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. Author manuscript; available in PMC: 2022 Sep 1.
Published in final edited form as: Neuroscience. 2021 Jul 22;471:20–31. doi: 10.1016/j.neuroscience.2021.07.013

L5-6 spinal nerve ligation-induced neuropathy changes the location and function of Ca2+ channels and Cdk5 and affects the compound action potential in adjacent intact L4 afferent fibers

Kimberly Gomez 1,a, Alberto Vargas-Parada 1, Paz Duran 2,a, Alejandro Sandoval 3, Rodolfo Delgado-Lezama 1, Rajesh Khanna 4, Ricardo Felix 2
PMCID: PMC8384716  NIHMSID: NIHMS1727914  PMID: 34303780

Abstract

Voltage-gated Ca2+ (CaV) channels regulate multiple cell processes, including neurotransmitter release, and have been associated with several pathological conditions, such as neuropathic pain. Cdk5, a neuron-specific kinase, may phosphorylate CaV channels, altering their functional expression. During peripheral nerve injury, upregulation of CaV channels and Cdk5 in the dorsal root ganglia (DRG) and the spinal cord, has been correlated with allodynia. We recently reported an increase in the amplitude of the C component of the compound action potential (cAP) of afferent fibers in animals with allodynia induced by L5-6 spinal nerve ligation (SNL), recorded in the corresponding dorsal roots. This was related to an increase in T-type (CaV3.2) channels generated by Cdk5-mediated phosphorylation. Here, we show that CaV channel functional expression is also altered in the L4 adjacent intact afferent fibers in rats with allodynia induced by L5-6 SNL. Western blot analysis showed that both Cdk5 and CaV3.2 total levels are not increased in the DRG L3-4, but their subcellular distribution changes by concentrating on the neuronal soma. Likewise, the Cdk5 inhibitor olomoucine affected the rapid and the slow C components of the cAP recorded in the dorsal roots. Patch-clamp recordings revealed an increase in T- and N-type currents recorded in the soma of acute isolated L3-4 sensory neurons after L5-6 SNL, which was prevented by olomoucine. These findings suggest changes in CaV channels location and function in L3-4 afferent fibers associated with Cdk5-mediated phosphorylation after L5-6 SNL, which may contribute to nerve injury-induced allodynia.

Keywords: calcium channels, Cdk5, olomoucine, spinal nerve ligation, allodynia, neuropathic pain

INTRODUCTION

Neuropathic pain occurs as a result of injury or disease of the nerves and represents a global public health challenge. The expression of diverse proteins, including voltage-gated calcium (CaV) channels is altered in neuropathic pain. These channels are expressed in sensory neurons where they play essential roles in excitability and neurotransmitter release. CaV channels mediate calcium influx in response to membrane depolarization allowing ions entering the cell, coupling the electrical signals in the cell surface to intracellular processes. They have been classified into low voltage-activated (LVA or T-type) and high voltage-activated (HVA) channels, a class that includes the L-, N-, P/Q- and R-types, which can be distinguished using pharmacological approaches (Catterall et al., 2005; Zamponi et al., 2015). The CaVα1 subunit protein contains the ion-conducting pore, the gating apparatus, and is responsible for voltage sensing and binding of channel-specific drugs and toxins.

Spinal nerve injury in animal models induces mechanical allodynia, a characteristic clinical sign of neuropathic pain (Campbell and Meyer, 2006). Interestingly, in allodynia induced by ligation of spinal nerves L5 and L6 (L5-6 SNL) (Kim and Chung, 1992), the lesion can affect both injured and intact axons. Although initial studies focused mainly on the lesion’s local sites and the affected sensory neurons, evidence suggests molecular and functional changes in adjacent SN, which mostly contain intact axons (Liu et al., 2019). Indeed, the expression of diverse nociception-related proteins is altered in neurons in L4 dorsal root ganglia (DRG) after L5-6 SNL, associated with increased excitability of the uninjured nerves, which is thought to also participate in the development and maintenance of neuropathic pain (Campbell and Meyer, 2006; Liu et al., 2019; Ma et al., 2003; Yang et al., 2018). For this reason, there is a growing interest in studying the possible changes in the functional expression of ion channels in general, and CaV channels in particular, in intact sensory neurons adjacent to injured SN.

In sensory neurons of animals subjected to L5-6 SNL, both T-type and HVA CaV channels contribute to the onset and maintenance of mechanical allodynia. It is known that the expression of the CaVα2δ−1 auxiliary subunit of HVA channels, is altered following damage to the sensory nerves (Bauer et al., 2009; Dolphin, 2018). The main action of CaVα2δ−1 is to favor the trafficking of the channels towards the plasma membrane (Dolphin, 2018; Felix et al., 2013). Likewise, T-type (CaV3.2) channels also participate in nociceptive signaling (Altier and Zamponi, 2004; Snutch and Zamponi, 2018; Todorovic and Jevtovic-Todorovic 2013; Waxman and Zamponi, 2014). Interestingly, an upregulation of these channels has been shown in the L4 DRG neurons of rats with L5 SNL-induced allodynia (Liu et al., 2019). Although the precise molecular mechanism is yet to be defined, it might be related to transcriptional and post-translational modifications. The increase in CaV3.2 expression in the early phase of L5 SNL may involve the transcription factor Egr-1, while in the late stages the upregulation of the deubiquitinating enzyme USP5, that protects the channels from proteasomal degradation, may contribute to CaV3.2 overexpression in L4 DRG (Tomita et al., 2020). Interestingly, knockdown of USP5 augments CaV3.2 ubiquitination reducing protein levels and decreasing T-currents in DRG neurons. Also, uncoupling USP5 from CaV3.2 channels by Tat peptides induces analgesia in neuropathic pain (García-Cabello et al., 2014).

