N-type calcium channels play an essential role in primary afferent neurotransmission in the spinal cord dorsal horn.11,36 Indeed, these channels were first identified in dorsal root ganglion (DRG) neurons.20,42 The use of selective peptide blockers including ω-conotoxin GVIA6,46 has furthered understanding of the importance of N-type channels23,24 and their distribution.1,22 Their targeting for chronic pain therapy is also well-established.51,57 Although clinical use of ziconotide (ω-conotoxin MVIIA) is limited because of its intrathecal route of administration and side effects, nevertheless, it validates the role of N-type channels in pain pathophysiology and pharmacotherapy.45
CaV2.2 channels form a complex with auxiliary β and α2δ subunits, which are important for channel trafficking and function.18 The α2δ-1 isoform is prominent in primary afferent pathways and is up-regulated following neuropathic injury.4,34,40 Indeed, genetic ablation of either CaV2.228,47 or α2δ-144 suppresses various sensory modalities in chronic pain models.
Despite the importance of CaV2.2 channels in primary afferent transmission in nociceptive pathways, examination of their distribution and trafficking as well as altered expression following nerve injury has been hindered by the lack of reliable antibodies recognising native N-type calcium channels, which have been validated using knockout tissue. Furthermore, previous studies using antipeptide antibodies have reported conflicting results.12,29,60,61 For this reason, we previously developed CaV2.2 constructs containing exofacial epitope tags, in a position not affecting channel function.9 We then generated a knockin mouse line carrying the hemagglutinin (HA) tag in this position in the Cacna1b gene, to examine the distribution of native CaV2.2 protein in the intact nervous system.41 We previously found a dramatic effect of α2δ-1 ablation on CaV2.2_HA distribution, with the loss of cell surface CaV2.2, particularly in small peptidergic nociceptive sensory neuron somata and terminals.41
Here, we have examined the effect of partial sciatic nerve ligation (PSNL) on CaV2.2_HA distribution in sensory neurons and spinal cord and provide novel insights into cellular pathophysiological mechanisms after nerve injury. We have compared CaV2.2_HA distribution, ipsilateral and contralateral to nerve injury, both in DRG neuronal cell bodies and in their terminals in the dorsal horn, and we have then examined the effect of α2δ-1 knockout on this. We have further used several markers of different DRG subtypes, to examine their coexpression with CaV2.2_HA. This includes the glial cell line–derived neurotrophic factor (GDNF) family ligand receptor (GFRα1), which is present in certain low-threshold mechanoreceptors (LTMRs) and is up-regulated following nerve injury.5,27 Glial cell line–derived neurotrophic factor is a DRG trophic factor, which is analgesic in neuropathic pain.7
Our key finding is that CaV2.2_HA is up-regulated, ipsilateral to PSNL, in medium/large DRG neurons, where it shows increased association with GFRα1. In parallel, we observe increased CaV2.2_HA in ipsilateral medial/central deep dorsal horn, where GFRα1 is correspondingly up-regulated. The increased CaV2.2_HA in DRGs and deep dorsal horn is α2δ-1 dependent, whereas the elevation in GFRα1 is not, indicating that it represents increased CaV2.2_HA trafficking to these mechanoreceptor terminals, which may result in elevated neurotransmission.
2.1. Partial sciatic nerve ligation
The CaV2.2_HA mouse line was generated by Taconic Artemis on the C57BL/6 background, as described in detail previously.41 The α2δ-1−/− C57BL/6 mouse line described previously21,44 was crossed, as heterozygotes, with the Cav2.2_HA knockin mice to generate double-transgenic Cav2.2_HAKI/KI α2δ-1−/− mice. Wild-type (WT) mice were C57BL/6. Both male and female mice were used in this study. Mice were housed in groups of no more than 5 on a 12 h: 12 h light: dark cycle; food and water were available ad libitum. All experimental procedures were covered by UK Home Office license, had local ethical approval, and followed the guidelines of the International Association for the Study of Pain.62
Surgery was performed based on a method described previously.44,50 Mice were maintained under 2% vol/vol isoflurane (Baxter, Northampton, United Kingdom) anesthesia delivered in a 3:2 ratio of nitrous oxide and oxygen. Under aseptic conditions, the left sciatic nerve was exposed through blunt dissection of the biceps femoris above the trifurcation of the nerve. Approximately half of the nerve was ligated with a nonabsorbable 7-0 braided silk thread (Ethicon, VetTech, United Kingdom). The surrounding muscle and skin was closed with absorbable 6-0 vicryl sutures (Ethicon, VetTech) and topical lidocaine cream (5% wt/wt) applied to the skin. Sham surgery was performed in an identical manner, omitting the nerve ligation step. After surgery, the mice were allowed to recover. Foot posture and general behavior of the operated mice were monitored throughout the postoperative period. While blind to genotype, mechanical hypersensitivity was tested 14 days after surgery to confirm that the operated mice used for the study displayed neuropathic responses.
