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. Author manuscript; available in PMC: 2016 May 31.
Published in final edited form as: Cell Rep. 2016 May 5;15(7):1376–1383. doi: 10.1016/j.celrep.2016.04.039

Chloride Homeostasis Critically Regulates Synaptic NMDA Receptor Activity in Neuropathic Pain

Lingyong Li 1,4, Shao-Rui Chen 1,4, Hong Chen 1, Lei Wen 1, Walter N Hittelman 2, Jing-Dun Xie 1,3, Hui-Lin Pan 1
PMCID: PMC4871741  NIHMSID: NIHMS778547  PMID: 27160909

SUMMARY

Chronic neuropathic pain is a debilitating condition that remains difficult to treat. Diminished synaptic inhibition by GABA and glycine and increased NMDA receptor (NMDAR) activity in the spinal dorsal horn are key mechanisms underlying neuropathic pain. However, the reciprocal relationship between synaptic inhibition and excitation in neuropathic pain is unclear. Here we show that intrathecal delivery of K+-Cl− cotransporter-2 (KCC2) using lentiviral vectors produces a complete and long-lasting reversal of pain hypersensitivity induced by nerve injury. KCC2 gene transfer restores Cl− homeostasis disrupted by nerve injury in both spinal dorsal horn and primary sensory neurons. Remarkably, restoring Cl− homeostasis normalizes both presynaptic and postsynaptic NMDAR activity increased by nerve injury in the spinal dorsal horn. Our findings indicate that nerve injury recruits NMDAR-mediated signaling pathways through disrupting Cl− homeostasis in spinal dorsal horn and primary sensory neurons. Lentiviral vector-mediated KCC2 expression is a promising gene therapy for treating neuropathic pain.

Keywords: neuropathic pain, cation-chloride cotransporters, gene therapy, synaptic transmission, synaptic plasticity

Graphical abstract

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eTOC Blurb

Li et al. find that spinal KCC2 gene transfer induces sustained KCC2 expression and restores chloride homeostasis disrupted by nerve injury in both dorsal horn and primary sensory neurons. KCC2 gene transfer completely and persistently eliminates neuropathic pain and normalizes pre- and postsynaptic NMDA receptor activity increased by nerve injury.

INTRODUCTION

Chronic neuropathic pain is a major, debilitating clinical problem that remains difficult to treat. Because all of the existing analgesics for treating neuropathic pain, including anti-depressants, opioids, and gabapentinoids, have limited efficacy and often produce intolerable adverse effects (Finnerup et al., 2015; Sommer, 2015), the development of highly effective treatments with minimal off-target effects is urgently needed. GABAergic and glycinergic interneurons, which are densely distributed in the spinal dorsal horn, are the basis of the gate control theory of pain (Melzack and Wall, 1965). Normal synaptic inhibition by GABA and glycine critically depends on the coordinated activities of two functionally distinct cation-chloride cotransporters: Na+-K+-2Cl− cotransporter-1 (NKCC1) and K+-Cl− cotransporter-2 (KCC2). KCC2, encoded by Slc12a5, is the dominant neuronal Cl− extrusion mechanism, whereas NKCC1 normally raises intracellular Cl− levels above equilibrium and opposes the action of KCC2 (Payne et al., 1996; Rivera et al., 1999). Thus, changing the intracellular Cl− concentration can profoundly alter the strength and polarity of GABA- or glycine-mediated responses. Neuropathic pain caused by peripheral nerve damage, spinal cord injury, diabetic neuropathy, and chemotherapy is associated with reduced KCC2 activity or increased NKCC1 activity in spinal dorsal horn neurons (Boulenguez et al., 2010; Chen et al., 2014d; Coull et al., 2003; Jolivalt et al., 2008; Zhou et al., 2012). Nerve injury also increases NKCC1 phosphorylation and activity in dorsal root ganglion (DRG) neurons to reduce presynaptic inhibition (Chen et al., 2014a; Modol et al., 2014). However, it is unclear whether and to what extent restoring Cl− homeostasis at the spinal cord level leads to long-term reduction in neuropathic pain.