More recently we reported that the exacerbated expression of Cdk5 in L5-6 SNL increased the functional expression and changes the subcellular localization of CaV3.2 channels in sensory neurons and that the inhibition of the kinase altered the compound action potential (cAP) recorded in the L5 dorsal roots (Gomez et al., 2020a). Here, we show that the augmented expression of Cdk5 in rats with mechanical allodynia induced by L5-6 SNL changes the cell location and increases the functional expression of T- (CaV3.2) and N-type channels, which results in alterations in the cAP of adjacent intact L4 fibers. This finding is relevant for our understanding of the molecular mechanisms underlying neuropathic pain.

EXPERIMENTAL PROCEDURES

Western blot analysis

Total protein extracts were obtained from animals that were sacrificed by decapitation, and the L3-4 spinal nerves, DRG and dorsal roots were extracted and kept in lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 1% SDS, 0.5% sodium deoxycholate, 0.1% Triton X100, 1 mM PMSF and complete proteinase inhibitor). Tissues were homogenized at 4°C, maintained on ice and then centrifuged at 14,000 rpm (10 min at −4°C). The supernatants were collected for protein quantification using the Bradford assay. Denaturing electrophoresis in polyacrylamide gels (10%) with sodium dodecyl sulfate (SDS-PAGE) employing 60 μg of proteins was then performed. Samples were heated (95°C for 5 min) and loaded onto gels, transferred to polyvinylifluoride membranes and blocked with 5% skimmed milk in PBS-0.05% Tween 20 (PBST) [pH 7.4] for ~2 h at room temperature (RT). Membranes were washed with PBST and incubated with the mouse anti-CaV3.2 antibody (1:500 dilution; H-300: sc-25691, Santa Cruz Biotechnology), the anti-Cdk5 antibody (1:3000; C-8: sc-173, Santa Cruz Biotechnology), the anti-p35 antibody (1:1000; sc-820, Santa Cruz Biotechnology) as well as the anti-β actin antibody (1:10000, GeneTex), and incubated overnight at 4°C. Secondary horseradish peroxidase-conjugated antibodies (anti-rabbit, 1:10000 or anti-mouse, 1:5000) were used in concurrence with the chemiluminescence detection system (Amersham Pharmacia GE Healthcare). Signals were quantified as the optical density index using the Image Studio 5.2 software (LI-COR Biosciences). The optical density index of the bands was normalized to that of β-actin. The densitometric quantification was performed using the ImageJ software (NIH).

Induction and evaluation of mechanical allodynia

Behavioral tests were carried out using female Wistar rats (~150 g) maintained with free access to food and water in a controlled environment (22°C and a 12 h light-dark cycle). Experiments were performed following the guide on ethical guidelines for experimental pain investigations in conscious animals (Zimmermann, 1983). Also, our studies were approved by the Institutional Animal Care and Use Committee (Cinvestav-IPN) and complied with the Official Mexican Standard (NOM-062-ZOO-1999), and were conducted per the regulations of the University of Arizona’s College of Medicine Institutional Animal Care and Use Committee, and the NIH-published Guide for Care and Use of Laboratory Animals, as well as the ethical regulations sanctioned by the International Association for the Study of Pain. Animals were anesthetized, and the left spinal nerves L5-6 were exposed and ligated in a region distal to the DRG (Kim and Chung, 1992). In the sham-operated animals, the left spinal L5-6 nerves were exposed but not ligated. To evaluate mechanical allodynia, von Frey filaments (Stoelting) were used to determine the threshold of removal of the hind limb (50% paw withdrawal threshold) using the up-and-down method (Chaplan et al., 1994). On days 1, 3, 7, and 14 after surgery, animals are evaluated to corroborate the presence of mechanical allodynia as described elsewhere (Gomez et al., 2020a).

Recording of compound action potentials (cAPs)

The L4 spinal nerves-dorsal root ganglion-dorsal root (SN-DRG-DR) preparations (of about 1 cm long) were obtained from control (sham) and SNL rats 14 d after surgery, as described previously (Gomez et al., 2020a). Animals were subjected to a laminectomy that allowed the DRG’s dissection in continuity with the spinal nerves and the dorsal roots. Tissues were collected in Hartmann solution containing (in mM): 117 NaCl, 3.6 KCl, 2.5 NaHCO2, 1.2 MgCl2, 25 glucose, 1 CaCl2 bubbled with a gas mixture containing 95% O2 and 5% CO2. The recordings were obtained at RT (~22°C) by placing the SN-DRG-DR preparation into a recording chamber. The distal end of the spinal nerve was suctioned with a glass electrode connected to an electrical stimulator of rectangular pulses, while the proximal end of the dorsal root was suctioned with a glass electrode connected to a DC amplifier. cAPs were evoked by stimulation with 300 μs current pulses and recorded at a gain x 1000 with DC-3 kHz filters. One hundred ms traces were digitized with a Digidata interface (Molecular Devices) at 50 kHz. The cAPs threshold (T) was estimated as the necessary current to evoke a cAP with an occurrence of 50%. Once the cAP recordings were stable, preparations were stimulated at different intensities (3 and 50 x threshold, T), to evoke the cAPs from distinct primary afferent fibers. For each stimulation intensity, 30 traces were recorded at a frequency of 0.2 Hz in the absence or the presence of olomoucine and/or mibefradil (100 and 0.5 μM, respectively).