For immunohistochemistry, on days 14 or 15 after surgery, mice were deeply anaesthetized with an intraperitoneal injection of pentobarbitone (Euthatal, Merial Animal Health, Harlow, United Kingdom; 600 mg/kg), perfused transcardially with saline containing heparin, followed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) at a flow rate of 2.5 mL·min−1 for 4 minutes. Lumbar 4 DRGs and the lumbar enlargement of the spinal cord were dissected out. Following dissection, the spinal cord was postfixed for 2 hours, whereas the DRGs did not undergo extra fixation. Tissue was washed with PB, cryoprotected by incubation in PB with 20% sucrose overnight, and finally mounted in Optimal cutting temperature (OCT) compound (VWR International, Lutterworth, United Kingdom) before storing at −80°C, until sectioning. Dorsal root ganglia and spinal cord were sectioned at 15 and 20 μm, respectively, using a cryostat, placing the sections sequentially in series of 6 slides, so the distance between any section and the next on any slide is 90 or 120 µm in each case. Slides were stored at −80°C until processed.
For immunofluorescence labelling of DRGs, sections were blocked with 10% goat serum in PBS containing 0.3% Triton X-100 for more than 1 hour at room temperature (RT), followed by incubation with the unconjugated goat Fab antimouse IgG (H + L) (0.1 mg/mL in PBS, Jackson ImmunoResearch Lab, Stratech Ltd, Ely, United Kingdom, catalogue number 115-007-003) for 1 hour at RT to reduce nonspecific binding of antirat antibody to endogenous IgG in mouse tissue, washed in PBS, 0.1% Triton X-100 (PBS-T), and then incubated with rat monoclonal anti-HA antibody (Roche, catalogue number 11867423001, 1:100), for 2 to 3 days at 4°C in 5% goat serum, 0.3% Triton X-100 in PBS. Following extensive washing in PBS-T, immunolabelled samples were fixed in 4% paraformaldehyde in PBS for 30 minutes at RT, washed in PBS-T and incubated for 1 to 2 days at 4°C with the goat antirat conjugated with Alexa Fluor 488 (Invitrogen, Thermo Fisher Scientific, Oxford, United Kingdom, catalogue number A11006, 1:500). After washing, sections were treated with the nuclear stain DAPI (Molecular Probes, catalogue number D106, 0.5 μM) and mounted in VectaShield (Vector Laboratories, 2BScientific Ltd., Upper Heyford United Kingdom, catalogue number H-1000). When costaining HA with the goat antibody against GFRα1 (R&D Systems, Bio-techne, Abingdon , United Kingdom, catalogue number AF560, 1:200), the procedure was the same except that horse serum was used instead of goat serum in the blocking and antibody solution, the goat Fab antimouse was omitted, and the secondary antibodies were donkey antigoat conjugated with Alexa Fluor 488, and the donkey antirat highly cross-adsorbed antibody conjugated with Alexa Fluor 594 (Thermo Fisher Scientific, catalogue numbers A11055 and A21209 respectively, both used at 1:500).
For spinal cord immunohistochemistry, sections were incubated with rat monoclonal anti-HA antibody (as above) and costained with rabbit anti-calcitonin gene-related peptide (CGRP), IB4 conjugated with FITC (Sigma, catalogue numbers C8198 and L2895), or with goat anti-GFRα1. Some sections were labelled for α2δ-1, as described previously,44 with the following modifications: after heat-induced epitope retrieval (10 mM citrate buffer, pH 6.0, 0.05% Tween 20, 95°C for 10 minutes), the sections were washed, blocked with 10% goat serum in PBS containing 0.3% Triton and treated with the unconjugated goat Fab anti-mouse IgG (H + L) (0.1 mg/mL in PBS) for 1 hour at RT. Mouse monoclonal antidihydropyridine receptor (α2-1 subunit) antibody (Sigma, D219, 1:100) was applied for 2 or 3 days at 4°C. After extensive washes, the samples were incubated with biotin-conjugated goat antimouse Fab fragment (1:500, Jackson Immuno Research Lab, catalogue number 115-067-003), overnight at 4°C, followed by washes and Streptavidin-AlexaFluor-488 overnight at 4°C (1:500, Invitrogen, catalogue number S32354).
2.3. Confocal image acquisition and analysis
Immunostaining was visualized using an LSM 780 (Carl Zeiss UK Ltd., Cambourne, United Kingdom) confocal microscope. Images were acquired with constant settings in each experiment from at least 3 sections per DRG or 7 per spinal cord from at least 3 mice unless otherwise stated. Only intact tissue sections, unfolded and with uniform staining, were selected for imaging and analysis. For DRG sections, multiple images were acquired with a 63× 1.4NA objective (0.7 μm optical section) covering the whole area of the section containing neurons using the tiling mode with a 5% overlap and stitched with Zen software (Zeiss). For analysis, using ImageJ software (Schneider et al., 2012), in every intact DRG neuron with a visible nucleus, we selected 2 different types of regions of interest (ROI). Using images with temporarily enhanced brightness and contrast, solely to aid visualization of the circumference of even dimly stained cells, first we drew a 10 pixel-wide line (0.9 µm) following the perimeter of the cell from which we recorded the length as an estimation of the size of the cell (small <61 µm, medium 61-94 µm, or large >94 µm) and the mean membrane intensity. Next, we selected an ROI for the area inside the first ROI, excluding the plasma membrane and the nucleus, to record the mean intracellular intensity. Regions of interest outside each section were used as background and deducted from sample measurements.
To determine the proportion of DRG neurons expressing CaV2.2_HA, GFRα1 or both, cells with staining above a threshold (3× the SD of the contralateral side from each animal) were selected using the multipoint tool of ImageJ and merged with the list of ROIs around the perimeter of each section, as described above, to quantify the different populations according to staining and size.
