Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 May 29;23(9):2678-2689.
doi: 10.1016/j.celrep.2018.04.107.

NMDA Receptor Activation Underlies the Loss of Spinal Dorsal Horn Neurons and the Transition to Persistent Pain After Peripheral Nerve Injury

Affiliations
Free PMC article

NMDA Receptor Activation Underlies the Loss of Spinal Dorsal Horn Neurons and the Transition to Persistent Pain After Peripheral Nerve Injury

Perrine Inquimbert et al. Cell Rep. .
Free PMC article

Abstract

Peripheral nerve lesions provoke apoptosis in the dorsal horn of the spinal cord. The cause of cell death, the involvement of neurons, and the relevance for the processing of somatosensory information are controversial. Here, we demonstrate in a mouse model of sciatic nerve injury that glutamate-induced neurodegeneration and loss of γ-aminobutyric acid (GABA)ergic interneurons in the superficial dorsal horn promote the transition from acute to chronic neuropathic pain. Conditional deletion of Grin1, the essential subunit of N-methyl-d-aspartate-type glutamate receptors (NMDARs), protects dorsal horn neurons from excitotoxicity and preserves GABAergic inhibition. Mice deficient in functional NMDARs exhibit normal nociceptive responses and acute pain after nerve injury, but this initial increase in pain sensitivity is reversible. Eliminating NMDARs fully prevents persistent pain-like behavior. Reduced pain in mice lacking proapoptotic Bax confirmed the significance of neurodegeneration. We conclude that NMDAR-mediated neuron death contributes to the development of chronic neuropathic pain.

Keywords: NMDA receptor; chronic pain; disinhibition; dorsal horn; excitotoxicity; nerve injury; neuropathic pain.

Conflict of interest statement

DECLARATION OF INTERESTS

J.S. is now an employee of Biogen. This work was completed before he joined the company. The company did not have a role in the design, conduct, analysis, interpretation, or funding of the research. All other authors declare no competing interests.

