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. Author manuscript; available in PMC: 2025 Dec 18.
Published in final edited form as: J Neurophysiol. 2025 Nov 21;134(6):2027–2035. doi: 10.1152/jn.00379.2025

Activity of protein kinase C controls the efficacy of cannabinoid receptor type 1 in the medial prefrontal cortex after neuropathic pain

Dongman Chao 1,2, Yong Liu 1, Tao Cheng 1, Quinn H Hogan 2, Bin Pan 1,*
PMCID: PMC12709621  NIHMSID: NIHMS2125089  PMID: 41269674

Abstract

Many patients with chronic pain conditions suffer from depression. In our previous report, we found that afferent noxious inputs after painful nerve injury compromise activity-dependent endocannabinoid (eCB) signaling in the medial prefrontal cortex (mPFC), resulting in depression. Since protein kinase C (PKC) and cAMP/protein kinase A (PKA) pathways can regulate eCB receptor type-1 (CB1R)-mediated synaptic transmission, we explored the possible roles of PKC and PKA in causing reduced eCB signaling in neuropathic pain (spared nerve injury, SNI). Four weeks after SNI, rats developed both depression and hypersensitivity of the plantar skin to punctate mechanical stimulation. Radioligand-based assay of CB1R binding in the harvested mPFC tissue showed no change after SNI in both male and female rats. Slice electrophysiological recordings from the mPFC showed that SNI did not alter the effects of a PKA agonist (forskolin) or blocker (H-89) on evoked inhibitory postsynaptic currents (eIPSCs). However, the PKC blocker chelerythrine (CHEL) fully prevented the inhibitory effect of the CB1R agonist (WIN-55212–2) on eIPSCs, whereas PKC activation with phorbol 12-myristate 13-acetate reversed the reduced effect of WIN-55212–2 on eIPSC and miniature IPSC after SNI. Together, these data suggest that PKC activity controls the efficacy of CB1Rs in mPFC after neuropathic pain.

Keywords: Protein kinase C, Cannabinoid receptor type 1, Medial prefrontal cortex, Neuropathic pain

New & Noteworthy:

Our research reveals that protein kinase C (PKC) activity in the medial prefrontal cortex (mPFC) is essential for cannabinoid receptor type 1 (CB1R)-mediated suppression of GABAergic transmission. After neuropathic pain, reduced PKC activity diminishes CB1R efficacy, despite unchanged receptor expression. Restoring PKC activity rescues CB1R function. These findings identify PKC as a critical modulator of endocannabinoid signaling in chronic pain, suggesting new therapeutic targets for pain-associated affective disorders.

1. Introduction:

Chronic pain is a major public health challenge that is inadequately addressed. The medial prefrontal cortex (mPFC) is a key brain area in the pain system, receiving ascending nociceptive input and contributing to the affective and executive aspects of pain (1). It also exerts important top-down control of pain sensation by projections that innervate the periaqueductal gray (PAG), which is the primary relay center for descending pain modulation (2–4). We have previously shown that chronic pain alters endocannabinoid (eCB) signaling in the mPFC, such that the analgesic efficacy of cannabinoid receptor type 1 (CB1R) is reduced during the chronic phase of neuropathic pain (5). In this study, we explore the underlying mechanisms of this reduced efficacy of mPFC CB1Rs in neuropathic pain.

CB1R is a member of the superfamily of G-protein-coupled receptors (GPCRs), which consist of seven transmembrane helices. Major downstream mediators of CB1R include the G proteins of the G(i/o) family, which inhibit adenylyl cyclase in most tissues and cells, and regulate ion channels, including calcium and potassium ion channels (6). Additionally, CB1R activation by cannabinoids such as delta 9-tetrahydrocannabinol (delta 9-THC) can increase the activity of protein kinase C (PKC) (7). In cultured telencephalic neurons, CB1R agonist can activate both extracellular signal-regulated kinases (ERK1/2) by activating Gi/o and phospholipase C-PKCε by activating Gq (8). Regarding upstream regulation of CB1R signaling, we have previously found that the cAMP/protein kinase A (PKA) pathway controls CB1R-mediated presynaptic vesicle release in ventral tegmental area (9), while others have observed inhibition by PKC phosphorylation of CB1Rs expressed in a pituitary tumor cell line (10). To expand our understanding of the modulation of CB1R regulation in the mPFC, the experiments we report here examine the regulation of CB1Rs in the rat mPFC in the context of painful nerve injury, specifically examining the roles of PKA and PKC as possible mediators of the reduced CB1R efficacy after painful nerve injury.