Recording of Ca2+ currents

DRG neurons were acutely dissociated, as described elsewhere (Choe et al., 2011). Briefly, L3 and L4 DRG were obtained from sham and SNL animals 14 d after surgery. Cells were collected and enzymatically digested in a solution containing neutral protease/collagenase type I and incubated for 60 min at 37°C under agitation. Dissociated neurons were centrifuged and washed with DMEM containing antibiotics, neural growth factor, and fetal bovine serum (Hyclone) before plating onto poly-D-lysine- and laminin-coated glass coverslips. Thirty min after, DMSO and olomoucine (Sigma Aldrich) at a final concentration of 0.1% and 100 μM, respectively, were added to the culture medium for 48 h. Ca2+ currents (ICa) were recorded according to the whole-cell patch-clamp technique with a HEKA EPC-10 USB amplifier and data were acquired and analyzed with Patchmaster and Fitmaster software (HEKA). All experiments were performed at RT (~22°C) using 2–4 MΩ resistance recording pipettes. The current density was calculated as peak ICa divided by membrane capacitance (Cm).

Electrophysiological recordings were performed on sensory neurons that are characterized by their small diameter. Consistent with this, the cells subjected to recording presented small membrane capacitance values, between 10–35 pF, corresponding to a diameter of approximately 17–33 μm. On the other hand, it is known that sensory neurons, in addition to the low threshold component (T-type channels), express two high activation threshold current main components, one sensitive to dihydropyridines and the other sensitive to ω-conotoxin (Choe et al., 2011; Gandini et al., 2014; Gandini et al., 2015). Therefore, to isolate and the HVA component of the current TTA-P2 (1 μM) was added to the bath recording solution, and to isolate specifically the N-type component, the contribution of other HVA and the T-channel subtypes were blocked using nifedipine (10 μM, L-type); ω-agatoxin GIVA (200 nM, P/Q-type); SNX-482 (200 nM, R-type); and TTA-P2 (1 μM, T-type). On the other hand, to isolate the contribution of the T-type channel currents, all HVA components of the total current were blocked using nifedipine (10 μM); ω-agatoxin GIVA (200 nM); ω-agatoxin GIVA (200 nM); and SNX-482 (200 nM).

The bath solution (~310 mOsm) for the recording of the HVA currnets consisted of (in mM): 110 N-methyl-D-glucamine, 10 BaCl2, 10 TEA-Cl, 10 HEPES, 10 D-glucose [pH 7.4]. The intracellular recording solution (~310 mOsm) consisted of (in mM): 150 CsCl2, 10 HEPES, 5 Mg-ATP, 5 BAPTA [pH 7.2]. Activation of N-current was measured using a Vh of −60 mV with 200-ms voltage steps applied at 5-s intervals (in 10 mV increments) from −70 to 60 mV. The current density was calculated as the peak ICa divided by Cm. Steady-state inactivation was determined by applying a 1500-ms conditioning prepulse (−100 to 30 mV in 10 mV increments), after which the voltage was stepped to 10 mV for 200-ms; a 15-s interval separated each voltage step.

The bath solution (~310 mOsm) for the recording of the T-type currents consisted of the following (in mM): 2 CaCl2, 152 TEA-Cl, and 10 HEPES [pH 7.4]. The intracellular recording solution (~310 mOsm) consisted of (in mM) 135 TEA-Cl, 10 EGTA, 40 HEPES, and 2 MgCl2 [pH 7.2]. Channel current activation was measured from a holding potential (Vh) of −90 mV by applying 50-ms voltage steps (5 mV increments) from −70 to 60 mV. Channel current inactivation was determined by applying a 1500-ms conditioning prepulse (−110 to 20 mV in 10 mV increments). The voltage was stepped to −30 mV for 20 ms; a 40-ms interval with a Vh of −90 mV separating each voltage step. To isolate the N-type currents, the contribution of other HVA and the T- channel subtypes were blocked using nifedipine (10 μM, L-type); ω-agatoxin GIVA (200 nM, P/Q-type); SNX-482 (200 nM, R-type); and TTA-P2 (1 μM, T-type).

Statistical Analysis

Data represent mean ± S.E.M. The statistical significance of differences between two sets of data was established according to the Student’s t-test. Analysis of variance (ANOVA) was also performed for multiple comparisons. Bonferroni’s post hoc test was used for pairwise comparisons. A p-value < 0.05 was considered statistically significant. Experiments using antibodies were performed at least in triplicate. Asterisks indicate statistical significance.