Figures

Figure 1.
Figure 1.. NMDAR-Mediated Glutamatergic Transmission Causes Nerve-Injury-Induced Apoptosis
(A) Schematic illustration of SNI and the relevant neuroanatomy. DRG, dorsal root ganglion. (B) Apoptotic cell profiles 7 days after SNI. H33342, Hoechst 33342 (bisbenzimide). Scale bar, 10 μm. (C) Distribution of apoptotic profiles within the dorsal horn, shown in overlays of 10 sections per mouse (n = 8 mice). (D) Time course of apoptosis induction. Apoptotic profiles were counted in 10 sections per mouse (n = 8). p < 0.001 for surgery and time in a two-way ANOVA. ***p < 0.001 in Sidak’s test following the ANOVA. (E) Apoptosis in uninjured mice and the ipsilateral and contralateral dorsal horns of mice 7 days after SNI (n = 8). p < 0.001 in a one-way ANOVA. ***p <0.001 in Dunnett’s test following the ANOVA. Apoptosis in the white matter was not significantly different in Student’s t test. (F and G) Stereotaxic injection of AAV8-GFP-Cre into the spinal cord of Grin1flox/flox mice eliminated NMDAR function. (F) GFP expression (left) and in situ hybridization of Grin1 mRNA (right) 3 weeks after injection of the vector into the left dorsal horn of the lumbar (L4) spinal cord. Scale bars, 100 μm (left) or 200 μm (right). (G) Excitatory currents evoked by NMDA (100 μM) in Grin1flox/flox mice injected with AAV8-GFP or AAV8-GFP-Cre. SNI was performed 2 or 3 weeks after the vector injection. Two weeks after the nerve injury, we compared current amplitudes in the dorsal horn of mice injected with AAV8-GFP (n = 5 neurons) or AAV8-GFP-Cre (n = 11). ***p <0.001 in Student’s t test. Currents recorded in Grin1flox/flox mice injected with AAV8-GFP did not differ from those in uninjured C57BL/6 mice (n = 4) or C57BL/6 mice after SNI (n = 6). (H) Apoptotic profiles 7 days after SNI in the dorsal horn of Grin1flox/flox mice injected with AAV8-GFP or AAV8-GFP-Cre (n = 6 mice). ***p < 0.001 in Student’s t test. Error bars indicate SEM. See also Figures S1 and S5.
Figure 2.
Figure 2.. Grin1 Deletion Protects against the Loss of Dorsal Horn Neurons
(A) Representative photographs of the L4 dorsal horn of uninjured C57BL/6 mice and 28 days after SNI. Neurons were immunostained for NeuN. Scale bar, 100 μm. (B) Stereological counts of neurons (NeuN) in laminae I+II and laminae III+IV of the dorsal horn of uninjured mice (n = 6) and after SNI (n = 8). (C) Representative photographs of the L4 dorsal horn of Grin1flox/flox mice 28 days after SNI. Scale bar, 100 μm. (D) Stereological counts of neurons in the dorsal horn of Grin1flox/flox mice injected with AAV8-GFP (n = 6) or AAV8-GFP-Cre (n = 5). Neurons were counted 28 days after SNI. *p < 0.05; **p < 0.01; ***p < 0.001 in Student’s t tests. Error bars indicate SEM. Schematic drawings indicate the counting ROIs (gray). See also Table S1.
Figure 3.
Figure 3.. Neuroprotection Preserves Spinal Inhibition
(A) GFP expression in GABAergic dorsal horn neurons of Gad1-GFP mice. Some GABAergic neurons also produced glycine. Scale bars, 100 μm (large panels) or 10 μm (small panels). (B) Stereological counts of GABAergic neurons in the L4 dorsal horn of uninjured Gad1-GFP mice and 28 days after SNI (n = 6 mice). (C) Viaat-Venus mice expressed the fluorescent protein in both GABAergic and glycinergic neurons. Scale bars, 100 μm (large panels) or 10 μm (small panels). (D) Stereological counts of inhibitory interneurons in laminae III+IV of uninjured Viaat-Venus mice (n = 5) and 28 days after SNI (n = 6). (E–I) Miniature IPSC recordings. (E) Representative traces of total mIPSCs in C57BL/6 mice. Bar graphs show current frequency and peak amplitude in uninjured mice (n = 17 neurons) and 2 weeks after SNI (n = 25). (F) Comparison of total mIPSCs in the spinal cord of Grin1flox/flox mice injected with AAV8-GFP (n = 8 neurons) or AAV8-GFP-Cre (n = 12). Currents were recorded 2 weeks after SNI. (G) Frequency and peak amplitude of GABAergic mIPSCs in uninjured C57BL/6 mice (n = 12) and 2 weeks after SNI (n = 12). (H) GABAergic mIPSCs in Grin1flox/flox mice injected with AAV8-GFP (n = 5) or AAV8-GFP-Cre (n = 4). GABAergic currents were recorded in the presence of strychnine. (I) Glycinergic mIPSCs, recorded in the presence of bicuculline, did not differ between uninjured C57BL/6 mice (n = 9) and 2 weeks after SNI (n = 11). *p < 0.05; **p < 0.01; ***p < 0.001. Student’s t test was used for all comparisons. Error bars indicate SEM. See also Figure S2 and Table S1.
Figure 4.
Figure 4.. Surviving Neurons Maintain the Expression of Gad, Glyt2, and Viaat
(A–D) Transcript and protein levels of (A) Gad1, (B) Gad2, (C) Glyt2 (Slc6a5), and (D) Viaat (Slc32a1) were unchanged in the L4 dorsal horn of mice 28 days after SNI (n = 3 or 4 biological replicates). qPCR results are presented as mean fold change of ΔCT ± SD after normalization to Gapdh and protein levels as mean fold change ± SEM after normalization to β3-tubulin (Tubb3). Images show representative western blot results. (E) ChIP analysis of H3K27ac at the promoter regions for Gad1, Gad2, Actb, and Arc (n = 4). His-tone modifications 28 days after SNI are shown, compared to uninjured mice. *p < 0.05. Student’s t test was used for all comparisons. Error bars indicate SEM. See also Figure S3 and Table S2.
Figure 5.
Figure 5.. Eliminating Functional NMDARs in the Dorsal Horn Blocks the Transition from Acute to Chronic Neuropathic Pain
(A and C) Withdrawal responses to (A) mechanical (von Frey filaments) or (C) cold stimulation (acetone evaporation) after sham surgery (n = 7 mice) or SNI (n = 10) in Grin1flox/flox mice injected with AAV8-GFP-Cre, and SNI in Grin1flox/flox mice injected with AAV8-GFP (n = 10). Behavioral outcomes were compared using twoway ANOVAs. p < 0.001 for treatment and time in the responses to stimulation with von Frey filaments; p < 0.01 for treatment and p < 0.001 for time in the acetone test. Asterisks and pound signs indicate the results of Bonferroni’s tests following the ANOVAs. (B and D) Areas under the curve (AUCs) were first compared for the total test duration after SNI (p < 0.001 for Frey filaments, B, and acetone test, D, in one-way ANOVAs). Separately, we compared AUCs in the acute phase of the first 7 days after SNI (p < 0.001 for both test modalities), during the transition from acute to persistent pain (p < 0.001 for the stimulation with von Frey filaments and p < 0.05 for the acetone test), and for persistent pain after 21 or 49 days, respectively (p <0.001 for von Frey filaments and p < 0.01 for the acetone test). Asterisks and pound signs indicate the results of Tukey’s tests following the ANOVAs. *p < 0.05, **p < 0.01, and ***p < 0.001 for the comparison of AAV8-GFP-Cre + SNI and AAV8-GFP + SNI; #p < 0.05, ##p < 0.01, and ###p < 0.001 for the comparison of AAV8-GFP-Cre + Sham and AAV8-GFP-Cre + SNI. Error bars indicate SEM. See also Figure S4.
Figure 6.
Figure 6.. Mice Lacking Proapoptotic Bax Are Protected against Nerve-Injury-Induced Disinhibition
(A) Apoptotic profiles in Bax+/+ and Bax−/− mice 7 days after SNI (n = 6). (B) Representative recordings of total mIPSCs. Bar graphs below show current frequency and peak amplitude in uninjured Bax+/+ mice and Bax+/+ mice 2 weeks after SNI (n = 18 neurons) and in uninjured Bax−/− mice (n = 13) and Bax−/− mice 2 weeks after SNI (n = 19). (C) GABAergic mIPSCs in uninjured Bax+/+ mice (n = 11) and Bax+/+ mice 2 weeks after SNI (n = 14) and in uninjured Bax−/− mice (n = 8) and Bax−/− mice 2 weeks after SNI (n = 9). *p < 0.05 and ***p < 0.001 in Student’s t tests comparing uninjured Bax+/+ mice and Bax+/+ mice after SNI; #p < 0.05, ##p < 0.01, and ###p < 0.001 for the comparison between Bax+/+ and Bax−/− mice after SNI. Error bars indicate SEM. See also Figure S5.
Figure 7.
Figure 7.. Disrupting the Intrinsic Apoptotic Pathway Reduces Neuropathic-Pain-like Behavior
(A and C) Withdrawal responses to (A) mechanical (von Frey filaments) or (C) cold stimulation (acetone evaporation) after SNI (n = 9). p < 0.001 for genotype and p < 0.05 for time when responses to the stimulation with von Frey filaments were compared in a two-way ANOVA. p < 0.001 for both genotype and time in the acetone test. Pound signs indicate the results of Bonferroni’s tests following the ANOVAs. (B and D) AUCs for the withdrawal responses to (B) mechanical or (D) cold stimulation were compared for the total test duration after SNI. Pound signs indicate the results of Student’s t tests. #p < 0.05, ##p < 0.01, and ###p < 0.001 in Student’s t tests. Error bars indicate SEM.