2. Materials and Methods

2.1. Animals

Male and female Sprague Dawley rats weighing 170–200g obtained from Taconic Biosciences (Hudson, NY, https://www.taconic.com/rat-model/sprague-dawley) were maintained and used according to the NIH Guide for the Care and Use of Laboratory Animals, and in compliance with federal, state, and local laws. All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committees of the Medical College of Wisconsin and the University of Texas Medical Branch. Animals were housed in a pathogen-free facility, 2 animals per ventilated cage, in a room maintained at 25±1 °C at 35 to 45% humidity, with a 12/12-hour (hr) day/night cycle. Animals had free access to food and water. At the termination of the study, euthanasia was performed by decapitation during deep isoflurane anesthesia.

2.2. Nerve injury model

Some rats were subjected to SNI surgery (11). Briefly, during anesthesia by inhalation of isoflurane (1–3%), an incision (~2 cm) was made on the lateral mid-thigh, and the underlying muscles were separated to expose the sciatic nerve. The tibial and common peroneal nerves were individually ligated with 6.0 sutures and cut distally to the ligature, and 2–3 millimeters (mm) of each nerve was removed distal to the ligation. The sural nerve was preserved, and contact with it was avoided. Muscle and skin were closed using 4.0 monofilament nylon sutures. Control rats received sham surgery in the form of skin incision, exposure of the nerve, and closure only.

2.3. Sensory behavioral tests

To evaluate the development of pain behaviors in rats with SNI, sensory testing of the plantar skin included evoking reflexive behaviors induced at threshold intensity by punctate mechanical stimulation (von Frey test), by noxious mechanical stimulation (pin), by dynamic non-noxious mechanical stimulation (brush), and by cold stimulation (acetone). Specifically, a noxious punctate mechanical pin stimulation test was performed using a standard 22-gauge spinal anesthesia needle that was applied to the lateral one-third of the plantar surface of the hind paw with enough force to indent the skin but not puncture it. This evoked either a brief withdrawal response typical of normal animals, or a sustained withdrawal with shaking, licking of the foot, and delayed placement back on the cage floor, which we term a hyperalgesia response (12) and is selectively associated with place avoidance, indicating the aversiveness of this specific behavior (13) The von Frey test was performed using a set of calibrated monofilaments (Patterson Medical, Bolingbrook, IL) and an up/down technique to identify the minimum force for triggering a withdrawal (14). Dynamic mechanical stimulation (brush) was performed with a camel hair brush (4 mm wide), which was applied to the lateral plantar skin of the hind paw by light stroking in the direction from heel to toe for 2 s (12), and the frequency of withdrawal identified. Sensitivity to cold was assessed using the application of acetone, which was expelled through tubing perpendicularly to form a convex meniscus on the end of the tubing that was touched to the lateral plantar skin without contact of the tubing with the skin, and the frequency of withdrawal identified (15). Testing was performed 3 weeks following surgery

2.4. [3H]CP55940 binding assay

[3H]CP55940 binding assay was performed using the Multiscreen Filtration System as previously described (16, 17). mPFC neuronal membranes (10 μg of protein) were prepared and incubated with [3H]CP55940 in TME buffer (total volume, 0.2 ml) containing 0.1% fatty acid-free bovine serum albumin for 1 h at room temperature, which was terminated by filtration, followed by washing the filters three times with 250 μl of cold bovine serum albumin-containing buffer. Radioactivity remaining on the filters was determined using scintillation counting. Nonspecific binding was defined as the amount of [3H]CP55940 bound in the presence of 10 μM Δ9-THC. Kd (equilibrium dissociation constant determined from saturation analyses) and Bmax (maximal binding site density) values were determined from saturation isotherms using nonlinear regression to fit the data to the single-site binding equation (Prism; GraphPad Software Inc., San Diego CA).