RESULTS

Expression and localization of CaV3.2 channels and Cdk5 in L3-4 DRG following L5-6 SNL

T-type CaV3.2 channels and Cdk5 seem to be relevant in developing and maintaining mechanical allodynia in the L5-6 SNL paradigm (Gomez et al., 2020a); therefore, here we investigated the functional expression of these proteins in the adjacent intact L3-4 DRG (He et al., 2016; Weiss et al., 2013). Initially, by western blot analysis in protein extracts from animals in the sham-operated and the L5-6 SNL conditions, a band of ~250 kDa was observed in the DRG, which corresponds to the channel protein’s expected molecular weight (Fig. 1A). In these experiments, the L5-6 SNL did not cause aparent changes in the expression of the channels 14 d after nerve ligation in total cell lysates from a preparation that included the spinal nerve (SN), the DRG, and the dorsal root (DR). However, when the lysates were obtained from the nerves, the ganglia or the dorsal roots separately, a differential distribution of the channel proteins was observed in L3-4 (Fig. 1B). In particular, a small non-significant decrease (~15%) in the CaV3.2 expression was observed in the spinal nerves of animals subjected to SNL in comparison to sham-operated controls. In contrast, a significant increase (~2-fold) in channel expression was observed in the DRG. Likewise, a small and non-significant increase (~20%) in the expression of the channel protein was also observed in the DR. In the case of Cdk5, a ~33 kDa band was observed in total cell lysates from the preparation that included the SN, the DRG, and the DR in both sham-operated and animals subjected to the nerve ligation. Figure 2A shows that L5-6 SNL did not affect the total expression of the kinase 14 d after nerve injury compared to what was observed in the controls (Fig. 2A). However, a significant increase in the Cdk5 expression was observed in the SN and the DRG of animals subjected to nerve ligation in comparison to sham-operated controls. Interestingly, this effect was accompanied by a significant decrease (of ~30%) in Cdk5 expression exclusively in the dorsal roots of rats subjected to L5-6 SNL (Fig. 2B).

Fig. 1.

Fig. 1.

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Effect of L5-6 spinal nerve ligation (SNL) on the relative expression and localization of CaV3.2. A) The upper panels show the comparative western blot analysis of CaV3.2 protein expression in sham-operated animals and rats with allodynia induced by the ligation of the spinal nerves L5-6. Channel proteins were detected in cell lysates from a preparation that includes the spinal nerves, the DRG, and the dorsal roots (region shown in the red rectangle) 14 d after SNL using specific antibodies to CaV3.2 channels. The lower panels show the corresponding protein band signal intensities of channel proteins normalized to β-actin levels. The results are representative of three independent experiments. B) Comparative western blot analysis of CaV3.2 protein expression in sham-operated animals and rats with mechanical allodynia induced by the ligation of the spinal nerves L5-6, in lysates from the spinal nerve (SN), the dorsal root ganglion (DRG) and the dorsal root (DR) (upper panel). Channel proteins were detected in lysates from L3-4 (B) 14 d after SNL using specific antibodies to CaV3.2 channels. The lower panel shows the comparison of protein band signal intensities of channel proteins normalized to β-actin. The bar charts summarize the results of six separate experiments, and the asterisks denote significant differences from the control condition. Two-way ANOVA was performed for multiple comparisons followed by Bonferroni’s tests for pairwise comparisons.

Fig. 2.

Fig. 2.

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Effect of the SNL on the relative expression and localization of Cdk5. A) The upper panel show the comparative western blot analysis of Cdk5 expression in sham-operated and animals with allodynia induced by the ligation of the spinal nerves L5-6. Channel proteins were detected in cell lysates from a preparation that includes the spinal nerves, the DRGs, and the dorsal roots 14 d after SNL using specific antibodies to the kinase. β-actin was used as a loading control. B) Comparative western blot analysis of Cdk5 expression in sham-operated and animals subjected to L5-6 SNL, in lysates from the spinal nerve (SN), the dorsal root ganglion (DRG), and the dorsal root (DR) (upper panel). Cdk5 expression was detected in lysates from L3-4, 14 d after SNL using specific antibodies. The lower panel shows the comparison of protein band signal intensities normalized to β-actin. The bar chart summarizes the results of six separate experiments, and the asterisks denote significant differences from the control condition. Two-way ANOVA was performed for multiple comparisons followed by Bonferroni’s tests for pairwise comparisons.

Next, we analyzed the possible effects of L5-6 SNL on the expression of p35, the physiological activator of Cdk5. As shown in Fig. 3A, Western blot analysis showed the expression of a 35 kDa protein in L3-4 spinal nerve lysates, both in sham-operated animals and in animals subjected to nerve ligation. The results of this analysis showed that L5-6 SNL did not affect the expression of the kinase activator 14 d after nerve injury compared to that observed in controls, in total lysates. However, as in the case of Cdk5, the data showed that the functional expression of p35 is differentially affected when the anatomical regions were analyzed separately. Accordingly, in animals subjected to L5-6 SNL, a significant increase in the relative expression of p35 was observed in SN and the DRG compared to sham-operated controls (Fig. 3B). Likewise, a decrease (of ~50%) in the expression of p35 was observed in the DR of rats subjected to L5-6 SNL (Fig. 3B). Taken together, these data support the idea that Cdk5/p35-mediated phosphorylation of CaV3.2 could favor channel membrane location and help guide channels to defined subcellular compartments (Gomez et al., 2020a).

Fig. 3.

Fig. 3.