In addition to diminished synaptic inhibition, increased N-methyl D-aspartate receptor (NMDAR) activity in the spinal dorsal horn plays a key role in the development of neuropathic pain (Chaplan et al., 1997; Chen et al., 2014c). Peripheral nerve injury increases spinal NMDAR activity, which impairs synaptic inhibition through calpain-mediated KCC2 proteolysis (Zhou et al., 2012). However, it is unclear whether the loss of Cl−-dependent synaptic inhibition accounts for the increased NMDAR activity in the spinal dorsal horn induced by nerve injury. To determine whether restoring Cl− homeostasis reduces the synaptic NMDAR activity in the spinal dorsal horn that is increased by nerve injury and, therefore, relieves neuropathic pain, we studied intrathecal KCC2 gene delivery in rat models. Gene therapy offers the potential to correct these sustained abnormal signaling pathways and is well suited for treating chronic pain. Lentiviral vectors in particular have the advantages of long-term transgene expression, low immunogenicity, and the ability to accommodate larger transgenes and transduce nondividing cells such as mature neurons (Nayak and Herzog, 2010; Wong et al., 2006).

We show that intrathecal KCC2 gene transfer is highly efficient at restoring Cl− homeostasis in both spinal dorsal horn and DRG neurons and produces complete and long-lasting relief of neuropathic pain. Strikingly, KCC2 gene transfer normalizes, at both pre- and postsynaptic sites, spinal NMDAR activity increased by nerve injury. Our study provides direct evidence that disrupted neuronal Cl− homeostasis plays a critical role in potentiated synaptic NMDAR activity in neuropathic pain. This information significantly advances our understanding of homeostatic synaptic plasticity and suggests a strategy for treating chronic neuropathic pain.

RESULTS

Intrathecal KCC2 Gene Transfer Abolishes Nerve Injury-Induced Pain Hypersensitivity

Nerve injury decreases the protein level and function of KCC2 in spinal dorsal horn neurons, thereby contributing to the loss of synaptic inhibition and the development of neuropathic pain (Coull et al., 2003; Zhou et al., 2012). We constructed lentiviral vectors encoding rat KCC2 protein (KCC2 vector) or enhanced green fluorescent protein (GFP; control vector) (Figure 1A). To determine whether KCC2 gene transfer at the spinal cord level is sufficient to reduce pain hypersensitivity induced by nerve injury, we used spinal nerve ligation (SNL) in rats, a commonly used animal model of neuropathic pain (Chaplan et al., 1994; Kim and Chung, 1992). We performed intrathecal injection of the KCC2 or control vector 2 weeks after SNL surgery. Tactile allodynia, measured with von Frey filaments, and mechanical hyperalgesia, tested with a nociceptive pressure stimulus, were measured at baseline before SNL, at the time of intrathecal vector injection, and every 2–3 days for 6 weeks after vector injection (Figure 1B).

Figure 1. Intrathecal KCC2 Gene Transfer Eliminates Nerve Injury-Induced Pain Hypersensitivity.

Figure 1

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(A) Schematics of lentiviral vectors engineered to express KCC2-green fluorescent protein (GFP) (KCC2 vector) or GFP alone (control vector). LTR: long terminal repeats; CMV: cytomegalovirus promoter.

(B) Experimental design for testing the effects of the KCC2 vector using a rat neuropathic pain model.

(C) Time course of changes in the mechanical nociceptive threshold after intrathecal injection of the KCC2 (n = 8 rats) or control (n = 7 rats) vector in SNL rats. BL: baseline. **P < 0.01, ***P < 0.001 (two-way ANOVA analysis followed by Tukey’s post hoc test).

