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. 2021 Apr 15;6(2):137–147. doi: 10.1089/can.2020.0004

Morphine Induces Upregulation of Neuronally Expressed CB2 Receptors in the Spinal Dorsal Horn of Rats

Patrick Grenier 1, Adam Sunavsky 1, Mary C Olmstead 1,2,*
PMCID: PMC8064965  PMID: 33912678

Abstract

Background: Cannabinoid receptors play a key role in regulating numerous physiological processes, including immune function and reward signaling. Originally, endocannabinoid contributions to central nervous system processes were attributed to CB1 receptors, but technological advances have confirmed the expression of CB2 receptors in both neurons and glia throughout the brain. Mapping of these receptors is less extensive than for CB1 receptors, and it is still not clear how CB2 receptors contribute to processes that involve endocannabinoid signaling.

Objectives: The goal of our study was to assess the effects of peripheral nerve injury and chronic morphine administration, two manipulations that alter endocannabinoid system function, on CB2 receptor expression in the spinal dorsal horn of rats.

Methods: Twenty-four male Sprague Dawley rats were assigned to chronic constriction injury (CCI), sham surgery, or pain naïve groups, with half of each group receiving once daily injections of morphine (5 mg/kg) for 10 days. On day 11, spinal cords were isolated and prepared for fluorescent immunohistochemistry. Separate sections from the deep and superficial dorsal horn were stained for neuronal nuclei (NeuN), CD11b, or 4′,6-diamidino-2-phenylindole (DAPI) to mark neurons, microglia, and cell nuclei, respectively. Double labeling was used to assess colocalization of CB2 receptors with NeuN or microglial markers. Quantification of mean pixel intensity for each antibody was assessed using a fluorescent microscope, and CB2 receptor expressing cells were also counted manually.

Results: Surgery increased DAPI cell counts in the deep and superficial dorsal horn, with CCI rats displaying increased CD11b labeling ipsilateral to the nerve injury. Surgery also decreased NeuN labeling in both regions, an effect that was blocked by morphine administration. CB2 receptors were expressed, predominantly, on NeuN-labeled cells with significant increases in CB2 receptor labeling across all surgery groups in both deep and superficial areas following morphine administration.

Conclusions: Our findings provide supporting evidence for the expression of CB2 receptors on neurons and reveal upregulation of receptor expression in the dorsal spinal cord following surgery and chronic morphine administration, with the latter producing a larger effect. Synergistic effects of morphine-cannabinoid treatments, therefore, may involve CB2-mu opioid receptor interactions, pointing to novel therapeutic treatments for a variety of medical conditions.

Keywords: gliosis, opiate, opioid, CCI, chronic pain

Introduction

The endocannabinoid system plays a major role in both affective responses and immune system function.1,2 Originally, these processes were believed to be mediated by different populations of cannabinoid receptors: CB1 receptors, highly expressed in the central nervous system (CNS), were linked to reward, motivation, and nociception,3,4 whereas the role of CB2 receptors, located exclusively in the peripheral nervous system,5 was limited to immune and inflammatory responses.6,7 Advances in molecular biology techniques8,9 (for review, see Zimmer10) revealed the existence of CB2 receptors in the CNS, with initial reports of localization on immune cells.11 More recently, CB2 receptors have been identified on neurons,12–14 although anatomical mapping and characterization of this population is less extensive than that of CB1 receptors.

