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. 2017 Nov;158(11):2222-2232.
doi: 10.1097/j.pain.0000000000001027.

Age-dependent plasticity in endocannabinoid modulation of pain processing through postnatal development

Charlie H-T Kwok  1 , Ian M Devonshire  1 , Amer Imraish  1 , Charles M Greenspon  1 , Stevie Lockwood  1 , Catherine Fielden  1 , Andrew Cooper  1 , Stephen Woodhams  1   2 , Sarir Sarmad  3 , Catherine A Ortori  3 , David A Barrett  3 , David Kendall  1 , Andrew J Bennett  1 , Victoria Chapman  1   2 , Gareth J Hathway  1
Affiliations

Age-dependent plasticity in endocannabinoid modulation of pain processing through postnatal development

Charlie H-T Kwok et al. Pain. 2017 Nov.

Abstract

Significant age- and experience-dependent remodelling of spinal and supraspinal neural networks occur, resulting in altered pain responses in early life. In adults, endogenous opioid peptide and endocannabinoid (ECs) pain control systems exist which modify pain responses, but the role they play in acute responses to pain and postnatal neurodevelopment is unknown. Here, we have studied the changing role of the ECs in the brainstem nuclei essential for the control of nociception from birth to adulthood in both rats and humans. Using in vivo electrophysiology, we show that substantial functional changes occur in the effect of microinjection of ECs receptor agonists and antagonists in the periaqueductal grey (PAG) and rostroventral medulla (RVM), both of which play central roles in the supraspinal control of pain and the maintenance of chronic pain states in adulthood. We show that in immature PAG and RVM, the orphan receptor, GPR55, is able to mediate profound analgesia which is absent in adults. We show that tissue levels of endocannabinoid neurotransmitters, anandamide and 2-arachidonoylglycerol, within the PAG and RVM are developmentally regulated (using mass spectrometry). The expression patterns and levels of ECs enzymes and receptors were assessed using quantitative PCR and immunohistochemistry. In human brainstem, we show age-related alterations in the expression of key enzymes and receptors involved in ECs function using PCR and in situ hybridisation. These data reveal that significant changes on ECs that to this point have been unknown and which shed new light into the complex neurochemical changes that permit normal, mature responses to pain.

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Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Figures