(D) Time course of changes in the tactile withdrawal threshold after intrathecal injection of the KCC2 (n = 8 rats) or control (n = 7 rats) vector in SNL rats. BL: baseline. **P < 0.01, ***P < 0.001 (two-way ANOVA analysis followed by Tukey’s post hoc test). Data are presented as means ± SEM.

(E) Effect of intrathecal injection of the KCC2 (n = 8 rats) or control (n = 7 rats) vector on the mechanical nociceptive threshold in sham control rats. Data are presented as means ± SEM.

(F) Effect of intrathecal injection of the KCC2 (n = 8 rats) or control (n = 7 rats) vector on the tactile threshold in sham control rats. Data are presented as means ± SEM.

Intrathecal injection of the control vector had no significant effect on tactile allodynia or mechanical hyperalgesia in SNL rats over the course of 8 weeks (Figure 1C,D). By contrast, a single intrathecal injection of the KCC2 vector abolished mechanical hyperalgesia in all rats. Two weeks after the KCC2 vector delivery, the nociceptive threshold had already returned to the pre-SNL (baseline) level, and this effect lasted for at least another 6 weeks (Figure 1C). KCC2 vector injection also fully reversed the thermal sensitivity of SNL rats (Supplementary Figure 1). To our surprise, intrathecal injection of KCC2 vectors only gradually reduced tactile allodynia over the first 5 weeks. Nevertheless, the tactile threshold of treated SNL rats completely returned to the pre-SNL (baseline) level 6 weeks after KCC2 vector injection; the effect lasted for at least another 2 weeks (Figure 1D). Intrathecal injection of the KCC2 vector had no significant effect on the tactile and nociceptive withdrawal thresholds in sham control rats (Figure 1E,F) and on the contralateral (non-injured) hindpaw of SNL rats (Supplementary Figure 2). These results indicate that intrathecal KCC2 gene transfer results in profound and enduring elimination of pain hypersensitivity caused by nerve injury.

Intrathecal KCC2 Gene Transfer Induces Efficient KCC2 Expression in Spinal Dorsal Horn and DRG Neurons

Immunocytochemical labeling and confocal microscopy revealed that the KCC2 vector induced transgene expression of KCC2 throughout the spinal dorsal horn 6 weeks after intrathecal injection (Figure 2A). We evaluated the in vivo neuronal transduction efficiency of the lentiviral vector by counting the number of NeuN (a specific neuronal marker)-positive neurons labeled with GFP in the spinal dorsal horn. Double immunolabeling with GFP and NeuN showed that ~90% of dorsal horn neurons in laminas I-III were co-localized with GFP in sham and SNL rats. Intrathecal injection of the KCC2 vector also resulted in ectopic KCC2 expression in the DRG. Approximately 94% of NeuN-positive DRG neurons were co-localized with GFP in sham and SNL rats (Figure 2B).

Figure 2. Transduction Efficiency in Spinal Dorsal Horn and DRG Neurons and KCC2 Expression Levels Induced by Intrathecal KCC2 Vector.

Figure 2

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(A) Low- and high-magnification confocal images show the distribution of GFP-positive spinal dorsal horn neurons (labeled with NeuN) in sham and SNL rats 6 weeks after intrathecal injection of the KCC2-GFP vector.

(B) Confocal images show the distribution of GFP-positive DRG neurons (labeled with NeuN) of sham and SNL rats treated with the intrathecal KCC2-GFP vector.

(C) Original gel images and quantification data show the protein level of KCC2 (140 kDa) in the dorsal spinal cords of sham and SNL rats 2, 4, and 6 weeks after intrathecal injection of the KCC2 (n = 6 samples from 6 rats in each group) or control (n = 6 samples from 6 rats in each group) vector. ***P < 0.001 compared with control vector-treated sham rats; #P < 0.001 compared with control vector-treated sham rats (two-way ANOVA analysis with Tukey’s post hoc test). Data are presented as means ± SEM.