One difficulty in identifying CB2 receptors in the CNS is that expression may be low or undetectable in basal conditions,7,15 but upregulated following injury or inflammation.16 For example, CB2 receptor expression increases in preclinical models of nerve injury,17 specifically in pain-processing regions such as the lumbar dorsal horn18; this may explain the enhanced analgesic effect of cannabinoid receptor agonists in chronic pain.19 Chronic morphine administration also induces upregulation of CB2 receptors, with a time course that coincides with the development of analgesic tolerance.20 Increases in CB2 expression following chronic pain or morphine administration likely involve proinflammatory changes in neuronal-glial interactions.21 Both manipulations are associated with increased infiltration and proliferation of glia that correlate with an upregulation of glial markers, including the microglial marker CD11b. CB2 receptor agonists attenuate the effect of activated microglia22 and inhibit astrocyte activation,23 suggesting that CB2 upregulation plays a neuroprotective role in these states.24

The hypothesis that CB2 receptors are neuroprotective has been difficult to evaluate because the majority of studies examining CB2 receptor expression in the CNS have focused on non-neuronal cells. We addressed this issue by assessing changes in CB2 receptor expression, and colocalization with microglial (CD11b) and neuronal nuclei (NeuN) markers, in the spinal dorsal horn following chronic morphine administration in pain naïve and chronic pain animals. Chronic pain was induced by chronic constriction injury (CCI) of the sciatic nerve. Changes in glial and neuronal CB2 receptor expression were assessed 10 days later, a period that coincides with peak spinal gliosis and hypersensitivity following nerve injury,25 and the development of analgesic tolerance following morphine administration.26 As in our previous studies,27 gliosis was assessed by quantifying CD11b expression, a well-characterized marker of monocytes that predominantly labels microglia in the CNS, both in activated and quiescent states.28–30 Both surgery and chronic morphine induced an upregulation in CB2 receptor expression in the spinal dorsal horn. Fluorescent colocalization showed that CB2 labeled cells were predominantly NeuN positive but CD11b negative, confirming a change in neuronal expression of CB2 receptors in the spinal cord in these two models.

Materials and Methods

Subjects

Twenty-four male Sprague Dawley rats (Charles River, Quebec, Canada) weighing 250–300 g at the start of the experiment were pair-housed on a 12 h:12 h reverse light cycle and allowed ad libitum access to food and water. Procedures were performed in accordance with guidelines set by the Canadian Council on Animal Care; experiments were approved by Queen's University Animal Care Committee.

Surgery

Animals were randomly assigned to neuropathic (NP) pain (CCI of the sciatic nerve), sham surgery, or pain-naïve groups. On surgery day, all animals received liquid acetaminophen (0.6 mL of 32 mg/mL dose, orally) and were anesthetized with isoflurane (5 L/min induction, 2–3 L/min maintenance). For CCI animals, an incision was made in the skin and scissors were used to bluntly dissect the muscle layers. The sciatic nerve was exposed and four loose ligatures were tied around the nerve using chromic gut 4.0 suture thread.31 Sham animals received skin and blunt muscle dissection without manipulation of the nerve. Pain-naïve animals received no surgery. Animals received acetaminophen (50 mg) following surgery and the next morning.

Drug treatment

Animals in each surgical group were randomly assigned to a treatment group, receiving morphine (5 mg/kg s.c.; Sandoz Canada, Inc., Boucherville, Canada) or saline (1 mL/kg) once daily for 10 days, beginning on surgery day. This dosing regimen induces opiate tolerance and spinal gliosis.27

Behavioral testing

Mechanical allodynia was assessed on days 4, 7, and 10 postsurgery (before injection) using calibrated von Frey filaments (Stoelting, Wood Dale, IL) to determine 50% withdrawal thresholds.32

Tissue collection and sectioning

On day 11 postsurgery, animals were anesthetized with sodium pentobarbital (75 mg/kg i.p.; MTC Pharmaceuticals, Cambridge, Canada) and transaortically perfused with 0.9% saline followed by 500 mL of 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). Spinal cords were extracted and postfixed for 30 min in 4% PFA, then transferred to 30% sucrose in 0.1 M PB for 48 h at 4°C. Spinal cords were snap-frozen in −50°C isopentane and stored at −80°C. The lumbar portion of each spinal cord was later isolated, and 30 μm sections were sliced on a cryostat and collected to assess CB2 colocalization with the microglial marker CD11b or the neuronal marker NeuN.