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Effect of SNL on the relative expression and localization of p35. A) The upper panels show the comparative western blot analysis of p35 expression in sham-operated animals and rats subjected to L5-6 SNL in total lysates. Proteins were detected in lysates from L4, 14 d after SNL using specific antibodies. The lower panels show the corresponding protein band signal intensities normalized to β-actin levels to obtain the kinase activator’s relative expression. B) Comparative western blot analysis of p35 expression in sham-operated and animals subjected to L5-6 SNL, in lysates from the spinal nerve (SN), the dorsal root ganglion (DRG), and the dorsal root (DR) (upper panel). The Cdk5 activator expression was detected in lysates from L3-4, 14 d after SNL using specific antibodies. The lower panel shows the comparison of protein band signal intensities normalized to β-actin. The bar chart summarizes the results of four separate experiments, and the asterisks denote significant differences from the control condition. Two-way ANOVA was performed for multiple comparisons followed by Bonferroni’s tests for pairwise comparisons.

Effect of SNL on the cAPs recorded in primary afferent fibers

Given that the expression and localization of both CaV3.2 channels and Cdk5 are altered during the development of mechanical allodynia in SNL animals (Gomez et al., 2020a), we next investigated whether the possible increase in the functional expression of the CaV3.2 channel, which occurs in parallel with the increase in the expression of Cdk5, might induce changes in the cAPs of primary afferent fibers recorded in the L4 dorsal root of animals subjected to L5-6 SNL. The presence of allodynia in the animal model used in our experiments has been validated elsewhere (Gomez et al., 2020a). Hence, we initially studied the T-type channels’ potential role in the sensory nerve fibers’ excitability. To this end, the cAPs evoked by stimulation of the L4 SN at 50 x T were recorded in the intact adjacent L4 DR in animals subjected to L5-6 SNL (Fig. 4A). The results showed that the cAPs recorded in the dorsal roots of sham-operated animals, as well as animals with L5-6 SNL, displayed a smaller area under the curve (AUC) after the bath application of the Cdk5 inhibitor olomoucine (a reduction of about 30%) in comparison to untreated (control) SN-DRG-DR segments (Fig. 4A; right panel). The latencies of the cAPs were not affected significantly by the inhibitor treatment.

Fig. 4.

Fig. 4.

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Contribution of T-type channels to the C component of the cAP. A) The upper panel shows a schematic diagram of the experimental setup. Extracellular recordings of cAPs were performed using 300 ms duration stimulation pulses at 50 x T applied at a frequency of 0.2 Hz and recorded from a preparation that included the spinal nerve, the dorsal root ganglion, and the dorsal root (L4 SN-DRG-DR) in sham and SNL animals. The lower panels show representative traces of cAPs recorded in C-type fibers from sham-operated and animals subjected to L5-6 SNL after the application of the Cdk5 antagonist olomoucine (Olo), as indicated. The bar chart on the right shows the comparison of the normalized area under the C component of cAPs (AUC) in the untreated condition and after the application of Olo. B) Representative traces of cAPs recorded in C-type fibers from animals subjected to L5-6 SNL. The bar chart on the right shows the comparison of the normalized area under the curve (AUC) of the C component of cAPs in the control condition (SNL) and after the application of mibefradil (SNL + Mib) and after the combination of Mib + Olo (SNL + Mib + Olo). In all cases, the bar charts summarizes the results of 3–7 independent experiments, and the asterisks denote significant differences from the control condition. Kruskal-Wallis test was performed followed by Dunns’ test.

Next, we sought to determine whether the change in the cAPs caused by olomoucine was related to a potential decrease in the effects of Cdk5 on CaV3 channel activity. To this end, the cAPs evoked by stimulation of the spinal nerves and recorded in the DR in animals with SNL were recorded in the presence of the CaV3 channel blocker mibefradil alone or in combination with olomoucine. The results showed that the AUC of the cAPs recorded in the nociceptive (C-type) fibers were significantly decreased (~22%) with respect to the sham control SN-DRG-DR segments in the presence of mibefradil (Fig. 4B). Interestingly, though the channel blocker caused a decrease in the amplitude of the C component of the cAPs, the combined administration of mibefradil and olomoucine caused a decrease in the AUC of ~30%, showing no additive effects (Fig. 4B; right panel). Therefore, mibefradil seems to exert an occlusive effect on the inhibition caused by olomoucine, i.e. the two drugs (Olo and Mib) are acting on the same target (the CaV3.2 channel). We also investigated whether the changes observed in the cAPs of sensory fibers were present in rapid-conducting Aα/β fibers. The results of these experiments indicated that in the SN-DRG-DR preparation, olomoucine decreased the cAPs recorded in nerves from sham-operated and L5-6 SNL animals (>30%) with respect to the control untreated SN-DRG-DR segments (Fig. 5A). The quantitative analysis of the cAPs showed no significant changes in the latency of the rapid fibers’ response.

Fig. 5.

Fig. 5.

