentin reduces field potentials in the rat spinal dorsal horn after the induction of LTP (Tanabe et al., 2006). We showed in this study that TBS-induced LTP was abolished by α2δ-1 genetic knockout and by disruption of the α2δ-1–NMDAR interaction with α2δ-1Tat peptide, suggesting that α2δ-1-bound NMDARs are indispensable for LTP induction in the spinal cord by TBS of primary afferents. We have shown that α2δ-1 is required for increased presynaptic NMDAR activity in the spinal dorsal horn in neuropathic pain (Chen et al., 2018, 2019). Thus, it is likely that presynaptic α2δ-1 is critically involved in TBS-induced synaptic plasticity by augmenting NMDAR activity. α2δ-1 expression is increased in neuropathic pain conditions, and excess α2δ-1 couples to NMDARs to potentiate their synaptic trafficking and activity in the spinal dorsal horn (Chen et al., 2018; Zhang et al., 2021). However, it is not likely that α2δ-1 upregulation is a prerequisite for its interaction with NMDARs. PKC-induced potentiation of NMDAR activity depends critically on α2δ-1 (Zhou et al., 2021), and PKC activity is obligatory for the induction and maintenance of spinal LTP induced by primary afferent stimulation (Yang et al., 2004). TBS-induced activity at primary afferent terminals and dorsal horn neurons may induce rapid NMDAR phosphorylation via PKC activation, and phosphorylated NMDARs could readily interact with α2δ-1 to form α2δ-1–NMDAR complexes in the spinal dorsal horn after TBS induction (Zhou et al., 2021). Therefore, α2δ-1 functions as an amplifier of nociceptive input after TBS of primary afferents by promoting synaptic trafficking and activation of NMDARs in the spinal dorsal horn.
Another major finding of our study is that TBS-induced synaptic plasticity in the spinal cord is largely cell type- specific. Spinal dorsal horn neurons are molecularly and functionally diverse, consisting of a network of excitatory and inhibitory neurons (Yasaka et al., 2010). Neurons in the lamina II can be broadly classified into excitatory (glutamatergic) and inhibitory (GABAergic and/or glycinergic) interneurons, which express VGluT2 and VGAT, respectively. VGluT2-expressing excitatory neurons in the spinal dorsal horn are involved in maintaining chronic pain induced by tissue inflammation and nerve injury (Wang et al., 2018), whereas VGAT-expressing neurons are normally involved in inhibiting nociceptive transmission in the spinal dorsal horn (Koga et al., 2017). Paired recording in spinal cord slices shows that the vast majority of lamina II neurons are excitatory glutamatergic interneurons (Santos et al., 2007). In the hippocampus, GABAergic neurons show mostly LTD in response to tetanic stimulation (McMahon and Kauer, 1997). In spinal cord slices from juvenile mice, primary afferent stimulation induces LTP in spinothalamic tract-projecting neurons but LTD in GAD67-tagged GABAergic neurons (Kim et al., 2015). We found in this study that TBS of primary afferents induced only LTP in VGluT2-expressing lamina II neurons. Because TBS of primary afferents can induce LTD in some VGAT-expressing neurons in the lamina II, the LTD recorded in unlabeled lamina II neurons was likely from VGAT-expressing inhibitory interneurons.