Similar articles

See all similar articles

Cited by 12 articles

See all "Cited by" articles

References

    1. Amir R, Kocsis JD, and Devor M (2005). Multiple interacting sites of ectopic spike electrogenesis in primary sensory neurons. J. Neurosci 25, 2576–2585. - PMC - PubMed
    1. Bao X, Pal R, Hascup KN, Wang Y, Wang WT, Xu W, Hui D, Agbas A, Wang X, Michaelis ML, et al. (2009). Transgenic expression of Glud1 (glutamate dehydrogenase 1) in neurons: in vivo model of enhanced glutamate release, altered synaptic plasticity, and selective neuronal vulnerability.J. Neurosci 29, 13929–13944. - PMC - PubMed
    1. Bráz JM, Sharif-Naeini R, Vogt D, Kriegstein A, Alvarez-Buylla A, Rubenstein JL, and Basbaum AI (2012). Forebrain GABAergic neuron precursors integrate into adult spinal cord and reduce injury-induced neuropathic pain. Neuron 74, 663–675. - PMC - PubMed
    1. Cheng L, Duan B, Huang T, Zhang Y, Chen Y, Britz O, Garcia-Camp-many L, Ren X, Vong L, Lowell BB, et al. (2017). Identification of spinal circuits involved in touch-evoked dynamic mechanical pain. Nat. Neurosci 20, 804–814. - PMC - PubMed
    1. Colloca L, Ludman T, Bouhassira D, Baron R, Dickenson AH, Yarnitsky D, Freeman R, Truini A, Attal N, Finnerup NB, et al. (2017). Neuropathic pain. Nat. Rev. Dis. Primers 3, 17002. - PMC - PubMed

Publication types

MeSH terms

Feedback