Spinal cord images (at least 7 per animal, unless otherwise stated) at low magnification were acquired using a 20× 0.8 NA objective (5-µm optical section) covering the whole dorsal half of each section also in tiling mode and were stitched with Zen software as for DRG sections. For analysis, using the same software, the mean intensity was recorded from a profile scan of a rectangular ROI of 50 × 300 µm placed across the superficial layers of the medial, central, and lateral part of the ipsilateral and contralateral dorsal horn of each section. The ratio of ipsilateral to contralateral per ROI was calculated to determine the relative level of change in fluorescence and then averaged per each animal. To quantify that data, the mean of the superficial (0-80 μm) or deep (140 and 300 μm) regions was extracted. The HA, CGRP, IB4, and GFRα1 data from different experiments were pooled according to genotype and presented as the mean ± SEM.
For high magnification examination of the spinal cord sections, a 63× 1.4 NA objective in Airyscan mode (0.2-µm optical sections) for HA, CGRP, IB4, and GFRα1 was used in tiling mode to generate multiple images covering the ipsilateral or contralateral dorsal horn of each section; superresolution images then underwent Airyscan processing and were stitched using Zen software. To quantify density of CaV2.2_HA immunoreactivity and that of the other markers, a square ROI (70 × 70 µm) in the deeper layers of the medial region or the superficial layers of the medial, central, and lateral dorsal horn was analysed with FIJI software 6. Each ROI was split into 2 or 3 channels (depending on number of markers used) and thresholded (4 × the SD of the deeper ROI or the combined 3 superficial ROIs from the contralateral side from each section) to create a mask per channel with the all the clusters between 110 and 2800 pixels (0.2-5 μm2) selected using the particle analyzer command. All the clusters were saved as list of ROIs and used in the original image to record their size and mean intensity. The corresponding masks for HA and the other marker/s were merged, and the overlapping clusters (>1%) were extracted using the plugin Binary Feature Extractor from the BioVoxxel Toolbox (http://www.biovoxxel.de), to obtain the associated clusters. Overall, 126 superficial and 42 deeper ROIs were analysed, from a total of 21 sections with 2 or 3 sections per mouse stained for HA, CGRP and IB4, or HA and GFRα1, from 2 CaV2.2_HAKI/KI -α2δ-1+/+ and 2 CaV2.2_HAKI/KI -α2δ-1−/− mice per experiment.
2.4. Statistical analysis
Data were analysed with GraphPad Prism 7 or 9 (GraphPad software, San Diego, CA) or Origin-Pro 2021 (OriginLab Corporation, Northampton, MA). Where error bars are shown they are SEM, “N” refers to number of mice or clusters, unless indicated otherwise. Statistical significance between 2 groups was assessed by Student t test or paired t test, as stated. Repeated-measures 2-way ANOVA followed by Šídák's multiple comparisons test was used to analyse the number of clusters according to cluster size (or density) and the side of the spinal cord from multiple sections. Details of statistical test results are given in Figure legends and in Supplementary Information (available at https://links.lww.com/PAIN/B762).
3.1. Effect of partial sciatic nerve ligation on distribution of CaV2.2_HA and α2δ-1 in dorsal root ganglion neurons in vivo
The use of CaV2.2_HA knockin mice has revealed that CaV2.2_HA is present both intracellularly and on the cell surface of DRG neuronal cell bodies.41 We confirmed this distribution in our current experiments using sections of dorsal root ganglia from 12- to 16-week-old CaV2.2_HAKI/KI mice that have undergone unilateral PSNL (Fig. 1A, i). Immunoreactivity for HA was absent from CaV2.2WT/WT mice (Fig. 1A, ii). In agreement with our previous quantification,41 the level of both cell surface and intracellular CaV2.2_HA was highest in small DRG cell bodies (Fig. 1B, i and ii), and it was very low in large DRG neuronal somata contralateral to the PSNL injury (Fig. 1B, i and ii).
Following PSNL, the CaV2.2_HA signal was significantly increased on the cell surface, ipsilateral to the nerve injury, compared with the contralateral side, particularly in large and medium DRG neurons (by 34.6% and 13.8%, respectively) but not in small DRG neurons (Fig. 1A, arrow, Fig. 1B, i). Furthermore, analysis of intracellular CaV2.2_HA showed that although intracellular CaV2.2_HA density was highest in small DRG neurons (Fig. 1A, 1B, ii), it was increased ipsilateral to PSNL relative to the contralateral side, only in large DRG neurons (by 17.1%, Fig. 1B, ii). There was no increase in CaV2.2_HA in sham-operated animals (Supplementary Fig. 1B compared with A, which also shows the data from individual DRG neurons for these experiments, available at https://links.lww.com/PAIN/B762). Together, these results indicate that PSNL produces an increase of CaV2.2_HA in large and medium DRG neurons ipsilateral to the injury, particularly on their cell surface.
3.2. Genetic ablation of α2δ-1 prevents the increase of CaV2.2_HA in dorsal root ganglion neurons ipsilateral to partial sciatic nerve ligation
It has been found in several studies that α2δ-1 is up-regulated following sensory nerve injury and is important for the development of neuropathic allodynia and mechanical hypersensitivity.4,30,40,44 We therefore examined the effect of α2δ-1 knockout on CaV2.2_HA distribution following PSNL. We observed that the CaV2.2_HA signal at the cell surface of DRG neurons was almost abolished in α2δ-1−/− mice (Fig. 1C), as we had found previously.41 Following PSNL in these mice, there was no appearance of CaV2.2_HA on the cell surface of the DRG neurons (Fig. 1C, ipsilateral) or any increase in intracellular CaV2.2_HA signal ipsilateral to PSNL (Figs. 1C and D). These results highlight the importance of α2δ-1 in the elevation of CaV2.2 in large and medium DRGs that we observed ipsilateral to PSNL.