2.5. Slice Preparation and Electrophysiology

At 31–34 days following SNI or control sham surgery, rats were anesthetized by isoflurane inhalation and decapitated. mPFC slices (300 μm) and whole-cell recordings were made as described previously (18). Slices were stored in artificial cerebrospinal fluid (aCSF) containing (in mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose at room temperature. All solutions were saturated with 95% O2 and 5% CO2. All recordings were performed at 32±1 °C by using an automatic temperature controller (Warner Instrument, Hamden, CT). Patch pipettes, ranging from 2–4 MΩ resistance, were formed from borosilicate glass (Cat#1B150–3, World Precision Instruments, Sarasota, FL). Recordings were made with an Axopatch 700B amplifier (Molecular Devices, Downingtown, PA). Signals were filtered at 2 kHz and sampled at 10 kHz with a Digidata 1440A digitizer and pClamp10 software (Molecular Devices). Series resistance (5–10 MΩ) was monitored before and after the recordings, and data were discarded if the resistance changed by 20%.

Evoked inhibitory postsynaptic currents (eIPSCs) were recorded in aCSF containing the glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX, 20 μM) and D-2-amino-5-phosphonovaleric acid (D-AP-5, 50 μM). The internal solution in the patch pipettes contained (in mM): 80 potassium gluconate, 60 KCl, 5 QX-314, 10 HEPES, 0.2 EGTA, 2 MgCl2, 4 MgATP, 0.3 Na2GTP, and 10 Na2-phosphocreatine (pH 7.2 with KOH). To examine the effects of WIN 55212–2 on IPSCs, IPSCs were evoked at 10-s intervals. Input-output (I/O) curves of IPSCs were generated using incremental stimulus intensities of 10–160 mA. For the recording of miniature IPSCs (mIPSCs), tetrodotoxin (TTX) was added in the aCSF to block action potentials.

2.6. PKC Enzymatic Activity and Protein Expression

Thirty-five days after SNI or sham surgery, rats were deeply anesthetized and decapitated. The mPFC was rapidly dissected on ice, frozen, and stored at −80 °C.

PKC activity assay.

Tissue was homogenized in ice-cold PKC assay buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100) containing protease and phosphatase inhibitors, and centrifuged at 14,000 × g for 15 min at 4 °C. Supernatants were collected for biochemical analyses. Kinase activity was quantified using a colorimetric PKC activity ELISA kit (Abcam, ab139437) following the manufacturer’s protocol. Equal protein (5–10 μg/well) was incubated on PKC substrate-coated plates in the presence of ATP, and phosphorylation was detected with a phospho-specific antibody and horseradish peroxidase/3,3’,5,5’-tetramethylbenzidine development. Absorbance was read at 450 nm (reference 620 nm). Technical duplicates were averaged for each animal, and activity was expressed as milli-units per mg protein.

Western blot.

Tissue was homogenized in radioimmunoprecipitation assay buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitors, and centrifuged at 14,000 × g for 15 min at 4 °C. The lysates were mixed with 2× Laemmli buffer, heated 5 min at 95 °C, and resolved by SDS-PAGE (8–12%). Proteins were transferred to PVDF membranes, blocked in 5% BSA/TBST, and incubated overnight at 4 °C with antibodies to phospho-PKC isoforms (α, β, δ; 1:1,000, Cell Signaling Technology) and GAPDH (1:2,000). HRP-conjugated secondary antibodies (1:2,000) were applied for 1 h, and bands were visualized with enhanced chemiluminescence. Densitometry was performed in ImageJ; each PKC signal was normalized to GAPDH and expressed relative to the mean of the sham group (= 100%).

2.7. Chemicals

All drugs were prepared as concentrated stock solutions and stored at −20 or −80 °C before use. WIN55 212-2 (WIN), Phorbol 12-myristate 13-acetate (PMA), H89 dihydrochloride (H89), chelerythrine chloride (CHEL), forskolin, and all common chemicals were purchased from Sigma-Aldrich (St Louis, MO) or Tocris Bioscience (Minneapolis, MN). H89, CHEL, CNQX-Na2 (Tocris) and D-AP-5 (Tocris) were dissolved in water. PMA, forskolin, and WIN were dissolved in dimethyl sulfoxide (DMSO). When these drugs were applied to slices, control slices were treated in the same concentration of the respective solvent for a similar exposure time. Drug concentrations and exposure durations (H89 10 μM; chelerythrine 10 μM) were chosen based on prior slice studies and our published work, and produced expected physiological effects here (see Results)

2.8. Statistics

Statistical analyses included unpaired and paired Student’s t-tests for two-group comparisons and two-way ANOVA with Sidak’s post hoc test for multi-group/time-course data. All tests, sample sizes (cells and animals), and p-values are reported in the Results and figure legends. Data are shown as mean ± SEM unless otherwise specified.