Fluorescent immunohistochemistry

Free-floating sections were washed 3×5 min in 0.1 M tris-buffered saline (TBS), then 1×5 min in 0.1 M TBS-triton (TBS-T). Tissue was blocked for 2 h at room temperature in 10% normal goat serum (NGS) and bovine serum albumin (BSA) in 0.1 M TBS-T. Tissue was incubated with primary antibodies in solution containing 1% NGS and BSA in 0.1 M TBS-T for 24 h at 4°C. Primary antibodies used for microglial colocalization with CB2: (1:500 anti-CB2, rabbit polyclonal, Cat. #ab45942, lot: GR3243679-1, Abcam, Cambridge, United Kingdom; 1:1000 anti-CD11b, raised in mouse, MLA257R, batch: 0404, AbD Serotec, Raleigh, NC); for neuronal colocalization with CB2: (1:500 anti-CB2, rabbit polyclonal, Cat. #ab45942, lot: GR3243679-1, Abcam; 1:500 anti-NeuN, mouse monoclonal, MAB 377, lot: LV1519148, Millipore, Burlington, MA).

The following day, sections were washed 2×5 min in TBS-T, 2×10 min in TBS-T, then incubated in the dark for 2 h with fluorescent-conjugated secondary antibodies in a solution of 5% NGS in 0.1 M TBS-T (1:200 anti-rabbit Alexa 488, 1:200 anti-mouse Alexa 594, Molecular Probes, Invitrogen, Canada). Sections were washed 3×10 min in 0.1 M TBS-T, then 1×10 min in TBS. Cell nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI, 1:2000, D1306; Life Technologies, Eugene, OR) for 10 min. Sections were washed 3×10 min in 0.1 M TBS, mounted, coverslipped with Aquamount (Polysciences, Inc., Warrington, PA), and stored at 4°C.

Fluorescent imaging and quantification

Slides were imaged using a fluorescent microscope (Leica DM 4000B). Whole sections were captured at 5×magnification for all three wavelengths (blue, green, and red) to assess global expression patterns in the lumbar spinal cord; higher magnification (40×) was used to assess expression levels in the superficial (laminae I/II) and deep (lamina V) dorsal horn for at least three sections from each animal on the ipsilateral and contralateral sides. Lateral differences within groups were assessed using independent t-tests. If differences were observed, ipsilateral and contralateral sides were compared separately across drug and surgery conditions.

Mean pixel intensity of CD11b, NeuN, and DAPI labeling was quantified using ImageJ software (National Institutes of Health). CD11b, NeuN, and DAPI expression levels were compared in the deep and superficial dorsal horn using a two-way analysis of variance (ANOVA) with surgery and drug as between subjects factors, followed by Sidak post hoc analysis (Prism 6.0; GraphPad Software, San Diego, CA).

CB2 immunoreactive cells were counted manually by two blinded counters and averaged for each image. Cell counts were analyzed by three-way ANOVA to compare regions of the dorsal horn, surgical group, and drug treatment. To determine cellular expression patterns of CB2 receptor labeling, channels were merged for each set of images for CB2 and CD11b and for CB2 and NeuN to assess microglial and neuronal expression, respectively.

Immunohistochemistry optimization

CB2 receptor antibody specificity was assessed by preincubating the antibody for 30 min at room temperature in an immunogenic blocking peptide (ab45941, lot:GR101946; Abcam) consisting of the amino acid sequence of the target binding site before it was added to the tissue. Several concentrations were assessed before tissue incubation (0, 100, 500, 1000, and 5000 ng/mL). As negative controls, individual sections from each group were set aside and labeled as described above, omitting either the primary or secondary antibody to show loss of labeling.