We found that nerve injury increased the occurrence of LTP in VGluT2-expressing, but not VGAT-expressing, lamina II neurons, suggesting that TBS-induced LTP is altered in a cell type-specific manner in the neuropathic pain condition. α2δ-1 is expressed mainly in excitatory neurons throughout the nervous system (Cole et al., 2005; Taylor and Garrido, 2008). Peripheral nerve injury causes α2δ-1 upregulation in dorsal root ganglion neurons (Li et al., 2004) and aberrant sprouting of myelinated primary afferents to the spinal lamina II (Pan et al., 2003). Nerve injury and TBS of primary afferents similarly potentiate α2δ-1-dependent presynaptic and postsynaptic NMDAR activity in the spinal dorsal horn. Nerve injury likely increases α2δ-1 expression in a large number of DRG neurons and dorsal horn excitatory neurons. Abnormal synaptogenesis after nerve injury involves α2δ-1-containing primary afferent terminals in the spinal lamina I-II (Yamanaka et al., 2021), which could explain why nerve injury increases the prevalence of TBS-induced LTP in VGluT2-expressing excitatory neurons. Also, the synaptic inhibitory tone is important for restraining the excitability of lamina II neurons induced by dorsal root stimulation (Ikeda et al., 2000). Nerve injury causes tonic activation of NMDARs in the spinal dorsal horn (Chen et al., 2014b, 2018), which diminishes synaptic inhibition in dorsal horn neurons by promoting proteolysis of potassium-chloride cotransporter-2 via calpain activation (Zhou et al., 2012). Therefore, the loss of synaptic GABAergic/glycinergic inhibition could also increase the occurrence of LTP in excitatory dorsal horn neurons after TBS induction. Nerve injury, via increased α2δ-1 expression and the recruitment of synapses formed by primary afferents and excitatory dorsal horn neurons, could sustain synaptic strength and a network of neural hypersensitivity (i.e., central sensitization), constituting a major mechanism of chronic neuropathic pain.
Our study reveals the significance of TBS-induced LTP in primary afferent synapses with spinal dorsal horn neurons in encoding nociception and in the development of persistent pain. We found in this study that TBS of the sciatic nerve in mice induced sustained pain hypersensitivity, lasting at least 3 weeks. Tetanic stimulation of the sciatic nerve induces hyperalgesia lasting 8 d in rats (Ying et al., 2006). Remarkably, the pain phenotype induced by TBS was associated with gabapentin preference, and pain hypersensitivity was readily reversed by NMDAR antagonists and gabapentin. Furthermore, TBS of the sciatic nerve increased the α2δ-1 interaction with NMDARs and their synaptic trafficking in the spinal cord. We also found that disrupting the α2δ-1–NMDAR interaction largely attenuated TBS-induced pain hypersensitivity. Therefore, α2δ-1-bound NMDARs are critically involved in the development of persistent pain induced by TBS of primary afferent nerves. Because TBS-induced pain hypersensitivity was effectively reversed by NMDAR antagonists and gabapentinoids, which are effective for treating neuropathic pain in animal models and in patients (Rowbotham et al., 1998; Pan et al., 1999; Chen et al., 2018, 2019; Zhang et al., 2021), it seems that TBS of primary afferents in mice predominantly caused a neuropathic pain-like phenotype. Also, TBS-induced LTP and pain hypersensitivity share similar signaling mechanisms, suggesting that TBS-induced LTP at the primary afferent–dorsal horn synapse in spinal cord slices is an appropriate in vitro model for studying molecular mechanisms involved in the development of chronic pain.
In conclusion, our study provides new insight into the functional significance of the theta rhythm of bursting discharge activity in LTP induction at primary afferent synapses with dorsal horn neurons. Our findings underscore the importance of the rhythmic bursting discharge activity of primary afferents in generating lasting synaptic plasticity and in the development of chronic pain. Remarkably, primary afferent activity-dependent synaptic potentiation and pain hypersensitivity require both α2δ-1 and NMDARs. This new knowledge advances our understanding of the molecular mechanism of synaptic plasticity in the development of neuropathic pain. Because both gabapentin and α2δ-1Tat peptide effectively blocked TBS-driven LTP in the spinal cord and persistent pain in our study, gabapentinoids and drugs targeting the α2δ-1 C terminus could be used at the time of major surgery or tissue/nerve injury for minimizing the development of chronic pain.
Footnotes
This study was supported by Grants NS-101880 and DA-041711 from the National Institutes of Health; and by the N.G. and Helen T. Hawkins Endowment. We thank Sarah Bronson in the Department of Scientific Publications at MD Anderson Cancer Center for proofreading the final version of the manuscript.
The authors declare no competing financial interests.
- Correspondence should be addressed to Hui-Lin Pan at huilinpan{at}mdanderson.org or Shao-Rui Chen at schen{at}mdanderson.org