(D) Original gel images and quantification data show ectopic KCC2 expression in the DRG of sham and SNL rats treated with the KCC2 or control vector 2, 4, and 6 weeks after intrathecal injection (n = 6 samples from 6 rats in each group). The red triangle indicates the KCC2 band position. GAPDH (~40 kDa) was included as a protein loading control. Data are presented as means ± SEM.

The KCC2 protein level in the dorsal spinal cord was significantly lower (about 30%) in SNL rats treated with control vectors compared with that in the sham controls (Figure 2C). Treatment with the KCC2 vector approximately doubled the amount of KCC2 proteins in the dorsal spinal cord of SNL rats compared with control vector-treated rats (Figure 2C). Western blots also showed that intrathecal injection of the KCC2 vector induced ectopic KCC2 expression in the DRG of both sham and SNL rats (Figure 2D). However, intrathecal injection of the KCC2 vector had no significant effect on the NKCC1 protein levels in the DRG and dorsal spinal cord (Supplementary Figure 3). These data indicate that intrathecal KCC2 gene transfer is highly efficient to induce sustained KCC2 expression in the spinal dorsal horn and DRG neurons.

Intrathecal KCC2 Gene Transfer Restores KCC2 Activity of Spinal Dorsal Horn Neurons Impaired by Nerve Injury

SNL reduces KCC2 activity by promoting KCC2 protein degradation in the spinal cord (Zhou et al., 2012). We examined whether intrathecal injection of the KCC2 vector could restore KCC2 activity in the spinal dorsal horn of SNL rats 5−6 weeks after injection. We recorded the reversal potential of GABA-mediated currents (EGABA), which reflects intracellular Cl− levels (Coull et al., 2003; Rivera et al., 1999), of dorsal horn neurons in spinal cord slices. In dorsal horn neurons from sham rats, EGABA was about −70 mV. In dorsal horn neurons from SNL rats treated with the control vector, there was a significant depolarizing shift (about 14 mV) in EGABA (Figure 3A,B). Treatment with the KCC2 vector fully restored EGABA in the spinal dorsal horn neurons of SNL rats (Figure 3A,B). These results suggest that intrathecal injection of the KCC2 vector fully restores nerve injury-reduced KCC2 activity in spinal dorsal horn neurons.

Figure 3. Intrathecal KCC2 Gene Transfer Restores EGABA in Spinal Dorsal Horn Neurons and DRG Neurons of SNL Rats.

Figure 3

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(A) Original traces of currents elicited by puff AMAP (holding potentials from −90 mV to −50 mV) to dorsal horn neurons of sham and SNL rats treated with KCC2 or control vector.

(B) Mean current-voltage plots and summary data show the EGABA of spinal dorsal horn neurons from sham and SNL rats 5−6 week after treatment with the KCC2 (sham, n = 11 neurons; SNL, n = 12 neurons) or control (sham, n = 12 neurons; SNL, n = 12 neurons) vector. *P < 0.05 compared with sham rats treated with control vector. #P < 0.05 compared with sham rats treated with control vector. One-way ANOVA analysis followed by Tukey’s post hoc test.

(C) Representative traces of GABA-elicited currents (holding potentials are shown on the left) in DRG neurons from sham or SNL rats treated with KCC2 or control vector.

(D) Mean current-voltage plots and summary EGABA data in DRG neurons from sham or SNL rats 5−6 week after treatment with the KCC2 (sham, n = 9 neurons; SNL, n = 10 neurons) or control (sham, n = 8 neurons; SNL, n = 7 neurons) vector. *P < 0.05 compared with sham rats treated with control vector; #P < 0.05 compared with sham rats treated with control vector. One-way ANOVA analysis followed by Tukey’s post hoc test. Data are presented as means ± SEM.