Results

Before surgery, all rats displayed maximum withdrawal threshold scores (15 g) in the von Frey test of mechanical allodynia. Scores of sham and pain-naïve animals remained at this upper cutoff across 10 days of testing with no variance within or between groups. In contrast, CCI rats showed a significant decline in mechanical withdrawal thresholds over 10 days postsurgery (F(2.12)=29.93, p<0.01), confirming the effectiveness of CCI surgery: mean (SEM) day 7=7.76 g (4.76) and day 10=2.47 g (1.10).

Representative images of DAPI labeling for each surgical group are shown in the top panels of Figure 1. Quantification of mean pixel intensity revealed no lateral differences in the deep (Fig. 1A) or superficial (Fig. 1B) dorsal horn, so data were pooled for subsequent analyses. In both areas of the dorsal horn, DAPI labeling was upregulated after surgery (deep [F(2,269)=47.50 p<0.01], superficial [F(2,265)=59.60, p<0.01]) and drug administration (deep [F(1,269)=5.950, p<0.05], superficial [F(2,265)=8.012, p<0.01]), although the pattern differed across conditions (drug x surgery interactions: deep [F2,269)=5.28, p<0.01], superficial [F(2,265)=6.58, p<0.01]). Post hoc analyses revealed increased labeling in deep and superficial areas of sham and CCI, compared to pain-naïve rats treated with saline (ps<0.01), and no difference between the two surgical groups. Morphine-treated rats exhibited a stepwise increase in labeling from pain-naïve to sham and from sham to CCI rats, although only the first comparison was statistically significant (p<0.05). In other words, the difference in labeling between pain-naïve and sham rats was larger in saline (p<0.01) than in morphine (p<0.05) groups.

FIG. 1.

FIG. 1.

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Top panels: Representative DAPI labeling of cell nuclei by fluorescent IHC is shown within the deep (left two columns) and superficial (right two columns) spinal dorsal horn of pain naïve (top row), sham (middle row), and CCI (bottom row) animals. Sections from rats treated for 10 days with saline or morphine are shown in the left and right columns of each set of panels. 40×magnification; scale bar=100 μm. (A, B) Bars represent mean (±SEM) pixel intensity of DAPI labeling in the deep and superficial dorsal horn of pain naïve, sham, and CCI animals treated with 10 days of saline or morphine. n=36–48 sections from four animals per group pooled from both sides. *p<0.05; ****p<0.0001. a.u., arbitrary units; CCI, chronic constriction injury; DAPI, 4′,6-diamidino-2-phenylindole; IHC, immunohistochemistry.

As shown in Figure 2, surgery induced an upregulation of CD11b expression ipsilateral to the injury in both the deep (F(2,63)=22.68, p<0.0001) and superficial (F(2,63)=15.05, p<0.01) dorsal horn. With the exception of labeling in the superficial dorsal horn of saline-treated rats, CD11b expression was significantly higher in CCI rats than in the other two groups (post hoc ps<0.01), which did not differ from each other (ps>0.05). Morphine decreased CD11b labeling in the deep (F(1,63)=4.25, p<0.05) but not the superficial (F(1,63)=1.14, p=0.2899) dorsal horn, although none of the post hoc analyses reached statistical significance (ps>0.05). There was no drug x surgery interaction in either the deep (F(2,63)=1.70, p=0.19 or superficial (F(2,63)=1.78, p=0.18) areas and no changes in CD11b expression across groups on the contralateral side (data not shown).

FIG. 2.

FIG. 2.

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Top panels: Representative CD11b labeling of microglia by fluorescent IHC within the deep (left two columns) and superficial (right two columns) spinal dorsal horn in pain naive (top), sham (middle), and CCI (bottom) animals. Sections from rats treated for 10 days with saline or morphine are shown in the left and right columns of each set of panels. 40×magnification; scale bar=100 μm. (A, B) Bars represent mean (±SEM) pixel intensity of CD11b labeling on the ipsilateral side in the deep and superficial dorsal horn of pain naïve, sham, and CCI animals treated with 10 days of saline or morphine. n=9–12 sections from four animals per group. **p<0.01, ***p<0.0001, ****p<0.0001.