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Inhibition of Cdk5 activity with olomoucine affects the fast component of the cAP. A) cAPs were extracellularly recorded from a preparation that included the spinal nerve, the dorsal root ganglion, and the dorsal root (SN-DRG-DRL5) in sham and SNL animals, obtained at a 3 × T stimulation. The bar charts on the right compare the normalized area under the fast component and the latency of the cAPs. Blockade of Cdk5 activity with olomoucine (Olo) decreases the area of the fast component in both sham and SNL animals. B) Olo treatment does not occlude the effect of mibefradil (Mib) on the fast component of the cAP. The blockade of CaV3 channels with Mib decreases the amplitude of the fast component recorded in L4. The combined use of Mib and Olo had an additive effect on the cAPs area (right panel). In all cases, the bar charts summarizes the results of 4–6 independent experiments, and the asterisks denote significant differences from the control condition. Kruskal-Wallis test was performed followed by Dunns’ test.

As mentioned earlier, previous work from our research group has shown that Cdk5 exerts stimulatory effects on CaV3 channels (Calderón-Rivera et al., 2015; Gomez et al., 2020a; Loya-Lopez et al., 2020). Consequently, we investigated whether the modification in the cAP caused by olomoucine was related to a possible reduction in these channels’ functional expression. To this end, cAPs evoked by stimulation of the SN and recorded in the DR in animals with L5-6 SNL were recorded in the presence of mibefradil alone or in combination with olomoucine. The results of this analysis showed that mibefradil caused a decrease in the amplitude of the fast component of cAP (of ~40%), while the combined administration of mibefradil and olomoucine had an additive effect (Fig. 5B). The latencies of the cAPs were not affected significantly by the treatments. Therefore, in this case, the CaV3 channel antagonist did not exert an occlusive effect on the effects of olomoucine, suggesting that both compounds have different action mechanisms. In this context, it is worth mentioning that previous reports have shown that Cdk5 may also affect other Ca2+ channels’ activity, particularly those of the N-type (Furusawa et al., 2014; Su et al., 2012).

Effect of SNL and olomoucine on the Ca2+ currents recorded in sensory neurons

Given that the SNL produces changes in the size and shape of the cAPs recorded in nociceptive afferent fibers, we next explored whether these alterations could be associated with changes in both T- and N-type Ca2+ channel functional expression. In an initial series of experiments, the animal model used for these recordings was validated by assessing the limb’s withdrawal in response to stimulation with von Frey filaments, and the effect of the nerve ligation on the development of mechanical allodynia on day 14 was evaluated. Hence, stimulation on the plantar surface of the left hind limb (ipsilateral) of control animals (sham) produced a paw withdrawal threshold of ~14 g. In contrast, the ligation of the spinal nerves decreased this withdrawal threshold (to < 4 g) in the ipsilateral limb in response to stimulation with von Frey filaments (Fig. 6A).

Fig. 6.

Fig. 6.

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Effect of SNL and olomoucine on the functional expression of T-type Ca2+ channels in DRG sensory neurons. A) Stimulation with von Frey filaments to the plantar surface of the left lower limb (ipsilateral) produced mechanical allodynia in animals subjected to L5/L6 SNL, as evidenced by a significant reduction in the paw withdrawal threshold from ~14 g in the sham-operated animals to < 3 g in animals 14 d after surgery (n = 8 and 10, respectively). B) Representative superimposed ICa traces recorded in DRG sensory neurons from sham or SNL animals in the presence of vehicle (DMSO) or olomoucine as indicated. Currents were evoked by applying voltage steps ranging from −70 to 60 mV from a Vh of −90 mV. C) Average I-V curves obtained in the presence of DMSO or Olo as in B (n = 8–9 recorded cells). D) Average normalized activation and steady-state inactivation curves as a function of membrane voltage obtained in DRG neurons as in B (n = 7–8 recorded cells). Normalized activation and inactivation curves were fit to Boltzmann functions.

We then assessed the functional consequences of using the Cdk5 inhibitor olomoucine (100 μM; 48 h) on the activity of the T-type channels of sensory neurons by whole-cell patch-clamp recordings. Here is worth recalling that T-type channel currents were isolated by blocking the HVA components of the total current using nifedipine (10 μM, L-type); ω-conotoxin GVIA (500 nM; N-type); ω-agatoxin GIVA (200 nM, P/Q-type) and SNX-482 (200 nM, R-type). As shown in Figures 6B and 6C, current density through T-type channels was significantly increased in the voltage range of −90 to 60 mV, in the the SNL (DMSO) condition, compared with the control condition. Likewise, the Cdk5 inhibitor’s presence prevented the stimulatory effect of the kinase on T-current density. This is consistent with our previous observation that the silencing of Cdk5 using specific siRNAs abrogates the kinase’s effect on the CaV3 channels heterologously expressed in HEK-293 cells (Calderón-Rivera et al., 2015; Gomez et al., 2020a). To investigate whether the effect of olomocine on T-type channels was mediated by alterations in channel gating, the voltage dependence of current activation and inactivation were next assessed. Normalized conductance-voltage and steady-state inactivation curves were generated for the control groups (sham and SNL animals plus vehicle) and SNL plus olomoucine (Fig. 6D). The results show that, although there was a change of about 10 mV in the inactivation curve in the depolarizing direction in neurons subjected to SNL in the presence of the vehicle (DMSO), the half-maximal activation and inactivation voltages (V½) in the sham controls did not differ significantly from that of the olomoucine-treated cells.