Figure 1.
Figure 1.
Changes in the expression of endocannabinoids (eCBs) and eCB-synthesising enzymes in the brainstem nuclei of the rat and human midbrain during postnatal development. (A and B) Mass spectrometry analysis of anandamide levels in the periaqueductal grey (PAG) and rostroventral medulla (RVM). Expression of anandamide increased during early postnatal development and reached maturity by postnatal day (P)21. (P < 0.05 in the PAG and RVM; 1-way analysis of variance [ANOVA].) (C and D) Mass spectrometry analysis of 2-arachidonoylglycerol (2-AG) levels in the PAG and RVM. No changes in the expression of 2-AG were observed in the PAG (C). Expression of 2-AG increased during early postnatal development and reached maturity by P21 in the RVM (D; P < 0.01; 1-way ANOVA). (E) Taqman real-time (RT) PCR analysis of the eCB-synthesising enzymes NAPE-PLD and DAGLα in the ventral PAG. Expression of NAPE-PLD mRNA increased during early postnatal development and reached maturity by P21 (P < 0.05; 1-way ANOVA). (F) Taqman RT-PCR analysis of NAPE-PLD and DAGLα in the RVM. No significant changes were observed. (G) Expression of NAPE-PLD mRNA was highest in the human infant midbrain compared with all other age groups tested (P < 0.01; 1-way ANOVA). Nine to 11 animals per age group used for mass spectrometry experiments. Three to 4 animals per age group for Taqman RT-PCR and immunohistochemical experiments. Four to 8 human midbrain tissue per age group used for TaqMan RT-PCR. Data shown here represent mean ± SEM. * and ** = P < 0.05 and P < 0.01, respectively, between age comparisons, 1-way ANOVA with Bonferroni multiple comparisons.
Figure 2.
Figure 2.
Changes in the expression of endocannabinoid receptors CB1, CB2, and GPR55 in the brainstem of the rat during postnatal maturation. (A) TaqMan real-time (RT) PCR analysis of CB1 receptor mRNA in the ventral periaqueductal grey (vPAG) of P10, P21, and P40 rats, no statistically significant changes were observed between the ages. (B) Fluorescent image (×20 magnification) and quantification (C) of CB1 immunoreactivity in the vPAG. White arrows denote CB1-specific terminal staining. The expression of CB1 receptors in the vPAG decreased as the animals aged, as immunoreactivity was highest at P10 (P < 0.0001; 1-way analysis of variance [ANOVA]). TaqMan RT-PCR analysis of CB1 receptors in the rostroventral medulla (RVM) of P10, P21, and P40 rats, no changes were observed between the ages. (E) Fluorescent image (×20 magnification) and quantification (F) of CB1 immunoreactivity in the RVM. White arrows denote CB1-specific terminal staining. The expression of CB1 receptors in the RVM increased as the animals aged (P < 0.01-0.001; 1-way ANOVA) (C and D) N = 3 to 5 animals per age group. CTR, control. Data shown here represent mean ± SEM. **, ***, and **** = P < 0.01, P < 0.001, and P < 0.0001, respectively, between age comparisons, 1-way ANOVA with Bonferroni multiple comparisons.
Figure 3.
Figure 3.
No changes in CB2 transcript levels in the periaqueductal grey (PAG) and rostroventral medulla (RVM) whilst the expression of the GPR55 gene is developmentally regulated. (A and B) TaqMan RT-PCR analysis oCB2 receptor mRNA in the PAG and RVM, respectively, no significant changes in were detected. (C and D) TaqMan RT-PCR analysis of GPR55 receptor mRNA in the PAG and the RVM of P10, P21, and P40 rats (P < 0.001; 1 way analysis of variance [ANOVA]). No changes were observed in the PAG, but GPR55 mRNA was transiently upregulated at P21 in the RVM (D). N = 3 to 5 animals per age group. Data shown here represent mean ± SEM. ** = P < 0.01, between age comparisons, 1-way ANOVA with Bonferroni multiple comparisons.
Figure 4.
Figure 4.
Changes in the expression of endocannabinoid receptors CB1 and CB2 in the human midbrain during postnatal maturation. (A and B) TaqMan real-time (RT) PCR analysis of CB1 and CB2 receptor mRNA, no significant changes in were detected. (C) In situ hybridisation images (×20 magnification) of CB1 receptors in preterm, term, infant, and P40 midbrain. (D) Quantification by cell-counting analysis showed that the expression of CB1 receptors is higher during early postnatal development compared with P40 (P < 0.01; 1-way analysis of variance [ANOVA]). (E) In situ hybridisation images of CB2 receptors. (F) No significant changes in the expression of CB2 receptors were observed throughout postnatal development. Four to 8 human midbrain tissue per age group was used for TaqMan RT-PCR and in situ hybridisation, respectively. Data shown here represent mean ± SEM. * and ** = P < 0.05 and P < 0.01, respectively, between age comparison, 1-way ANOVA with Bonferroni multiple comparisons.
Figure 5.
Figure 5.
Effect of intraperiaqueductal grey (PAG) and rostroventral medulla (RVM) microinjection of HU210 (CB1/2 receptor agonist, 4 μg/animal) on withdrawal reflexes to mechanical von Frey hair stimulation in P10, P21, and P40 rats. (A) Intra-PAG HU210 significantly decreased spinal reflex excitability compared with vehicle responses in all ages tested (P < 0.0001; 2-way analysis of variance [ANOVA]). This effect was more pronounced in P10 animals compared with P40s. (B) Intra-PAG HU210 significantly increased mechanical thresholds in P21 and P40 rats (P < 0.0001; 2-way ANOVA). This effect is more pronounced in P21 and P40 animals compared with P10. (C) Intra-RVM HU210 decreased spinal reflex excitabilities compared with vehicle responses in all ages tested (P < 0.0001, 2-way ANOVA). This effect was strongest in P21 animals. (D) Intra-RVM HU210 significantly increased mechanical thresholds compared with vehicle responses in all ages tested (P < 0.0001, 2-way ANOVA). This effect was strongest for P21 animals. Four to 8 animals per drug per age group. Data shown here represent the mean ± SEM. **** = P < 0.0001, between drug comparisons, 2-way ANOVA with Bonferroni multiple comparisons. # and #### = P < 0.05 and P < 0.0001, respectively, between age comparison, 2-way ANOVA with Bonferroni multiple comparisons.
Figure 6.
Figure 6.
Effect of intraperiaqueductal grey (PAG) and rostroventral medulla (RVM) microinjection of AM251 (CB1 receptor inverse agonist, GPR55 receptor agonist, 2.77 and 1.35 μg/animal) and lysophosphatidylinositol (LPI) (endogenous GPR55 agonist, 12 μg/animal) on withdrawal reflexes to mechanical von Frey hair stimulation in P10, P21, and P40 rats. (A) Intra-PAG AM251 and LPI only decreased spinal reflex excitability in P10 and P21 rats (P < 0.0001, 2-way analysis of variance [ANOVA]). There are significant age-related differences in AM251 and LPI responses between P40 and younger animals (P < 0.0001, 2-way ANOVA). (B) Intra-PAG AM251 and LPI did not change the mechanical thresholds in either P10 or P40 rats, but significantly increased it in P21 animals. There are significantly age-related differences in AM251 and LPI-mediated changes in mechanical withdrawal thresholds (P < 0.001, 2-way ANOVA). (C) Intra-RVM AM251 did not have an effect in P40 rats, but significantly reduced spinal reflex excitabilities in P10 and P21 animals. Lysophosphatidylinositol increased spinal reflex excitability in P40 rats, but decreased it in P10 and P21 animals. These age-related differences were significant (P < 0.0001, 2-way ANOVA). (D) Intra-RVM AM251 and LPI significantly increased mechanical thresholds in P10 and P21 rats, but had no effect in P40 animals. This age-related difference was significant (P < 0.0001, 2-way ANOVA). (E) Intra-PAG AM251 (2.77 and 1.35 μg) did not have an effect in P40, but significantly inhibited spinal reflex excitability in P21 animals (P < 0.0001, 1-way ANOVA). Coadministration of AM251 (1.35 μg) with the GPR55 receptor–specific antagonist ML193 (1 μg) reversed AM251-mediated inhibition of spinal reflex excitability in P21 animals. **, ***, and **** = P < 0.01, P < 0.001, and P < 0.0001, respectively, between drug comparisons, 2-way ANOVA and 1-way ANOVA with Bonferroni multiple comparisons. #, ### and #### = P < 0.05, P < 0.001 and P < 0.0001, respectively, between age comparison, 2-way ANOVA and 1-way ANOVA with Bonferroni multiple comparisons.
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