3.3. CaV2.2_HA is decreased in patches of superficial dorsal horn ipsilateral to partial sciatic nerve ligation, in parallel with loss of IB4 and CGRP
Next, we examined the effect of PSNL on the distribution of CaV2.2_HA, in the dorsal horn of the spinal cord, in parallel with other DRG subtype markers, CGRP and IB4 (Fig. 2). As we previously described,41 there is strong immunoreactivity for CaV2.2_HA in the superficial laminae I and II of the dorsal horn (Fig. 2A, i), and there was no signal in naive WT mice (Fig. 2B). This localization shares topographic distribution with the presynaptic marker CGRP, which is present in peptidergic nonmyelinated primary afferent C-fiber terminals in laminae I and II-outer (Fig. 2A, ii), and also with IB4, which is present in nonpeptidergic primary afferent C-fiber terminals, mainly in lamina II-inner (Fig. 2A, iii).
Following PSNL, we observed a patchy loss of staining for IB4, and to a lesser extent CGRP, ipsilateral to the nerve injury, which was paralleled by a loss of CaV2.2_HA (Fig. 2A, ipsilateral on right side of each section, i-iii, and merged image in iv; closed arrows). This irregular loss of IB4 and CGRP staining has previously been described in many studies, and it is believed to be due to deafferentation of neurotrophin-dependent terminals following nerve injury.2,38,55 For the first time, we can now see that CaV2.2_HA present in those terminals is also reduced. To quantify the observed signals, and the effect of PSNL, we took ROIs perpendicular to the pial surface in medial, central, and lateral regions of the dorsal horn (Fig. 2C) and quantified the fluorescence intensity profiles through the different laminae (Fig. 2D, data shown for the medial ROI), as described previously.41 We then determined the ratio of ROI intensity (ipsilateral/contralateral for each section) with respect to the PSNL injury (Fig. 2E). For both IB4 and CGRP, taking the mean intensity for each animal for the superficial layers (Supplementary Fig. 2B, C, available at https://links.lww.com/PAIN/B762), although there is an obvious patchy reduction in most cases, no significant overall reduction was observed, presumably because of the irregular and variable nature of the loss in this nerve injury model. The CaV2.2_HA signal showed a similar patchy loss of staining in the same areas as found for IB4 and CGRP (Fig. 2A, iv, solid arrow in merged image), suggesting that it may be present in the same terminals, as we previously concluded using dorsal rhizotomy and high-resolution microscopy.41
3.4. Superresolution analysis of distribution of CaV2.2-HA in superficial dorsal horn following partial sciatic nerve ligation: comparison with distribution of IB4 and CGRP
We then analysed superresolution Airyscan images taken from ROIs from the medial, central, and lateral regions of the superficial dorsal horn sections contralateral and ipsilateral to PSNL (ROI for medial region shown in Figs. 3A and B) and examined the size and intensity of CaV2.2_HA, CGRP, and IB4 clusters and their association (mask and data for medial ROI shown in Figs. 3C and D). CaV2.2_HA, together with either IB4 or CGRP, are found in rosette-like glomerular clusters in the superficial dorsal horn (images A and B, below Fig. 3B), as previously observed.41 Quantification of all the clusters from the combined ROIs contralateral to PSNL in superficial dorsal horn shows that 35.2% of IB4 and 34.1% of CGRP clusters were associated with CaV2.2_HA. Similarly, 22.5% and 19.1% of CaV2.2_HA clusters were associated with IB4 and CGRP, respectively (data determined from 15 ROIs, 3 from each side, in 5 sections from 2 mice; 2607 CaV2.2 clusters, 1657 IB4 clusters, and 1442 CGRP clusters). We also examined the effect of PSNL on the area and intensity of CaV2.2_HA, CGRP, and IB4 clusters with respect to their density of distribution. We found that only the density of CaV2.2_HA clusters (number of clusters/ROI) was decreased ipsilateral to nerve injury (Figs. 3C and 3D, i-ii), with no clear change in size profile (Fig. 3D, i) or intensity distribution (Fig. 3D, ii). Similarly, the density of both IB4 clusters (Fig. 3D, iii-iv) and CGRP clusters (Fig. 3D, v-vi) decreased ipsilateral to PSNL, again with no change in size profile (Fig. 3D, iii, v) or intensity distribution (Fig. 3D, iv, vi).
Taken together, these results show that the patchy reduction observed at low magnifications for CaV2.2_HA, IB4, and CGRP in the superficial dorsal horn, ipsilateral to PSNL, corresponds to a decreased number of glomerular clusters rather than a change in their size or intensity in response to the nerve injury.