Intrathecal KCC2 Gene Transfer Reverses the Depolarizing Shift in EGABA of DRG Neurons Induced by Nerve Injury

DRG neurons express NKCC1, but not KCC2 (Mao et al., 2012; Rivera et al., 1999). Nerve injury increases NKCC1 phosphorylation and causes a depolarizing shift of EGABA in DRG neurons (Chen et al., 2014a; Modol et al., 2014). Because intrathecal injection of the KCC2 vector induced ectopic KCC2 expression in DRG neurons, we determined whether this could offset NKCC1 activity increased by nerve injury in DRG neurons. DRG neurons are highly heterogeneous, and it was difficult to ensure that similar sensory neuron phenotypes were sampled in different groups. We therefore used isolectin B4 (IB4)-Alexa 594 dye, which can label a subgroup of live DRG neurons (Wu et al., 2004). The EGABA of IB4-positive DRG neurons acutely dissociated from sham rats treated with control vectors was −26 ± 2.1 mV. SNL caused a significant depolarizing shift in the EGABA of IB4-positive DRG neurons (Figure 3C,D). Treatment with the KCC2 vector caused a significant hyperpolarizing shift (about 10 mV) in the EGABA of IB4-positive DRG neurons from both SNL and sham control rats (Figure 3C,D). Our data suggest that induced KCC2 expression effectively counteracts increased NKCC1 activity to restore Cl− homeostasis in injured DRG neurons.

Intrathecal KCC2 Gene Transfer Normalizes Pre- and Postsynaptic NMDAR Activity Increased by Nerve Injury in Spinal Cords

Because treatment with the KCC2 vector fully reversed the depolarizing shift of EGABA in spinal dorsal horn and DRG neurons caused by nerve injury and because increased spinal NMDAR activity is critically involved in the development of neuropathic pain (Chaplan et al., 1997; Chen et al., 2014c; Sigtermans et al., 2009), we next examined whether restoring Cl− homeostasis via KCC2 gene transfer could reduce the increased spinal NMDAR activity in SNL rats. We obtained spinal cord slices from SNL rats 5–6 weeks after intrathecal injection of the control or KCC2 vector. We measured the postsynaptic NMDAR currents elicited by puff application of NMDA directly to the recorded dorsal horn neuron (Chen et al., 2014b). Treatment with the KCC2 vector significantly reduced the amplitude of the puff NMDAR currents of dorsal horn neurons in SNL rats to the level observed in sham control rats (P < 0.05; Figure 4A). In contrast, the AMPA receptor current elicited by puff application of AMPA to dorsal horn neurons did not differ significantly between KCC2 vector- and control vector-treated SNL rats (Figure 4A).

Figure 4. Intrathecal KCC2 Gene Transfer Normalizes Synaptic NMDAR Activity in the Spinal Dorsal Horn of SNL Rats.

Figure 4

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(A) Original current traces and summary data show currents elicited by puff application of 100 µM NMDA or 20 µM AMPA to spinal dorsal horn neurons in sham (n = 12 neurons), control vector-treated (n = 16 neurons), or KCC2 vector-treated (n = 18 neurons) SNL rats 5−6 weeks after injection. *P < 0.05 compared with sham group. One-way ANOVA analysis followed by Tukey’s post hoc test.

(B) Representative recording traces and cumulative plots show mEPSCs of spinal dorsal horn neurons recorded from control vector-treated or KCC2 vector-treated SNL rats before (baseline), with (AP5), and after (washout) bath application of 50 µM AP5.

(C) Summary data show baseline values and the AP5 effect on the frequency and amplitude of mEPSCs of spinal dorsal neurons from control vector-treated (n = 8 neurons) or KCC2 vector-treated (n = 9 neurons) SNL rats. *P < 0.05 compared with the baseline value. #P < 0.05 compared with the baseline value in the control vector-treated group. One-way ANOVA analysis followed by Tukey’s post hoc test.

(D) Original recording traces and mean data show the AP5 effect on the amplitude of EPSCs of spinal dorsal horn neurons monosynaptically evoked from stimulation of the dorsal root in control vector-treated (n = 11 neurons) or KCC2 vector-treated (n = 12 neurons) SNL rats 5−6 weeks after injection. *P < 0.05 compared with the baseline value. One-way ANOVA analysis followed by Tukey’s post hoc test. Data are presented as means ± SEM.