As with DAPI labeling, the effects of surgery on NeuN labeling (Fig. 3) interacted with drug condition in both the deep (F(2,132)=3.79, p<0.05) and superficial (F2,129)=5.34, p<0.01) areas. NeuN expression was decreased in CCI compared to pain-naïve rats in the deep (F(2,132)=5.22, p<0.01) and superficial (F(2,129)=9.35, p<0.01) dorsal horn, but only in saline-treated rats (deep: p<0.01; superficial: p<0.01). In addition, there was no effect of drug treatment on NeuN expression in the deep dorsal horn (F(1,132)=2.97, p=0.09), whereas morphine reduced NeuN labeling in the superficial dorsal horn (F(1,129)=5.37, p<0.05). Post hoc analyses showed significantly higher NeuN labeling in CCI rats treated with morphine, compared to CCI saline-treated rats (p<0.01) (Fig. 3B).

FIG. 3.

FIG. 3.

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Top panels: Representative NeuN labeling of neurons by fluorescent IHC within the deep (left two columns) and superficial (right two columns) spinal dorsal horn in naive (top), sham (middle), and CCI (bottom) animals. Sections from rats treated for 10 days with saline or morphine are shown in the left and right columns of each set of panels. 40×magnification; scale bar=100 μm. (A, B) Bars represent mean (±SEM) pixel intensity of NeuN labeling in the deep and superficial dorsal horn of pain naïve, sham, and CCI animals treated with 10 days of saline or morphine. n=18–24 sections from four animals per group pooled from both sides. **p<0.01, ****p<0.0001. NeuN, neuronal nuclei.

Figure 4 presents data from two separate immunolabeling experiments: the top panels show NeuN and CB2 receptor labeling; the bottom panels show CD11b and CB2 receptor labeling. Merged images from each experiment are presented in the right column of each panel. The CB2 antibody predominantly labeled NeuN-positive cells in both the deep and superficial dorsal horn, with few NeuN-positive cells that were not immunoreactive for CB2 and a small proportion of CB2 expressing cells that were NeuN-negative. Qualitative observations revealed that some non-neuronal cells co-expressed CD11b, but many did not and their morphology still appeared more neuronal than glial. NeuN-negative cells appeared morphologically similar to activated microglia that display retracted processes and amoeboid shape.

FIG. 4.

FIG. 4.

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Sample images from fluorescent colocalization experiments showing double-labeling in lumbar spinal cord sections of saline-treated animals. Top group: DAPI staining of cell nuclei (blue), NeuN labeling of neurons (red), CB2 receptor labeling (green), and merged images of NeuN and CB2 labeling. Bottom group: DAPI staining of cell nuclei (blue), CD11b labeling of microglia (green), CB2 receptor labeling (red), and merged images of CD11b and CB2 labeling. Within the top and bottom groups of panels, whole sections are shown at 5×magnification (top rows; scale bar=500 μm), followed by 40×magnification images in the deep (middle rows) and superficial (bottom rows) dorsal horn (scale bars=100 μm).

Figure 5 shows the results of CB2 receptor labeled cell counts; analysis of these data revealed that both surgery (F(2,260)=5.22, p<0.01) and morphine treatment (F(1,260)=54.85, p<0.01) induced a significant upregulation in CB2 labeling. The main effect of area (F(1,260)=1555.58, p<0.01) indicated that labeling was significantly higher in the superficial, compared to the deep, dorsal horn. None of the two-way or three-way interactions were statistically significant (ps>0.05). Post hoc tests confirmed that morphine increased CB2 receptor labeling in both the deep and superficial dorsal horn for each surgery group (ps≤0.05).

FIG. 5.

FIG. 5.