3.5. Effect of α2δ-1 knockout on the CaV2.2_HA distribution in the superficial dorsal horn
We next examined the effect of genetic ablation of α2δ-1 on the changes in CaV2.2_HA distribution in the dorsal horn following PSNL. We have previously shown that the signal for CaV2.2_HA in the dorsal horn was markedly reduced in CaV2.2_HAKI/KI/α2δ-1−/− mice, particularly in the superficial laminae,41 and this is confirmed here (Fig. 4A ii, compared with i). However, we found here that the patchy reduction in CaV2.2_HA following PSNL (Fig. 4A, i) in the superficial layers of the dorsal horn was still evident in α2δ-1−/− mice (Fig. 4A, ii). We further quantified the PSNL-mediated reduction of CaV2.2_HA in laminae I and II of the dorsal horn, to examine the effect of α2δ-1- knockout (KO), by means of the ROI ipsilateral:contralateral ratio profiles (Fig. 4B, α2δ-1+/+ [red] compared with α2δ-1−/− [black] intensity profiles), which show that the reduction in laminae I-II remains evident in all ROIs of α2δ-1−/− dorsal horn (Fig. 4B, i-iii). This is confirmed from the mean intensity ratios for laminae I-II, which showed a statistically significant reduction in the central and lateral ROIs of α2δ-1−/− mice (Fig. 4C). There were no evident differences between the results in male and female mice for either genotype (Fig. 4C and Supplementary Table 1, available at https://links.lww.com/PAIN/B762).
In parallel, the patchy reduction in IB4 and CGRP signal, ipsilateral to PSNL injury in the superficial ROIs also remained evident in α2δ-1−/− dorsal horn (Supplementary Fig, 2A, vi-vii, arrows, available at https://links.lww.com/PAIN/B762), although it was only statistically significant for IB4 (Supplementary Fig. 2B, C, available at https://links.lww.com/PAIN/B762), indicating that these PSNL-induced reductions were not α2δ-1 dependent. In Airyscan analysis of sections from α2δ-1−/− dorsal horn (Supplementary Fig. 3A-D, available at https://links.lww.com/PAIN/B762), we found the reductions in CaV2.2_HA, IB4, and CGRP cluster density ipsilateral to PSNL were still present in superficial dorsal horn (Supplementary Fig. 3D i-vi, available at https://links.lww.com/PAIN/B762), with no change in size (Supplementary Fig. 3D i, iii, v, available at https://links.lww.com/PAIN/B762) or intensity distribution (Supplementary Fig. 3D ii, iv, vi, available at https://links.lww.com/PAIN/B762).
Together, these results show that CaV2.2_HA is present in the glomerular nerve terminals in the superficial dorsal horn that undergo nerve injury–dependent deafferentation ipsilateral to PSNL, and this partial loss is not α2δ-1 dependent.
3.6. CaV2.2_HA is increased in deep dorsal horn of spinal cord ipsilateral to partial sciatic nerve ligation
Surprisingly, we observed a consistent and marked increase in CaV2.2_HA ipsilateral to PSNL, in the deep layers of the dorsal horn (layers IV-V, Fig. 2A, i; Fig. 4A, i, open arrows), particularly in the medial and central ROIs (Fig. 2E green profile; Fig. 4B, orange profiles, i, ii), which was less evident in the lateral ROI (Fig. 4B, iii). Quantification shows a statistically significant increase in the deep dorsal horn for all 3 regions (Fig. 4D). There were no evident differences between the results in male and female mice for either genotype (Fig. 4D and Supplementary Table 1, available at https://links.lww.com/PAIN/B762). By contrast, there was no parallel increase in CGRP or IB4 in the deep dorsal horn, indicating that the increase was not due to sprouting of these terminals into the deeper layers (Fig. 2A, ii-iv; Fig. 2E).
3.7. Ablation of α2δ-1 almost abolishes the partial sciatic nerve ligation–induced increase in CaV2.2_HA in deep dorsal horn
Ablation of α2δ-1 almost abolished the effect of PSNL on the increase of CaV2.2_HA in the deep dorsal horn (laminae IV-V) (Fig. 4A, ii). The ROI ipsilateral:contralateral ratio profiles show that the PSNL-induced increase in CaV2.2_HA distribution in the ipsilateral medial and central deep dorsal horn was lost or markedly reduced in α2δ-1−/− mice (Fig. 4B, black intensity profiles), in contrast to the increase observed ipsilateral to PSNL in α2δ-1+/+ deep dorsal horn (Fig. 4B, orange intensity profiles). Quantification confirmed that the PSNL-mediated increase is lost in the central and lateral ROIs and much reduced in the medial ROI (Fig. 4D). Together, these results indicate that the presence of α2δ-1 (which is elevated following PSNL, see next section) is key to the increase in CaV2.2_HA in the medial and central deep dorsal horn ipsilateral to PSNL.
3.8. Expression of α2δ-1 is increased ipsilateral to partial sciatic nerve ligation in deep dorsal horn
In parallel experiments, immunostaining for α2δ-1 confirmed its strong expression in the superficial layers of the dorsal horn and its up-regulation ipsilateral to PSNL injury (Fig. 5A, i). The specificity of staining is confirmed by its absence in α2δ-1−/− dorsal horn (Fig. 5A, ii). Interestingly, α2δ-1 up-regulation shows a minimum in layers I and II from the intensity profiles (Fig. 5B, i and ii, blue profiles), in parallel with the patchy reduction in CaV2.2_HA signal, particularly in the medial and central ROIs (Fig. 5B, blue compared with the dotted orange intensity profiles, repeated from Fig. 4B for comparison). In addition to the increase of α2δ-1 in the superficial layers (Fig. 5C), there is also a marked increase of the α2δ-1 signal in layers IV-V of the deep dorsal horn ipsilateral to PSNL (Fig. 5A, open arrow; Fig. 5B, blue intensity profiles), which is evident in the medial and central ROIs (Fig. 5B, i-ii; Fig. 5D).