Presynaptic NMDARs are not functionally active in the normal spinal dorsal horn. However, in the chronic pain condition, presynaptic NMDAR activity is increased, facilitating glutamate release from primary afferent terminals (Chen et al., 2014b). Because intrathecal KCC2 gene transfer induced KCC2 expression in DRG neurons, we determined whether this treatment also altered presynaptic NMDAR activity in the spinal dorsal horn. We recorded miniature excitatory postsynaptic currents (mEPSCs), which reflect quantal glutamate release from presynaptic terminals (Chen et al., 2014b). In the dorsal horn neurons of control vector-treated SNL rats, bath application of (2R)-amino-5-phosphonopentanoate (AP5), a specific NMDAR antagonist, rapidly reduced the frequency, but not the amplitude, of mEPSCs (Figure 4B,C). In dorsal horn neurons recorded from KCC2 vector-treated SNL rats, the frequency of mEPSCs was significantly lower than that observed in control vector-treated SNL rats. Furthermore, AP5 application had no significant effects on the frequency or amplitude of mEPSCs in dorsal horn neurons from KCC2 vector-treated SNL rats (Figure 4B,C).

In addition, we recorded EPSCs monosynaptically evoked by stimulation of the dorsal root, reflecting glutamate release from primary afferent terminals (Chen et al., 2014b; Li et al., 2002). In dorsal horn neurons from control vector-treated SNL rats, bath application of AP5 significantly reduced the amplitude of the evoked EPSCs (Figure 4D). However, in dorsal horn neurons from KCC2 vector-treated SNL rats, AP5 had no significant effect on the amplitude of evoked EPSCs (Figure 4D). Consistent with the lack of an effect of KCC2 overexpression on tactile and nociceptive thresholds in sham rats, KCC2 vector injection produced no effect on pre- or postsynaptic NMDAR activity of spinal dorsal horn neurons in sham rats (Supplementary Figure 4A–D). Together, these results indicate that restoring Cl− homeostasis by increasing KCC2 expression normalizes both pre- and postsynaptic NMDAR activity increased by nerve injury in the spinal dorsal horn.

DISCUSSION

Our study shows that lentiviral vector-mediated KCC2 gene transfer via intrathecal injection has a profound and long-lasting effect on pain hypersensitivity caused by nerve injury. Such a dramatic effect is likely due to the ability of lentiviral vectors to induce expression of KCC2 in both dorsal horn and DRG neurons and thereby restore the balance of synaptic inhibition and excitation at both pre- and postsynaptic sites. Our electrophysiological data confirm that induced KCC2 expression effectively restored Cl− homeostasis in both spinal cord horn and DRG neurons. Although NKCC1 inhibitors and KCC2 enhancers produce short-acting effects on neuropathic pain (Cramer et al., 2008; Lavertu et al., 2014), these agents do not have the ability to persistently restore Cl− homeostasis in the spinal dorsal horn and DRG neurons that is disrupted in neuropathic pain. Compared to these drug treatments, intrathecal gene delivery could more directly target abnormal chronic pain signaling in a tissue-specific manner (Storek et al., 2008). Also, gene therapy could provide sustained longterm analgesia without the need for repeated administrations, making it particularly suitable for treating chronic pain. Interestingly, we found a distinct time course for the reversal of mechanical hyperalgesia and tactile allodynia after intrathecal injection of the KCC2 vector. Mechanical hyperalgesia induced by SNL was fully reversed by KCC2 gene transfer within 2 weeks, but the effect of gene transfer on tactile allodynia was more gradual, taking several weeks to achieve the maximum effect. It is possible that the KCC2 vector may initially transduce more high-threshold nociceptive than low-threshold sensory neurons, which could explain its more rapid effect on hyperalgesia than on allodynia. Because intrathecal injection via lumbar puncture is a commonly used and minimally invasive clinical procedure, our findings represent a proof of principle that this therapeutic strategy can potentially be used to treat patients with intractable neuropathic pain and other neurological disorders associated with reduced KCC2 activity, such as epilepsy and spasticity (Boulenguez et al., 2010; Palma et al., 2006).

