Korean J Pain.  2023 Apr;36(2):163-172. 10.3344/kjp.22372.

Calcium/calmodulin-dependent protein kinase II is involved in the transmission and regulation of nociception in naïve and morphine-tolerant rat nucleus accumbens

• Xi KW
• Chen DD
• Geng X
• Bian Y
• Wang MX
• Bian H

• KMID: 2541025
• DOI: http://doi.org/10.3344/kjp.22372

Abstract

Background
Synaptic plasticity contributes to nociceptive signal transmission and modulation, with calcium/ calmodulin-dependent protein kinase II (CaMK II) playing a fundamental role in neural plasticity. This research was conducted to investigate the role of CaMK II in the transmission and regulation of nociceptive information within the nucleus accumbens (NAc) of naïve and morphine-tolerant rats.
Methods
Randall Selitto and hot-plate tests were utilized to measure the hindpaw withdrawal latencies (HWLs) in response to noxious mechanical and thermal stimuli. To induce chronic morphine tolerance, rats received intraperitoneal morphine injection twice per day for seven days. CaMK II expression and activity were assessed using western blotting.
Results
Intra-NAc microinjection of autocamtide-2-related inhibitory peptide (AIP) induced an increase in HWLs in naïve rats in response to noxious thermal and mechanical stimuli. Moreover, the expression of the phosphorylated CaMK II (p-CaMK II) was significantly decreased as determined by western blotting. Chronic intraperitoneal injection of morphine resulted in significant morphine tolerance in rats on Day 7, and an increase of p-CaMK II expression in NAc in morphine-tolerant rats was observed. Furthermore, intra-NAc administration of AIP elicited significant antinociceptive responses in morphine-tolerant rats. In addition, compared with naïve rats, AIP induced stronger thermal antinociceptive effects of the same dose in rats exhibiting morphine tolerance.
Conclusions
This study shows that CaMK II in the NAc is involved in the transmission and regulation of nociception in naïve and morphine-tolerant rats.

Keyword

Analgesics; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Morphine; Neuronal Plasticity; Nociception; Nucleus Accumbens; Pain; Peptides; Rats

Figure

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  Fig. 1 Antinociceptive effect of microinjection of 4, 8 or 12 µg of AIP into NAc in naïve rats. The left HWL are shown in A (noxious thermal stimulation) and C (noxious mechanical stimulation); the right HWLs are depicted in B (noxious thermal stimulation) and D (noxious mechanical stimulation). The data in the experiment are expressed as mean ± standard error of the mean. Two-way ANOVA was employed to analyze the statistical difference among experimental groups. ***P < 0.001 means relative to the control group. AIP: autocamtide-2-related inhibitory peptide, NAc: nucleus accumbens, HWL: hindpaw withdrawal latency.

• 

  Fig. 2 Changes in CaMK II expression after AIP intra-NAc administration. (A) p-CaMK II/t-CaMK II and GAPDH western blotting results of the saline control and AIP groups. (B) p-CaMK II/t-CaMK II ratios are displayed using histograms. The data in the experiment are expressed as the mean ± standard error of the mean. Two-tailed Student’s t-test was employed to assess the statistical difference among groups. Differences with *P < 0.05 are deemed statistically significant. CaMK II: calcium/calmodulin-dependent protein kinase, AIP: autocamtide-2-related inhibitory peptide, NAc: nucleus accumbens, p-CaMK II: phosphorylated CaMK II, t-CaMK II: total CaMK II, GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

• 

  Fig. 3 Morphine tolerance induced by intraperitoneal morphine injection. A indicates HWL in response to noxious thermal stimulation; B represents HWLs to noxious mechanical stimulation. Rats have received intraperitoneal morphine injection for 7 days, and the antinociceptive effects are evaluate 30 minutes after the first morphine administration on day 1 and 7. The data of the experiment are expressed as mean ± standard error of the mean. Two-tailed Student’s t-test was employed to assess the statistical difference among groups. Differences with ***P < 0.001 are deemed statistically significant. HWL: hindpaw withdrawal latency.

• 

  Fig. 4 Changes in CaMK II expression in morphine-tolerant rat NAc. (A) p-CaMK II/t-CaMK II and GAPDH western blotting results of the saline control and morphine tolerance groups. (B) p-CaMK II/t-CaMK II ratios are shown in histograms. The data of the experiment are expressed as mean ± standard error of the mean. Two-tailed Student’s t-test was employed to analyze statistical differences among groups. Differences with **P < 0.01 are deemed statistically significant. CaMK II: calcium/calmodulin-dependent protein kinase, NAc: nucleus accumbens, p-CaMK II: phosphorylated CaMK II, t-CaMK II: total CaMK II, GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

• 

  Fig. 5 Antinociceptive effect of microinjection of 4, 8 or 12 µg of AIP into NAc in rats showing morphine tolerance. The left HWL are depicted in A (noxious thermal stimulation) and C (noxious mechanical stimulation); the right HWL are shown in B (noxious thermal stimulation) and D (noxious mechanical stimulation). 4, 8 or 12 µg of AIP was injected into NAc 10 minutes prior to intraperitoneal injection of 10 mg/kg morphine. Time = 0 min, indicates the start of intraperitoneal morphine injection. The data of the experiment are expressed as mean ± standard error of the mean. Two-way ANOVA was employed to analyze statistical differences among groups. ***P < 0.001 means relative to the control group. AIP: autocamtide-2-related inhibitory peptide, HWL: hindpaw withdrawal latency, NAc: nucleus accumbens.

• 

  Fig. 6 Comparison of the antinociceptive effects imparted by intra-NAc microinjection of 4, 8 or 12 µg of AIP on the HWLs of naïve rats and rats showing morphine tolerance. A and B depict HWLs to noxious thermal stimulation; C and D represent HWLs to noxious mechanical stimulation. Left HWLs are presented in A and C; right HWLs are depicted in B and D. The antinociceptive effects are evaluate 30 minutes after drug injection. The data of the experiment are expressed as the mean ± standard error of the mean. Two-tailed student’s t-test was used to assess statistical differences among experimental groups. *P < 0.05, **P < 0.01 indicate compared with the control group. 4 A, 8 A or 12 A represent intra-NAc microinjection of 4, 8 or 12 µg of AIP to naïve rats; 4 A, 8 A or 12 A + morphine represent intra-NAc microinjection of 4, 8 or 12 µg of AIP to morphine-tolerant rats. AIP: autocamtide-2-related inhibitory peptide, HWL: hindpaw withdrawal latency, NAc: nucleus accumbens.

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