3. Results:

3.1. SNI produced pain behavior.

As intended, animals subjected to SNI developed behaviors indicative of pain, whereas animals receiving sham SNI did not. Specifically, at 31–34d after surgery, hyperalgesia response to pin was seen in 66.7±23.5% of applications in SNI animals vs. 1.1±3.3% in sham animals (P = 0.0003 with non-parametric Mann-Whitney test); von Frey mechanical threshold was 0.04±0.1g in SNI animals vs. 25±0g in sham animals (P < 0.001 with t-test); withdrawal from brush occurred in 70±18.9% of applications in SNI animals vs. 4.2±11.8% in sham animals (P < 0.001 with non-parametric Mann-Whitney test); and withdrawal from cold occurred in 60±14.1% of applications in SNI vs. to 0±0% in sham animals (P < 0.001 with non-parametric Mann-Whitney test).

3.2. CB1R density and agonist affinity are not changed after SNI.

We previously observed reduced CB1R-mediated GABA synaptic transmission in mPFC after painful nerve injury (SNI) (5). It is possible that reduced CB1R binding with eCB contributes to this process. Radioligand-based CB1R binding did not differ between SNI and ShamSNI in both male and female rats (Fig. 1A). There also were no differences between ShamSNI and SNI in either the density of receptors that were labeled by [3H]CP55940 as represented as Bmax (Fig. 1B) or the dissociation constant (Fig.1C). Because reduced CB1R binding could not account for the impaired signaling after SNI, we next examined PKA activity, a well-established regulator of CB1R-mediated presynaptic release in other brain regions (9), to determine whether altered PKA signaling explained the reduced efficacy of CB1Rs in mPFC.

Figure 1.

Figure 1.

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[3H]CP55940 binding assay. (A) [3H]CP55940 binding of rat mPFC membranes was determined. There was no difference between ShamSNI and SNI in the density of receptors that are labeled by [3H]CP55940 and represented as Bmax (B) and the dissociation constant (Kd) (C). Each symbol represents one rat in B and C. Data are mean ± SEM. Statistical comparisons were made with an unpaired t-test (Bmax, Kd) between Sham and SNI for each sex; no significant (ns) differences were detected (P > 0.05).

3.3. Regulation of eIPSCs by PKA is not changed after SNI.

CB1Rs activity regulates vesicle release by the cAMP/PKA signaling pathway in the midbrain ventral tegmental area (9). It is therefore possible that elevated cAMP/PKA signaling after painful nerve injury may contribute to the reduced efficacy of CB1Rs in the mPFC. To examine this, we tested the effects of the PKA agonist forskolin (FSK), and the PKA blocker H89, on recorded eIPSC amplitudes. FSK potentiated eIPSCs to the same degree in rats with ShamSNI and SNI (Fig. 2A, B), which indicates that painful nerve injury does not change the sensitivity of presynaptic vesicle release to PKA. H89 had no effects on the eIPSCs in both ShamSNI and SNI rats (Fig. 2C, D), which indicates that there is no constitutive PKA effect in either ShamSNI or SNI animals. Since neither receptor binding nor PKA modulation, both established mechanisms for regulating CB1R signaling, accounted for the phenotype, we then tested whether PKC activity might contribute to CB1R regulation of GABAergic transmission in the mPFC, given some reports have identified the involvement of PKC in CB1R-dependent synaptic transmission, including observations that the phytocannabinoid delta THC can activate PKC isolated from rat forebrain (7) and PKC can phosphorylate CB1Rs and thereby attenuate its actions (10)..

Figure 2.

Figure 2.

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cAMP/PKA signaling in CB1R-dependant synaptic transmission. (A) Time course and (B) average of effects of PKA agonist FSK on eIPSCs in rats with ShamSNI and SNI. (C) Time course and (D) average of effects of PKA blocker, H89, on eIPSCs in rats with ShamSNI and SNI. n in A and C represents the number of neurons recorded from 5 rats per group. Each symbol in B and D represents one neuron. Statistical comparisons were made using an unpaired t-test between the Sham and SNI groups. No significant differences (ns) were detected (P > 0.05).