Fast synaptic inhibition by GABA and glycine is essential for physiological processing of nociceptive transmission in the spinal dorsal horn. Although a loss of synaptic inhibition has been found in many neuropathic pain conditions (Boulenguez et al., 2010; Chen et al., 2014d; Coull et al., 2003; Jolivalt et al., 2008; Zhou et al., 2012), it remains unclear to what extent impaired Cl− homeostasis sustains chronic neuropathic pain. Our study, by inducing KCC2 overexpression, indicates that disrupted Cl− homeostasis contributes critically to synaptic disinhibition and chronic pain caused by nerve injury. The spinal dorsal horn neurons are highly heterogeneous, and it would be interesting to further study the effect of expressing KCC2 specifically in a subpopulation (either excitatory or inhibitory) of dorsal horn neurons on neuropathic pain.

DRG neurons, and possibly their central terminals, normally maintain a relatively high Cl− level via NKCC1 (Sung et al., 2000), a requirement for presynaptic inhibition (Rudomin and Schmidt, 1999). Presynaptic GABAA receptor activation, which is produced by the release of GABA from spinal interneurons, inhibits glutamatergic input from primary afferents to dorsal horn neurons (Yuan et al., 2009). In this study, we demonstrated that nerve injury induced a depolarizing shift of EGABA in DRG neurons and that intrathecal KCC2 gene transfer completely reversed this depolarizing shift. Thus, we conclude that ectopic KCC2 expression counteracts NKCC1 activity to restore presynaptic inhibition, which may contribute to the powerful and sustained effect of KCC2 gene transfer on neuropathic pain.

Another striking finding of our study is that intrathecal KCC2 gene transfer completely reversed pre- and postsynaptic NMDAR activity of spinal dorsal horn neurons potentiated by nerve injury, indicating that Cl− homeostasis mediates a crosstalk between synaptic inhibition and excitation. Increased NMDAR activity in the spinal dorsal horn causes central sensitization and represents a key mechanism of neuropathic pain (Chaplan et al., 1997; Chen et al., 2014b; Chen et al., 2014c). Diminished synaptic inhibition and augmented NMDAR activation in neuropathic pain have been recognized as separate phenomena, and their reciprocal relationship is unclear. We have previously shown that spinal NMDAR activity increased by nerve injury promotes KCC2 degradation through calpain activation to diminish synaptic inhibition (Zhou et al., 2012). On the other hand, blocking GABAA and glycine receptors at the spinal cord level can cause pain hypersensitivity, which can be blocked by NMDAR antagonists (Cao et al., 2011; Yamamoto and Yaksh, 1993). Our findings provide direct evidence for the critical role of impaired Cl− homeostasis in increased spinal NMDAR activation induced by nerve injury. Both pre- and postsynaptic NMDAR activity in spinal dorsal horn and DRG neurons may be large but latent, held in check by Cl−-dependent synaptic inhibition. Our study reveals that Cl− homeostasis plays a critical role in the reciprocal relationship between synaptic inhibition and NMDAR activity and that an imbalance in this relationship likely initiates a vicious cycle that sustains chronic neuropathic pain. The complete reversal of postsynaptic NMDAR activity in the spinal dorsal horn and presynaptic NMDAR activity at primary afferent terminals likely accounts for the potent and persistent effect of induced KCC2 expression on neuropathic pain. Nevertheless, it is unclear exactly how restoring Cl− homeostasis normalizes nerve injury-induced potentiation of synaptic NMDAR activity. Because there is no evidence for a direct interaction between KCC2 and NMDARs, we postulate that KCC2 overexpression-induced suppression of pre- and postsynaptic NMDAR activity in neuropathic pain probably results from the reduced excitability of DRG and spinal dorsal horn neurons.