Lastly, we evaluated the effects of olomoucine on the N-type channel currents in sensory neurons. As shown in Figures 7A and 7B, current density was significantly increased in the voltage range of −70 to 60 mV, in the SNL (DMSO) condition, compared with the control conditions. Likewise, the presence of olomoucine prevented the stimulatory effect of Cdk5 on the current density. To determine whether the effect of olomoucine on N-type channel currents was mediated by alterations in channel gating, again, the voltage dependence of current activation and inactivation were evaluated. As for T-type channels, normalized conductance-voltage and steady-state inactivation curves were generated for N-type currents in the control groups (sham and SNL animals plus vehicle), and SNL plus olomoucine (Fig. 7C). The results show that the half-maximal activation and inactivation voltages (V½) in the sham-treated controls were similar to those obtained for the olomoucine-treated cells. The slope factors (k) for the Boltzmann fits were also similar.

Fig. 7.

Fig. 7.

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Effect of the SNL and olomoucine on the functional expression of N-type Ca2+ channels in DRG sensory neurons. A) Representative superimposed IBa traces through N-type channels recorded in DRG sensory neurons from sham or SNL animals in the presence of vehicle (DMSO) or Olo as indicated. Currents were evoked by applying voltage steps ranging from −70 to 60 mV from a Vh of −70 mV. B) Average I-V curves obtained in the presence of DMSO or Olo as in A (n = 9–10 recorded cells). C) Average normalized activation and steady-state inactivation curves as a function of membrane voltage obtained in DRG neurons as in A (n = 7–9 recorded cells). Normalized activation and inactivation curves were fit to Boltzmann functions.

DISCUSSION

In the present report, the functional and cell localization changes of the T- (CaV3.2) and N-type (CaV2.2) channels and Cdk5 in intact L3-4 afferent fibers adjacent to the injured peripheral nerve were studied in rats subjected to L5-6 SNL. Our results show that both channels and kinase are altered in intact L3-4 DRG neurons after SNL, providing evidence of a novel molecular mechanism of neuropathic pain. Likewise, our data suggest that L5-6 SLN slightly modifies the slow and significantly affects the fast components of the cAPs recorded in the dorsal root L4, and that these changes might associated with the observed changes mediated by Cdk5 in the function and location of both types channels.

Since L4 intact afferent fibers combine with L5 axons subjected to Wallerian degeneration of injured nerves in the L5-6 SNL model, functional changes can also be induced in the L4 fibers (Liu et al., 2019; Wu et al., 2002). In this regard, it is acknowledged that the incidence of abnormal spontaneous activity is higher in L4 DRG than in non-injured nociceptors after L5 nerve injury (Ma et al., 2003; Xu et al., 2015). Although the mechanisms by which this occurs are still not well understood, it is known that the ion channels that regulate the excitability of neurons could be contributing to this process (Abdulla and Smith, 2001; Waxman and Zamponi, 2014). Recent studies have shown that the cell distribution and functional roles of ion channels in general and T-type (CaV3) channels in particular, may be altered in sensory neurons during the development of neuropathic pain in models of nerve injury (Altier et al., 2007; Bourinet et al., 2016; Gomez et al., 2020a; Jagodic et al., 2008). Interestingly, the uninjured nerves may play a role in peripheral sensitization and contribute to neuropathic pain (Chen et al., 2018; Waxman and Zamponi, 2014). For this reason, here we investigated possible changes in the function and location of the CaV3.2 channels in the intact L3-4 afferent fibers after L5-6 SNL. Previous studies have shown that CaV3.2 is the predominant channel subtype in L4 DRG neurons (Bourinet et al., 2005; Talley et al., 1999). Recently, Liu and colleagues (2019) showed evidence of the possible role of CaV3.2 channels in L4 DRG neurons in animals subjected to L5 SNL. These authors reported an upregulation of the channels with a consequent increase in the T-type currents and changes in the activation of the currents in L4 DRG neurons from rats with L5 SNL-induced allodynia (Liu et al., 2019). Though the mechanisms of this regulation were not investigated, they could be related to post-translational modifications of the channels (He et al., 2016; Weiss, 2020). In this context, CaV3 channels are known to be regulated by various protein kinases, such as CaMKII and Cdk5 (Calderón-Rivera et al., 2015; Djouhri, 2016; Gomez et al., 2020a; 2020b; Loya-López et al., 2020).

However, it is not known whether this regulation of CaV3 channels by protein kinases occurs in DRG neurons during neuropathic pain (Gomez et al., 2020b). Recent studies from our research group showed that the exacerbated expression of Cdk5 in animals with mechanical allodynia induced by L5-6 SNL increases functional expression and changes the localization of CaV3.2 channels in L5-6 DRG (Gomez et al., 2020a). Here, we show that a similar effect occurs in intact L3-4 DRG neurons. Likewise, the AUC’s quantitative analysis directly showed the CaV3 channels’ contribution to the cAPs in nociceptive fibers. Our data also shows that fast conducting fibers, which in physiological conditions generally process tactile information and are also important in tactile allodynia after nerve injury (Djouhri, 2016; Xu et al., 2015), are indeed affected in animals subjected to L5-6 SNL. In particular, these results show what is to our knowledge, the first evidence for a significant increase in the amplitude of the cAPs in fast conducting fibers. This effect could be associated with additional fiber recruitment after the L5-6 SNL as a consequence of amplified phosphorylation of the CaV3 channels by Cdk5. Due to their low activation threshold, these channels function near the resting membrane potential, providing a window current that allows passive Ca2+ entry and contributing to neuronal excitability. Although a substantial fraction of these channels is inactivated at rest, their recovery from inactivation during brief periods of hyperpolarization may generate rebound APs (Cain and Snutch, 2010; Canto-Bustos et al., 2014). Together, these data unveil a novel mechanism of CaV3 channel regulation in intact L4 fibers and might contribute to explain mechanical allodynia induced by nerve injury.