3.4. CB1R-mediated inhibition of eIPSCs is PKC-dependent.

We have previously observed the reduced CB1R-mediated inhibition of GABA synaptic transmission in the mPFC after SNI (5), which could be the result of PKC signaling. To test this, we first confirmed our prior findings by showing reduced effects of the CB1R agonist WIN on eIPSC after SNI (Fig. 3A, B). We further observed that the PKC inhibitor CHEL (10 μM) alone has no effect on eIPSC (Fig. 3C, D). However, CHEL fully blocks the inhibitory effect of WIN on eIPSC (Fig. 3E, F). To quantify the sensitivity of this interaction, we examined the concentration-dependent effect of CHEL on WIN-induced eIPSC depression (Fig. 3G). Partial attenuation was evident at sub-micromolar concentrations of CHEL, with a best-fit IC50 of 0.34 μM (Fig. 3H). These data indicate that PKC activity supports CB1R efficacy in a dose-dependent manner, such that even modest PKC inhibition markedly weakens CB1R-mediated suppression of GABA release.

Figure 3.

Figure 3.

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CB1R-dependant synaptic transmission is PKC activity-dependent. (A) Time course and (B) average of effects of CB1R agonist WIN-55212–2 (WIN) on eIPSCs in rats with ShamSNI and SNI. (C) Time course and (D) average of effects of PKC blocker chelerythrine (CHEL) on eIPSCs in rats with ShamSNI and SNI. (E) Time course and (F) average of effects of PKC blocker CHEL on depression effects of WIN on eIPSCs in rats with ShamSNI and SNI. (G) Time course of effects of CHEL with different concentrations (0, 0.1, 0.3, 1, and 10 μM) on eIPSCs in naïve rat slices. (H) Concentration-response relationship for CHEL inhibition of WIN-induced eIPSC depression, plotted as relative WIN-induced depression (% of control, 0 μM CHEL). Data were fitted with a sigmoidal dose-response curve, yielding an IC50 of 0.34 μM. n in A, C, E, and G represents the number of neurons recorded from 5 rats per group. Each symbol in B, D, and F represents one neuron. Statistical comparisons were made using an unpaired t-test between the Sham and SNI groups. ***P<0.001.

To confirm that the reduced CB1R efficacy after SNI was not due to global alterations in PKC expression or catalytic activity, we next measured phosphorylated PKC levels and enzymatic activity in mPFC tissue lysates. Neither the total PKC activity (Fig. 4A) quantified by ELISA nor the expression of major phosphorylated PKC isoforms (α, β, and δ) determined by Western blot differed between sham and SNI rats (Fig. 4B–E). These results indicate that chronic nerve injury does not affect phosphorylated PKC abundance or kinase activity in the mPFC, supporting the conclusion that the reduced CB1R function observed after SNI might be local changes in PKC signaling at presynaptic GABAergic terminals rather than a global loss of PKC.

Figure 4.

Figure 4.

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PKC activity and phosphor-protein expression in the mPFC are unchanged after SNI. (A) PKC enzymatic activity from mPFC homogenates 35 days after SNI or sham surgery. No difference was detected between groups. (B) Representative Western blots showing phospho-PKC pan, α, β, and δ isoforms and GAPDH, and (C, D, E) summary quantification normalized to GAPDH (mean ± SEM; n = 8 for ELISA, n = 7 for Western blot; unpaired t-test, ns: not significant).

3.5. PKC activation reverses SNI-induced suppression of CB1R-evoked presynaptic GABA release

If PKC is a critical component reducing CB1R-mediated synaptic transmission in the mPFC, it is possible that loss of PKC activity after SNI contributes to the reduced effectiveness of CB1R as an inhibitor of GABA release. When we activated PKC with PMA alone, there was no effect on eIPSC in slices from ShamSNI and SNI (Fig. 5A, B). However, PMA treatment eliminated SNI-induced loss of sensitivity of eIPSC to WIN (Fig. 5C&D), supporting PKC signaling loss as a contributor to reduced CB1R control of inhibitory synaptic function after painful nerve injury.

Figure 5.

Figure 5.

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Activation of PKC reverses reduced CB1R-dependent synaptic transmission in chronic neuropathic pain. (A) Time course and (B) average of effects of PKC agonist PMA on eIPSCs in rats with ShamSNI and SNI. Each symbol in D represents one neuron. No significant differences were detected with the unpaired t-test. (C) Effects of PKC agonist PMA on depression effects of WIN on eIPSCs in rats with ShamSNI and SNI. (D) Summary of effects of PMA on eIPSCs. n in A, C, and E represents the number of neurons recorded from 5 rats per group. Each symbol in D represents one neuron. *P<0.05 by 2-way ANOVA followed by Tukey post hoc test.