These data show that α2δ-1 is extensively increased in the ipsilateral dorsal horn following PSNL in mice, as previous studies have demonstrated for other nerve injury models. Here, we also note that there is a differential up-regulation according to the laminae, with a lower increase in the superficial layers and greater relative up-regulation in the deeper layers, particularly in the central and medial regions, in parallel with the changes observed for CaV2.2_HA.
3.9. Elevation of CaV2.2-HA in deep dorsal horn following partial sciatic nerve ligation parallels an increase of GFRα1
Next, we focussed specifically on the medial deep layers of the dorsal horn, in which a strong increase in CaV2.2_HA was observed ipsilateral to PSNL. It has been found previously that the GDNF family receptor α (GFRα1, α2 and α3) proteins that bind GDNF, neurturin, and artemin, respectively, are present in DRG neurons56 and are differentially regulated in DRGs following nerve injury.5,27 Of particular note, GFRα1 expression is increased in large DRG neurons,5 with a corresponding widespread elevation in the dorsal horn,27 whereas the same pattern is not shown for GFRα2 and GFRα3.27 Therefore, we examined whether there was an increase in GFRα1 immunoreactivity ipsilateral to PSNL, in parallel with the increase in CaV2.2_HA (Fig. 6A). We found that GFRα1 was elevated particularly in the medial and central deep dorsal horn (Fig. 6A, i, arrow; Fig. 6B), and furthermore this increase remained present in α2δ-1−/− mice (Fig. 6A, iv; Fig. 6B). In parallel, these experiments confirmed the increase in CaV2.2_HA in the medial deep dorsal horn following PSNL in α2δ-1+/+ mice (Fig. 6A, ii, arrow; Fig. 6C) and the loss of this effect in α2δ-1−/− mice (Fig.6A, v; Fig. 6C).
3.10. Increased co-expression of CaV2.2-HA and GFRα1 in dorsal root ganglion neurons following partial sciatic nerve ligation
Having identified an increased co-expression of GFRα1 with CaV2.2_HA in the deep dorsal horn following PSNL, we then examined their expression in individual DRG neurons. An increase in immunoreactivity for GFRα1 was previously found in large-diameter DRG neurons in a different nerve injury model.5 Here, we found that following PSNL, the proportion of large DRG neurons immunopositive for GFRα1 was increased from 37% on the contralateral side to 57% on the ipsilateral side (Fig. 7A, ii, v; Fig. 7B), whereas there was no significant increase in medium or small DRG neurons (Fig. 7B). Unlike the increase of CaV2.2_HA in large DRG neurons (Fig. 7A, i, iv), the elevated GFRα1 level in DRG neurons following PSNL was not dependent on the presence of α2δ-1 (Supplementary Fig. 4, available at https://links.lww.com/PAIN/B762).
We further found that the proportion of DRG neurons that coexpressed CaV2.2_HA and GFRα1 was significantly increased ipsilateral to PSNL only for large DRG neurons, from 4% on the contralateral side to 19% on the ipsilateral side (Fig. 7A, iii, vi; Fig. 7C).
3.11. Superresolution analysis of distribution of CaV2.2-HA in deep dorsal horn following partial sciatic nerve ligation: comparison with distribution of GFRα1
Because there was a 4.75-fold increase in the coexpression of CaV2.2_HA and GFRα1 in individual large DRG neurons, we next examined whether there was also an increase in the coexpression in their terminal field in the deep dorsal horn. Therefore, we analyzed the association between GFRα1 and CaV2.2_HA in Airyscan images taken from dorsal horn sections contralateral and ipsilateral to PSNL (Fig. 8A). In the medial deep dorsal horn, we found a strong association between CaV2.2_HA and GFRα1 ipsilateral to PSNL (Fig. 8B, ii compared with i). This was clearly evident in the enlarged clusters (Fig. 8C ii compared with i). Analysis showed that 48.8% of CaV2.2_HA clusters were associated with GFRα1, and 28.0% of GFRα1 clusters were associated with CaV2.2_HA ipsilateral to PSNL (Fig. 8C, ii), whereas there was very little observed association on the contralateral side (Fig. 8C, i). Expanding on the low-resolution results in Figure 6, we find that the density of CaV2.2_HA clusters was increased in the deep dorsal horn, ipsilateral to PSNL (Fig. 8D, i and ii), with no change in size profile (Fig. 8D, i) or intensity distribution (Fig. 8D, ii). Further analysis of all combined data for CaV2.2_HA clusters in deep dorsal horn confirmed these conclusions (Supplementary Fig. 5, available at https://links.lww.com/PAIN/B762). In parallel, there was an increase in density of GFRα1 clusters (Fig. 8D, iii-iv), again with no marked change in size profile (Fig. 8D, iii) or intensity distribution (Fig. 8D, iv). We observed no evidence for increased expression of CaV2.2_HA within cell bodies in the dorsal horn following nerve injury.