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Immunoreactive cells expressing CB2 receptors within the deep and superficial dorsal horn in pain naïve, sham, and CCI animals after 10 days of saline or morphine treatment. Bars represent mean (±SEM) of manual counts of two blinded individuals, with data pooled from both sides. n=18–24 sections from four animals per group. *p=0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Preincubation of the antibody with its immunizing peptide, which shared the amino acid sequence of the target binding site on the CB2 receptor, resulted in complete loss of labeling, even at the lowest concentration (100 ng/mL), confirming that the antibody was selectively targeting the appropriate binding site (Fig. 6B–D, F–H). Additionally, omission of the primary (Fig. 6A, E) or secondary antibodies caused a complete loss of cell labeling.

FIG. 6.

FIG. 6.

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Loss of labeling by CB2 primary antibody omission and immunizing peptide block. Representative sample images are shown at 5×(A–D) and 40×(E–H) magnification in the superficial dorsal horn of sections with the primary antibody omitted (A, E) or sections incubated overnight with the optimal concentration of CB2 primary (1:250; B–D and F–H). Some sections (C, D, and G, H) were incubated with antibody that had been preincubated (30 min) with immunizing peptide corresponding to the amino acid binding sequence targeted by the antibody.

Discussion

As expected, both surgery and chronic morphine administration dramatically altered the expression of neuronal and microglia cells in the spinal cord dorsal horn of rats. Surgery, itself, increased total cell counts, inferred from DAPI labeling, although only CCI animals displayed an upregulation of CD11b ipsilateral to the site of injury. Higher cell counts in this group, therefore, likely reflect an infiltration and proliferation of microglia in the dorsal horn following nerve injury. Further studies specifically labeling activated microglia using CD68 or assessment of relative expression of CD11b:CD45 through flow cytometry could provide a more thorough characterization of microglia phenotypes under these conditions. An increase in DAPI labeling in sham-lesioned rats suggests that surgical procedures, in the absence of CCI, may trigger changes in spinal cord cell populations that are independent of microglia. In both areas of the dorsal horn, saline-treated rats displayed a stepwise decline in NeuN labeling, from naïve to sham to CCI groups. This points to a surgery-induced loss of neurons, although confirmation of this idea would require the use of additional molecular markers. The loss of NeuN-expressing cells (i.e., neurons) following nerve injury was reduced in rats that received chronic morphine treatment. This suggests that morphine may provide neuroprotective effects against neuronal loss in postsurgical conditions, at least in superficial layers of the dorsal spinal horn.

A primary goal of our study was to examine how pain- and morphine-induced alterations in neuronal and microglia populations in the spinal cord relate to CB2 receptor expression. Despite initial controversy,6 there is a general consensus that CB2 receptors are expressed on neurons in both rodents and primates.33 Our findings add to this evidence in that the CB2 antibody in our experiment predominantly labeled receptors on neurons, matching previous evidence for minimal colocalization of CD11b (OX-42) and CB2 receptor labeling34 and results using different antibodies in other brain regions.35 The fact that CB2 receptors were expressed primarily on neurons may explain why we did not see a significant increase in CB2 receptor expression in chronic pain animals: surgery caused a significant loss of NeuN labeling such that upregulation of CB2 receptor expression was lower than would be expected if CB2 receptors were co-expressed with the microglial marker CD11b. In addition, the significant increase in CB2 receptor expression following morphine administration was independent of surgery condition and occurred in both regions of the dorsal spinal cord. This fits with evidence for increased CB2 mRNA expression in a variety of brain regions following chronic opioid treatment.36