Our data also indicate that the L5-6 spinal nerves’ ligation affects the slow component of the cAPs. When the amplitude of this component is analyzed using mibefradil and olomoucine, the T-type channel blocker can occlude the effect of the Cdk5 inhibitor. This result suggests that both compounds share a common mechanism of action. In contrast, when analyzing the amplitude of the fast component of the cAPs in the presence of mibefradil and olomoucine, the effects of the channel blocker and the kinase inhibitor are additive, suggesting that in this case, both compounds are acting through different mechanisms. Interestingly, previous reports have shown that HVA channels (mainly of the N-type) are located in primary afferent fibers and their presynaptic terminals within the spinal cord’s superficial dorsal horn and may also play a role in neuropathic pain (Bourinet et al., 2014; Chew and Khanna, 2016; Jurkovicova-Tarabova and Lacinova, 2019). Indeed, the intracellular trafficking of these channels towards the plasma membrane and its presynaptic location may be increased after neuropathic pain injury (Cizkova et al., 2002; Yang et al., 2018). Although this has been related to an up-regulation of the CaVα2δ−1 auxiliary subunit (Bauer et al., 2009; Dolphin, 2018), it could also be explained by Cdk5-mediated phosphorylation of the channels as we shall discuss below.

The CaV2.2α1 pore-forming subunit of the N-type channels shows important functional diversity in different cell types. This likely occurs due to the association of CaV2.2α1 with different proteins and/or alternative RNA splicing. Of note, cell-specific splicing of exon 37 may act as a molecular switch governing channel coupling to different pain pathways, with exon 37a differently regulating the transmission of thermal and mechanical stimuli during hyperalgesia (Altier et al., 2007). In this context, it would be interesting to investigate whether the differential regulation of the nociceptive pathways by alternative splicing of N-type channels is related to their interaction with Cdk5. Likewise, considering that the N-type channels play essential roles in nociception, their inhibition is a promising strategy for treating chronic pain (Altier and Zamponi, 2004; Bourinet et al., 2014; Chew and Khanna, 2018; Jurkovicova-Tarabova and Lacinova, 2019). This could be achieved by directly blocking the channel, inhibiting its trafficking to the cell membrane, or modifying the signaling pathways that regulate it, including that of Cdk5 (Alles and Smith 2018; Altier et al., 2007; Altier and Zamponi, 2004; Su et al., 2012).

Previous studies have also shown that the CaVα2δ−1 subunit may play a role in the pathophysiology of neuropathic pain (Li et al., 2004; 2006; Gomez et al., 2019). There are several reports on the analgesic properties of gabapentinoides, ligands for CaVα2δ (Li et al., 2004; Perret and Luo 2009; Waxman and Zamponi, 2014), and the increase in this protein in the DRG and neurons of the spinal cord in animal models of chronic pain (Luo et al., 2001; Bauer et al., 2009; 2010). As with the T-type channel proteins, CaVα2δ−1 also seems to change its distribution in nociceptive afferent fibers (Aδ and C) following SNL, but in this case, the protein seems not to be concentrated in the neuronal soma but acquires a predominantly presynaptic location that would allow its association with channels involved in neurotransmitter release (Bauer et al., 2009; 2010). This synaptic channel redistribution and the phosphorylation of the ion-conducting CaV2.2α1 subunit may help understand the exacerbated activity of Ca2+ channels in mechanical allodynia that occurs in SNL. Taken together, the changes in the distribution and function of Cav3.2 (the major isoform of the T-type channels expressed in DRG neurons and directly implicated in nociceptive signaling) shown here, significantly expand our knowledge on the molecular events that occur in the intact nerves adjacent to the injured nerves and contribute to a better understanding of the pathophysiology of neuropathic pain. A broader and more detailed understanding of the molecular actors involved in pain pathways will undoubtedly contribute to the design of more and better strategies for treating chronic pain.

Highlights:

CaV3 channel location is altered in L4 adjacent intact afferent fibers after L5-6 SNL

CaV3.2 channel distribution changes by concentrating on the neuronal soma

T- and N-type channels in L3-4 sensory neurons are targets of Cdk5

CaV3 channel phosphorylation by Cdk5 may contribute to nerve injury-induced allodynia

ACKNOWLEDMENTS

Expert technical assistance was provided by M. Urbán and G. Raya-Tafoya.

FUNDING

This work was supported by funds from Conacyt (5098) to RDL. RK is supported by grants from NINDS (NS0987720, NS120663) and NIDA (DA042852). Doctoral fellowships from Conacyt to KG, PD and AVP is gratefully acknowledged.

Footnotes

DECLARATION OF INTEREST

RK is the co-founder of Regulonix LLC, a company developing non-opioids drugs for chronic pain. In addition, RK has patents US10287334 and US10441586 issued to Regulonix LLC. The other authors declare no competing financial interest.

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