By contrast, in the superficial dorsal horn, we found that there was little association between GFRα1 and CaV2.2_HA, and this was not increased following PSNL (detailed in Supplementary Fig. 6A-C, available at https://links.lww.com/PAIN/B762), in agreement with their lack of coexpression in small DRG neuronal cell bodies (Fig. 7). Ipsilateral to PSNL, there was a marked reduction of CaV2.2_HA clusters, in agreement with data in Figure 3. By contrast, the density of GFRα1 clusters was increased in the superficial dorsal horn, ipsilateral to PSNL (Supplementary Fig. 6C, available at https://links.lww.com/PAIN/B762).
In parallel analysis of sections from α2δ-1−/− dorsal horn (Supplementary Fig. 7A-E, available at https://links.lww.com/PAIN/B762), the increase in GFRα1 cluster density remained present in the deep dorsal horn (Supplementary Fig. 7E, iii-iv, available at https://links.lww.com/PAIN/B762), in agreement with Figure 6. By contrast, in the same ROIs, there was no increase in CaV2.2_HA cluster density in the medial deep dorsal horn, ipsilateral to PSNL (Supplementary Fig. 7E, i-ii, available at https://links.lww.com/PAIN/B762).
Together, these results show a close association between the increase in both CaV2.2_HA and GFRα1 in the medial deep dorsal horn ipsilateral to PSNL. However, because the increase in CaV2.2_HA (but not GFRα1) is α2δ-1 dependent, it likely involves an increase in CaV2.2_HA trafficking into terminal fields in which GFRα1 is also elevated.
CaV2.2 channels play an essential presynaptic role in neurotransmitter release in primary afferent terminals8,11 and are a therapeutic target in treatment of neuropathic pain. Indeed, N-type CaV channel blockers alleviate chronic pain in both animal models and humans.37,43,51 However, until now, it has not been possible to examine accurately the distribution of endogenous N-type channels, and the effect of an animal model of chronic pain, on tissue expression and distribution of the relevant endogenous N-type channels.
In this study, we have examined the effect of a chronic neuropathic pain model (PSNL) on the distribution of native N-type CaV2.2 channels in DRGs and dorsal horn of the spinal cord, using a knockin mouse, in which CaV2.2 contains an HA epitope tag to aid its identification. This tag does not affect channel function.9 In our previous study using these mice, we showed that CaV2.2_HA is strongly expressed on the cell surface of DRG neurons, particularly of the small CGRP-positive nociceptors, and in parallel, there is strong expression in the dorsal horn of the spinal cord, mainly in laminae I and II, corresponding to the primary afferent C-nociceptor glomerular terminals.41
CaV2.2 channels have been estimated to comprise 20 to 50% of the total calcium current in DRG somata, depending on species, developmental stage, culture conditions, and DRG neuron subtype.15,39,44,48,49 For example, Murali et al.39 found 40% N-type calcium current in small DRGs and 20% in larger DRGs. Despite the proviso that DRG neurons placed in culture may undergo rapid changes in ion channel cell surface expression,19 nevertheless, we found greater expression of CaV2.2_HA in small and medium, relative to large DRG neurons, both here and in our previous study.41 Furthermore, it has been found that primary afferent-evoked synaptic currents in laminae I and II, mainly originating from small peptidergic and nonpeptidergic DRG neurons, are 74% dependent on N-type channels,3 highlighting the preferential synaptic localization of these channels in vivo.
4.1. Effect of partial sciatic nerve ligation on distribution of CaV2.2_HA in dorsal root ganglion neuron subtypes
Despite the importance of N-type calcium channels in neuropathic pain transmission,11,52 there are few studies examining the effect of different types of neuropathic injury on CaV2.2 calcium channel levels or distribution in DRG cell bodies or primary afferent terminals. In this study in mice, we found that PSNL induced an increase of DRG CaV2.2_HA expression, particularly on the cell surface, in medium and large DRG cell bodies, but not in small DRGs, in which expression is already high. Turning to the spinal cord, an anticipated finding was that there is a patchy loss of staining for CaV2.2_HA in superficial laminae ipsilateral to the PSNL injury. This was paralleled by patchy loss of CGRP and IB4, which has been extensively documented previously by others,2,55 and attributed to resorption of neurotrophin-dependent terminals.38
4.2. Increase of CaV2.2_HA in deep medial dorsal horn following partial sciatic nerve ligation and colocalization with GFRα1
An important and unexpected finding of this study is the clear increase in CaV2.2_HA in the deep medial and central layers IV-V of the dorsal horn, ipsilateral to nerve injury. Here, the elevation of CaV2.2_HA showed a very similar, although less extensive, pattern to the increase of the GDNF receptor GFRα1, following sensory nerve injury (Fig. 6). There was an increase in the association of CaV2.2_HA and GFRα1 clusters in the ipsilateral deep dorsal horn (Fig. 8), and an increased colocalization of up-regulated CaV2.2_HA and GFRα1 in large DRG neurons (Fig. 7). Our findings with respect to GFRα1 are similar to those found in a previous study, in which sciatic nerve injury caused a widespread increase in nerve fiber labelling for GFRα1 immunoreactivity in the dorsal horn, particularly in the deeper medial third to half of this region.27