3.6. Enhanced WIN’s effects by PKC activation on IPSC are PKC-dependent

Evoked synaptic transmission reflects transmitter release only during induced activity. To provide additional information on altered transmitter release during resting phases of neuronal activity, we additionally examined spontaneous and miniature IPSCs (19, 20). As miniature currents represent quantal release of neurotransmitter, a change in amplitude is interpreted as an altered postsynaptic function, whereas a change in frequency represents an altered presynaptic neurotransmitter release, according to the quantal theory of neurotransmitter release (21–23). We found that PMA alone did not affect mIPSC frequency (Fig. 6A&B) or amplitude (Fig. 6C&D). After SNI, WIN showed less effect (compared to ShamSNI) on the frequency of mIPSCs, which was reversed by application of PMA (Fig. 6E&F), whereas the amplitude of mIPSCs was not sensitive to PMA, WIN, or nerve injury (Fig. 6G&H).

Figure 6.

Figure 6.

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PKC activity on CB1R-dependant presynaptic transmission. (A) Time course and (B) average of effects of PKC agonist PMA on frequency of mIPSCs in rats with ShamSNI and SNI. (C) Time course and (D) average of effects of PKC agonist PMA on amplitude of mIPSCs in rats with ShamSNI and SNI. (E) Time course and (F) average of effects of PMA on WIN’s depressive effects on frequency of mIPSCs in rats with ShamSNI and SNI. (G) Time course and (H) average of effects of PMA on WIN’s effects on amplitude of mIPSCs in rats with ShamSNI and SNI. (I) Representative traces of mIPSC from rats 35 d after SNI with WIN (top) and WIN+PMA (bottom). n represents the number of neurons recorded from 5 rats per group. In B and D, no significant differences (ns) were detected with the unpaired t-test. In F and H, *P<0.05 by 2-way ANOVA followed by Tukey post hoc test.

3.7. cAMP/PKA signaling and PKC signaling are critical in CB1R-mediated depression of inhibitory synaptic transmission.

Our data so far indicate that PKC activity is critical for CB1R-mediated depression of inhibitory synaptic transmission in mPFC. A remaining question is whether PKA is also critical in CB1R-mediated depression of inhibitory synaptic transmission. We explored this question with the PKA inhibitor, H89. In contrast to the depression of IPSC amplitude in response to WIN preceded by PMA that we previously observed (Fig. 5B, C), H89 blocked the effect of WIN to suppress eIPSC response in slices from both ShamSNI and SNI (Fig. 7A&B), which indicates that cAMP/PKA signaling is downstream of activation of CB1Rs in mPFC (Fig. 8A&B).

Figure 7.

Figure 7.

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PKA in CB1R/PKC-dependent presynaptic transmission. (A) Time course and (B) average of effects of H89 on eIPSCs in rats with ShamSNI and SNI. n represents the number of neurons recorded from 5 rats per group. In B, no significant difference (ns) was detected with the unpaired t-test.

Figure 8.

Figure 8.

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Model of the interaction of CB1Rs, PKC, and PKA after pain. (A) In healthy conditions, normal PKC activity maintains the proper function of CB1Rs and consequent depressed GABA release by CB1R activation. (B) In chronic pain, reduced PKC activity impairs CB1R function, weakening its ability to inhibit GABA release. This results in less suppression of presynaptic GABA transmission during CB1R activation, leading to greater inhibitory input onto postsynaptic pyramidal neurons compared to normal CB1R function.

4. Discussion

In this study, we showed that PKC signaling is essential for CB1R-mediated depression of inhibitory synaptic transmission in the mPFC, and changed PKC activity after SNI contributes to the reduced efficacy of CB1R-mediated synaptic transmission. In designing this study, we first ruled out more established mechanisms that might explain reduced CB1R efficacy. Neither CB1R density/affinity nor PKA modulation, both previously established regulators of CB1R signaling, accounted for the changes observed after SNI. Our data then revealed that PKC activity, which has been less studied in this context, is in fact critical for maintaining CB1R-mediated inhibition of GABA release in the mPFC. This stepwise approach highlights that the impairment of CB1R signaling in chronic pain is not due to canonical binding or PKA pathways, but rather to diminished PKC support of CB1R function.