As a secondary measure of spinal CB2 receptor expression, immunoreactive cells were counted manually. Morphologically, the majority of CB2 labeling appeared to be somatic, with much of the receptor labeling in the cytosol and membrane. Merging channels from the two separate experiments that co-labeled CB2 receptors and neurons (via NeuN) or CB2 receptors and microglia (via CD11b) revealed large proportions of CB2 labeled cells co-expressing NeuN, whereas there was little co-localization of CB2 receptors with CD11b, regardless of surgery or drug conditions. A small proportion of neurons did not express CB2 receptors and a slightly larger proportion of CB2-positive cells did not express NeuN, although their morphology still appeared more neuronal than glial. Moreover, some NeuN-negative cells appeared morphologically similar to activated microglia that display retracted processes and an amoeboid shape. These general observations match studies reporting a lack of CB2 receptor expression in CD11b-positive cells, and increased CB2 receptor labeling on specific neuronal subtypes within the CNS, including a loss of CB2 labeling following preincubation with the immunogenic peptide.35

Finally, we should note that extensive steps to characterize the CB2 receptor antibody were undertaken. Descriptions of the antibody labeling from the manufacturer describe some neuronal localization at the membrane and within the soma, and distribution within apical dendrites of pyramidal cells. In our experiments, there was a complete loss of labeling when the primary CB2 receptor antibody was omitted (Fig. 6) and preincubation of the antibody with its immunizing peptide, which shared the amino acid sequence of the target binding site on the CB2 receptor, resulted in a concentration-dependent decrease in labeling. This confirms that the antibody was selectively targeting the appropriate binding site,37,38 although there is a small possibility that the same sequence could be expressed by another nontarget protein. This seems unlikely given the protein length and conformational location. In addition, western blot data, provided by the manufacturer, showed the expected single dark band at 45 kDa. At the same time, we recognize that further control conditions, including one in which we employ another CB2 receptor antibody that targets a different epitope of the antigen,39 would increase confidence that our methodology was selectively targeting the CB2 receptor protein. In some studies, genetic deletion of the coding region for the CB2 receptor has been used to characterize antibody specificity, but the homology between mouse and rat CB2 is not complete,35 and questions have been raised as to whether the CB2 receptor is completely absent in these mice.40 Thus, the use of these genetically modified mice may not provide pertinent information for characterization of this antibody.

Conclusions

In sum, our finding that CB2 receptors are localized on NeuN-labeled cells in the rat spinal cord extends previous evidence for neuronal expression of CB2 receptors in other brain regions.33 If, as proposed, endocannabinoids have neuroprotective effects,24,40 these could be mediated, at least partially, through a population of CB2 receptors we identified, as CB2 receptor activation in the spinal cord inhibits neuronal responses to inflammation41 and reduces NP pain.42 Although we did not test it directly, the upregulation of CB2 receptors we observed after morphine administration may reflect opioid-cannabinoid interactions,43 which are manifested as CB2-mu opioid receptor synergy in measures of anti-nociception,44 analgesia, and tolerance.45 Given that CB2 receptors play a role in a variety of functions ranging from emotional processing46 to cognition,47,48 uncovering mechanisms of CB2 receptor activation in the central nervous system may provide new insights into therapeutic treatments for a variety of disorders.

Acknowledgments

We thank Katia Befort and Eric Dumont for helpful comments on data interpretation and article preparation. Queen's University is situated on traditional Anishinaabe and Haudenosaunee Territory.

Abbreviations Used

ANOVA

analysis of variance

BSA

bovine serum albumin

CCI

chronic constriction injury

CNS

central nervous system

DAPI

4′,6-diamidino-2-phenylindole

IHC

immunohistochemistry

NeuN

neuronal nuclei

NGS

normal goat serum

NP

neuropathic

PB

phosphate buffer

PFA

paraformaldehyde

TBS

tris-buffered saline

TBS-T

TBS-triton

Author Disclosure Statement

None of the authors have any personal or financial conflicts of interest to declare.

Funding Information

This work was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada to M.C.O. (203707) and an NSERC graduate fellowship to P.G.

Cite this article as: Grenier P, Sunavsky A, Olmstead MC (2021) Morphine induces upregulation of neuronally-expressed CB2 receptors in the spinal dorsal horn of rats, Cannabis and Cannabinoid Research 6:2, 137–147, DOI: 10.1089/can.2020.0004.

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