In summary, we found that intrathecal KCC2 gene delivery produces complete and long-lasting relief from nerve injury-induced neuropathic pain by restoring Cl− homeostasis in spinal dorsal horn and DRG neurons. Our findings also indicate that nerve injury increases pre- and postsynaptic NMDAR activity at the spinal cord level by disrupting Cl− homeostasis. This information significantly advances our understanding of homeostatic synaptic plasticity and provides a promising gene therapy strategy for treating intractable neuropathic pain.

EXPERIMENTAL PROCEDURES

Viral Vector Constructs

The full-length coding sequence of GFP or rat KCC2 was cloned into the lentiviral vector pLenti6/V5- DEST. The viral vector preparation is described in detail in the Extended Experimental Procedures.

Rat Model of Neuropathic Pain and Behavioral Assessments of Nociception

Adult male Sprague-Dawley rats (9−10 weeks old) were used for this study. All of the experimental procedures were approved by the Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center. Spinal nerve ligation and intrathecal injections were performed as previously described (Chen et al., 2014b; Kim and Chung, 1992). Tactile allodynia was tested using von Frey filaments and the “up-down” method (Chaplan et al., 1994). The mechanical nociceptive threshold was tested using a noxious pressure stimulus as previously described (Chen et al., 2014b; Zhou et al., 2012). See Extended Experimental Procedures for details.

Western Blotting and Immunocytochemistry

Western blotting and immunocytochemical labeling were performed using standard methods described in Extended Experimental Procedures.

Electrophysiological Recordings

Lumbar spinal cord slices were prepared from rats as described previously (Li et al., 2002; Zhou et al., 2012). Spinal dorsal horn neurons were visualized and selected for whole-cell recording of NMDAR activity. We used the Cl−-impermeable gramicidin-perforated recording method to record EGABA of spinal dorsal horn and DRG neurons, as described previously (Chen et al., 2014b; Sung et al., 2000). Details are provided in Extended Experimental Procedures.

Statistical Analysis

Results are expressed as means ± standard errors of the mean. The behavioral and biochemical data were analyzed using two-way ANOVA analysis with Tukey’s post hoc test. The electrophysiological data were analyzed using one-way ANOVA analysis with Tukey’s post hoc test. P < 0.05 was considered statistically significant. See Extended Experimental Procedures for details.

Supplementary Material

1
2

Highlights.

  • Intrathecal delivery of KCC2 in lentiviral vectors eliminates neuropathic pain

  • KCC2 gene transfer restores spinal cord KCC2 function impaired by nerve injury

  • KCC2 ectopic expression counteracts NKCC1 activity in primary sensory neurons

  • Restoring Cl− homeostasis normalizes spinal cord synaptic NMDA receptor activity

Acknowledgments

This work was supported by the National Institutes of Health (R01 NS073935 and R01 DE022015) and by the N.G. and Helen T. Hawkins Endowment (to H.-L.P.).

Footnotes

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SUPPLEMENTAL INFORMATION

Supplemental Information includes Extended Experimental Procedures and two supplementary figures.

AUTHORSHIP CONTRIBUTIONS

L.L. performed vector design and production, immunoblotting and behavioral testing. S.-R.C. conducted animal surgery, immunocytochemical labeling and electrophysiological recordings. H.C. prepared spinal cord slices and performed some electrophysiological recordings. L.W. performed DRG neuron dissociation and recording. W.N.H. assisted with confocal microscopy. J.-D.X. conducted some behavioral testing. L.L., S.-R.C., and H.-L.P. participated in data analysis. H.-L.P. conceived the project and wrote the manuscript with input from the other authors.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

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NIHMS778547-supplement-1.docx (11.8KB, docx)
2
NIHMS778547-supplement-2.pdf (558.5KB, pdf)

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