Activation of CB1Rs can inhibit adenylyl cyclase (AC) via Gi/o proteins, which results in reduced PKA activity (24, 25). This Gi/o-dependent reduction in AC and PKA activity suppresses transmitter release in different brain areas (9, 26, 27), which might be contributed by inhibiting presynaptic voltage-gated calcium channels (27, 28). Consistent with those reports, we found that PKA activity is critical in CB1R-mediated synaptic transmission in mPFC.

Other than PKA’s critical role in regulating synaptic transmission, PKC activity can also regulate synaptic transmission. Its activity can regulate presynaptic vesicle release. In cortical and hippocampal cultured neurons, PKC is critical in diacylglycerol-enhanced presynaptic vesicle release (29). In the human dentate gyrus, activation of PKC enhanced the excitatory presynaptic transmission (30). Consistent with those reports, we found that PKC activity is critical in CB1R-mediated synaptic transmission in mPFC.

Activation of CB1Rs can influence PKC activity. Cannabinoid, delta 9-THC, can increase the activity of PKC in rat forebrain at concentrations of 10 mM and above (7). In hippocampus, THC treatment induced a transient increase in PKC signaling activity (31). With cultured telencephalic neurons, CB1R agonist can activate extracellular signal-regulated kinases by activating Gi/o and PLC-PKCε by activating Gq (8). In renal cells, WIN might increase Na+/K+-ATPase activity through the PKC pathway (32). We found elevated eCB signaling in the first 2 weeks after painful nerve injury (5), which might cause activation of PKC in the early phase of SNI. However, prolonged activation of PKC can lead to degradation of PKC. Prolonged exposure to PMA leads to the chronic activation of PKCα and PKCδ, thus activating a negative feedback loop leading to their degradation (33). It is possible that elevated PKC activity in the early phase of neuropathic pain results in reduced PKC activity in the chronic phase of neuropathic pain. Although our bulk biochemical assays did not detect changes in PKC activity or protein levels after SNI, these negative results are likely due to the small fraction of GABAergic neurons or GABAergic terminals in the mPFC. Such localized alterations would be masked in whole-tissue preparations, highlighting the greater sensitivity of our slice electrophysiology approach to reveal functional changes in PKC-dependent CB1R signaling. Further studies will be needed to address this.

PKC can also modulate the efficacy of CB1Rs. In AtT-20 cells transfected with rat CB1Rs, the activation of an inwardly rectifying potassium current and depression of P/Q type calcium channels by cannabinoids were prevented by stimulation of PKC, which took effects by phosphorylating a single serine (Ser317) of a fusion protein incorporating the third intracellular loop of CB1 (10), which indicates that PKC might reduce efficacy of CB1Rs. On the contrary, activated PKC could augment CB1-mediated inhibition of cAMP accumulation by phosphorylation of serine residues (Ser402, Ser411, and Ser415) in N18TG2 neuroblastoma cultured cells (34). In this study, with in situ brain slice recordings, we found that PKC activity is critical in the normal function of CB1Rs in regulating presynaptic vesicle release in mPFC, and reduced PKC activity in the chronic phase of neuropathic pain caused reduced CB1R efficacy. Further studies will be needed on the role of PKC activity in the efficacy of CB1Rs.

It is important to note that the reduced CB1R efficacy observed here does not result from tonic activation of CB1Rs by elevated endocannabinoids. As shown previously, 2-AG levels in the mPFC normalize during the chronic phase of SNI, and CB1Rs are not tonically engaged under baseline conditions (5). This interpretation is further supported by the finding that CB1R antagonists such as AM251 potentiate GABAergic transmission only when CB1Rs are tonically active, as in monoacylglycerol lipase knockout mice with persistently elevated 2-AG levels, but not in normal tissue (35). These results reinforce the view that the loss of CB1R efficacy after SNI reflects receptor desensitization associated with altered PKC signaling, rather than persistent eCB tone.

5. Conclusion

In conclusion, we found that PKC signaling is critical in CB1R-dependent depression of presynaptic GABA release in the mPFC, and reduced PKC activity contributes to the reduced CB1R efficacy after painful nerve injury.

Grants:

This work is supported by a grant (R01NS112194) to B. P. from the National Institutes of Health at Bethesda.

Footnotes

Disclosures: All authors declare no conflicts of interest.

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