Glial cell-line-derived neurotrophic factor family ligands (GDNF, neurturin, artemin, and persephin) interact with the GFRα receptor family (1-4, respectively) together with their coreceptor, the tyrosine kinase, Ret (REarranged during Transfection). Ret is a transmembrane protein, whereas the GFRα receptors are glycosyl phosphatidyl–inositol (GPI) anchored, as is α2δ-1.13 From single-cell RNA-sequencing studies in adult mouse DRG neurons,53 3 clusters of large-diameter LTMRs were identified; of which, 2 were Ret positive. Ret-positive A-LTMRs have large neuronal somata, and their central endings are in deep layers of the dorsal horn.10,32 The A-LTMR DRGs give rise to the major tactile receptors in skin,32 and under pathological conditions such as neuropathic injury, A-LTMRs can also mediate the sensation of pain induced by touch, termed mechanical allodynia.16,33 The increase in Ret following sensory nerve injury has been found previously to be mainly in primary afferents.26
Together, these results indicate that the increase in CaV2.2_HA expression and cell surface trafficking in cell bodies of medium and large DRG cell bodies ipsilateral to PSNL is paralleled by a similar increase in the presumed terminals of these DRGs in the deep dorsal horn. These medium and large DRG neurons and their central projections are likely to represent Aβ LTMRs that contain GFRα1,5 which is also up-regulated in these neurons following sensory nerve injury.27
4.3. Importance of α2δ-1 in redistribution of CaV2.2_HA
The α2δ-1 auxiliary subunit associated with CaV1 and CaV2 calcium channels has been shown to be important for calcium channel trafficking in expression systems9 and in vivo.41 In both rats and mice, α2δ-1 protein is expressed in all DRG neurons, with highest expression in the somata of small DRG neurons.4,41 α2δ-1 plays a major role in primary afferent pain pathways and is up-regulated in all injured DRGs following neuropathic injury.4,17,31,35,40 Furthermore, genetic ablation of the Cacna2d1 gene, encoding α2δ-1, caused a marked delay in the development of neuropathic mechanical hypersensitivity,44 and overexpression of α2δ-1 mimics features of neuropathic injury.31 Our previous results using mice in which α2δ-1 is globally ablated, crossed with CaV2.2_HAKI/KI mice, have emphasised an essential role of α2δ-1 in trafficking CaV2.2_HA, both to the plasma membrane of DRG neuron cell bodies and to their primary afferent terminals in the dorsal horn.41 For this reason, we compared the effect of PSNL on CaV2.2_HA distribution, in both α2δ-1+/+ and α2δ-1−/− mice. We found that α2δ-1 knockout prevented the PSNL-induced increase in CaV2.2_HA in medium and large DRG cell bodies, ipsilateral to the ligation (Figs. 1B and C), and it also prevented or markedly reduced the PSNL-induced increase in CaV2.2_HA in the medial and central deep dorsal horn (Fig. 4D). By contrast, the reduction of CaV2.2_HA, CGRP, and IB4 in the superficial laminae, ipsilateral to PSNL, was not affected by α2δ-1 knockout. Therefore, the resorption of neurotrophin-dependent terminals that underlies this patchy loss of glomerular synapses2,38,55 is a neuroanatomical consequence of the nerve injury that is independent of α2δ-1.
Furthermore, the increase in GFRα1 in DRGs and in deep dorsal horn ipsilateral to PSNL was not reduced by α2δ-1 knockout. This differential lack of effect of α2δ-1 knockout on the increase iGFRα1, relative to its effect on the CaV2.2_HA increase ipsilateral to PSNL in large DRG neurons and in the deep dorsal horn, strongly suggests that the increase in CaV2.2_HA in the deep dorsal horn is due to an increase in α2δ-1–mediated trafficking of the channel complex to the cell surface and into terminal zones, with a consequent reduction in its intracellular degradation (for review see Ref. 18). In agreement with this, no increase in Cacna1b mRNA has been reported following nerve injury in multiple studies, which have reported a consistent increase in expression of Cacna2d1 mRNA.14,35,54,58,59
In contrast to these results using a highly specific anti-HA antibody that shows no signal in WT tissue, all previous studies examining the distribution of CaV2.2 in both DRGs and dorsal horn, following several different nerve injury models, have used antipeptide antibodies against intracellular epitopes. These studies have produced varying results, using both immunohistochemistry and western blotting.12,29,60,61 In 2 studies, an elevation of CaV2.2 immunoreactivity was observed in the dorsal horn superficial layers,12,61 and none revealed any elevation of CaV2.2 in deeper dorsal horn. In general, the use of antipeptide antibodies, which are often of relatively low affinity and have not been previously validated in knockout mouse tissue, may produce false-positive or false-negative results, especially when directed against low-abundance proteins such as ion channels.25
In conclusion, the use of CaV2.2_HA knockin mice has provided novel insights into alterations in CaV2.2 distribution and trafficking following PSNL. Notably, we find that CaV2.2_HA is up-regulated, ipsilateral to PSNL, particularly in large GFRα1-positive DRG neurons, and in parallel, there is an increase of CaV2.2_HA in the ipsilateral medial and central deep dorsal horn, where GFRα1 is also up-regulated. The up-regulation of CaV2.2_HA is α2δ-1 dependent, whereas the increase in GFRα1 is not, indicating that there is an increase in CaV2.2_HA trafficking into these mechanoreceptor terminals, which is likely to mediate increased neurotransmission.
Conflict of interest statement
The authors have no conflict of interest to declare.
Appendix A. Supplemental digital content
Supplemental digital content associated with this article can be found online at https://links.lww.com/PAIN/B762.
Supplemental video content
A video abstract associated with this article can be found at https://links.lww.com/PAIN/B763.
This work was supported by a Wellcome Trust Investigator award to A. C. Dolphin (098360/Z/12/Z).
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