Morphine administration induced anxiolytic-like effects on day 1, which was significantly decreased on day 8 of the repeated injection
The light/dark box test is based on an approach-avoidance conflict between exploration of novel environments and avoidance of a brightly lit open space. In this test, a higher time spent in the lightbox is interpreted as lower anxiety in the rats. The results revealed a significant interaction between the morphine treatment and days of testing on total time spent in the lightbox [F (1, 14) = 66, P < 0.001]. The main effects for both factors on total time spent in the lightbox were also significant; for drug [F (1, 14) = 88, P < 0.001], and for days of testing [F (1, 14) = 139, P < 0.001]. According to the post hoc Tukey’s test, morphine induces anxiolytic-like effects on day 1 compared with the saline-treated group (P < 0.001). However, morphine after the repeated injections induces anxiety revealed by a significant decrease in lightbox time spent and locomotion compared with the saline-treated control group on day 8 (P < 0.001).
The results also revealed a significant interaction between both factors on the number of locomotor activity in the light box [F (1, 14) = 57, P < 0.001]. The main effects for both factors, including the drug and days of testing on locomotion in the light box were also significant; for drug [F (1, 14) = 91, P < 0.001], and for days of testing [F (1, 14) = 18, P < 0.001]. The results revealed no significant interaction between both factors on rearing behavior in the light box [F (1, 14) = 0.7, P > 0.05]. The main effect of days of testing on rearing behavior in the light box was also not significant [F (1, 14) = 4.4, P > 0.05], but the main effect of drug on rearing behavior in the light box was significant [F (1, 14) = 14, P < 0.01]. The results also indicated a significant interaction between the morphine treatment and days of testing on crossing between light and dark boxes [F (1, 14) = 39, P < 0.001]. The main effects for both factors on crossing or transitions between both boxes were also significant; for drug [F (1, 14) = 20, P < 0.001], and for days of testing [F (1, 14) = 58, P < 0.001] (Fig. 1).
Naloxone precipitated withdrawal expressions confirmed morphine dependence after 8 days of repeated administration of the drug
On day 8 of the repeated injection, naloxone-precipitated withdrawal symptoms were assessed to confirm dependence to morphine in two independent experimental groups. The results of independent sample t-test revealed significant increases in the number of all withdrawal graded signs, including locomotor activity [t (14) = 5.1, P < 0.001], rearing [t (14) = 9.6, P < 0.001], jumping [t (14) = 6.2, P < 0.001], writhing [t (14) = 9.8, P < 0.001], rubbing [t (14) = 11.3, P < 0.001], and shaking [t (14) = 5.6, P < 0.001], as well as the total withdrawal score [t (14) = 9.9, P < 0.001] in morphine-treated group compared with saline-treated control group (Fig. 2).
The Tlr1 gene expression decreased but the Tlr4 gene expression did not significantly alter in the PFC after repeated injection of morphine
We examined gene expression of toll-like receptor 1 (Tlr1) and Tlr4 in the PFC after 8 days of the repeated injection of morphine. Analysis of the qPCR results revealed that the Tlr1 gene expression significantly decreased [t (14) = 3.8, P < 0.01] but no significant alteration was detected for Tlr4 gene expression [t (14) = 0.4, P > 0.05] in the PFC in morphine-treated group compared with saline-treated control group (Fig. 3).
The Il1, Tnfα, and Il6 gene expression increased but gene expression of their receptors except for Il6r decreased in the PFC after repeated injection of morphine
We examined gene expression of proinflammatory cytokines, including Il1, Tnfα, Il6 and their respective receptors, including Il1r, Tnfr, and Il6r in the PFC after 8 days of the repeated injection of morphine. Analysis of the qPCR data revealed significant increases in the gene expression of Il1 [t (14) = 13.04, P < 0.001], Tnfα [t (14) = 5.4, P < 0.001], and Il6 [t (14) = 5.3, P < 0.001] compared with the saline-treated control group. The results also revealed that the gene expression of Il1r [t (14) = 6.2, P < 0.001] and Tnfr [t (14) = 5.5, P < 0.001] significantly decreased but the Il6r gene expression [t (14) = 4.7, P < 0.001] significantly increased in morphine-treated group compared with the saline-treated control group (Fig. 4).
Repeated injection of morphine had no effect on Erk1 gene expression but significantly increased the p38 gene expression and decreased Jnk3 gene expression in the PFC
Protein kinases are considered as key modulators of the cellular adaptations associated with opioid tolerance and dependence (Liu and Anand 2001). We examined the expression of mitogen-activated protein kinases (MAPK), including Erk1, p38α, and Jnk3 MAPKs in the PFC after 8 days of repeated administration of morphine. Analysis of the qPCR results revealed a significant increase in p38α gene expression [t (14) = 13.5, P < 0.001] but a significant downregulation in Jnk3 gene expression [t (14) = 6.2, P < 0.001] in rats treated with morphine compared with saline-treated group. However, no group difference was detected for Erk1 gene expression between the experimental groups [t (14) = 1.9, P > 0.05] (Fig. 5).
Repeated injection of morphine increased gene expression of Creb1 and Nfkb but decreased the Fos gene expression in the PFC
A growing body of researches shows the effects of chronic morphine treatment on gene expression are mediated by alterations in transcription factors (Bali and Kenny 2019; Robison and Nestler 2011). We assessed the expression of cAMP response element-binding (Creb1), nuclear factor- kappa B (Nfkb) and Fos proto-oncogene, AP-1 transcription factor subunit (Fos) in the PFC after 8 days repeated injection of morphine. The gene expression results revealed significant upregulation in the gene expression of Creb [t (14) = 12.2, P < 0.001] and Nfkb [t (14) = 6.3, P < 0.001] in the PFC after repeated injection of morphine compared with the saline-treated group. However, the Fos expression was significantly downregulated in the PFC after repeated injection of morphine compared with the saline-treated group Fos [t (14) = 4.2, P < 0.001] (Fig. 6).
Repeated injection of morphine increased expression of pre-miRNAs, including Let-7c1, mir-133b, and mir365 in the PFC
A growing body of evidence supports that chronic morphine treatment affects miRNAs expression, which in turn can influence tolerance and addiction to morphine. We assessed the expression of three pre-miRNAs, including Let-7c1, mir-133b, and mir-365 in the PFC on day 8 of the repeated injection. The qPCR results revealed significant increases in the expression of Let-7c1 [t (14) = 10.4, P < 0.001], mir-133b [t (14) = 10.1, P < 0.001], and mir-365 [t (14) = 10.9, P < 0.001] in the PFC after repeated injection of morphine compared with the saline-treated group (Fig. 7).