The Decrease In The Anxiolytic Effect of Morphine After Repeated Use of The Drug In Rats is Associated With Inammatory Cytokine Signaling Pathways In The Prefrontal Cortex

We aim to examine anxiety-like behaviors and expression of specic genes complicated in neuroinammation in the prefrontal cortex (PFC) after repeated use of morphine. A group of male Wistar rats received injections of morphine (10 mg/kg) twice a day for eight days while a control group received saline (1 ml/kg) instead of morphine. On days 1 and 8, anxiety-like behaviors were evaluated using a light/dark box test. On day 8, opioid dependence was conrmed by measuring the behavioral expression of morphine withdrawal precipitated with naloxone. Expression of neuroinammation genes were also evaluated at mRNA levels in the PFC on day 8. The results revealed that morphine induced anxiolytic-like effects on day 1, which signicantly decreased after the repeated injection of the drug on day 8. The results also revealed that repeated morphine injection signicantly increased the mRNA level of Il1, Tnfα, and Il6 but decreased Il1r and Tnfr while increased Il6r in the PFC. The gene expression results also revealed a signicant decrease in Tlr1 but not in Tlr4 in the PFC of morphine-dependent rats. Although Erk1 expression had no signicant alteration but p38 increased and Jnk3 decreased signicantly in the PFC in morphine-dependent rats. Creb and Nfkb signicantly increased but Fos expression decreased. Let-7c, mir-133b, and mir-365 also signicantly increased in the PFC in morphine-dependent rats. We conclude that the alteration in neuroinammatory pathways at gene expression level in the PFC may party underlie neuroadaptive changes leading to the decrease in anxiolytic effect of morphine in dependent rats. equal variance tests. A mixed between-within subject’s ANOVA with two factors, including “the drug” with two levels (saline and morphine) and “days of testing” with two levels (days 1 and 8) was performed for analyzing anxiety-like behaviors. Post hoc Tukey’s test was used for pairwise comparisons. An unpaired two-tailed Student t-test was used to analyze naloxone precipitated withdrawal symptoms and gene expression data. P < 0.05 was considered as a statistically signicant level. Data analysis and plotting data were done using GraphPad Prism version 9.0. The corresponding author is responsible for data sharing upon reasonable request.


Introduction
Opioids, such as morphine mainly are used for pain control but their prolonged use not only increases the risk of analgesic tolerance but also induces dependence (Ueda and Ueda 2009;Watkins et al. 2009).
Acute morphine administration has also anxiolytic-like effects ( Since the PFC is directly and indirectly affected by repeated use of morphine (Koob and Volkow, 2010;Quirion et al., 1983), it may have important roles in morphine side effects, including cognitive impairment and anxiety.
At the molecular level, opioids modify gene expression pro le in the brain reward regions, such as the PFC, and these alterations are considered as the origins of the structural and functional remodeling of the reward pathway after chronic use of drugs (Ahmadi et  Li 2020). However, little is known on the role of in ammatory cytokines and immune signaling in different brain areas on anxiety-like behaviors induced by prolonged use of morphine.
This study aimed to examine gene expression of toll-like receptors, proin ammatory cytokines, and their receptors, and downstream signaling molecules in the PFC after repeated morphine injection to assess their involvement in anxiety-like behaviors in rats. Different microRNAs (miRNAs) have also been identi ed to be affected with chronic use of morphine (Barbierato et al. 2015;Huang et al. 2019). Based on target gene prediction for miRNAs, we also examined the expression of pre-miRNAs for let7-C1, mir-133b, and mir-365, which have a putative role to modulate the expression of in ammatory cytokine receptors.

Subjects
Sixteen male Wistar rats (Rattus norvegicus) weighing 280 ± 20 g were used. Rats were kept in a colony room under constant temperature (22 ± 2 °C) and 12-12 h light/dark cycle (light off at 19:00). The animals had free access to food and water except during the behavioral experiments. All efforts were made to minimize the number of animals used and their suffering. All procedures were done according to the Guide for the Care and Use of Laboratory Animals (2011) prepared by the National Academy of Sciences' Institute for Laboratory Animal Research. The Research Ethics Committee (REC) at the University of Kurdistan approved the study protocol (IR.UOK.REC.1398.021).

Drug treatment
Animals were randomly assigned to either a control saline-treated or a morphine-treated group. Morphine sulfate (Temad, Daroopakhsh Co., Tehran, Iran) was dissolved in physiologic saline and the morphinetreated group received a subcutaneous injection of morphine (10 mg/ml/kg) twice a day for eight consecutive days. The control group received saline (1 ml/kg) instead of morphine during the eight days of the treatment period.
Light and dark box test of anxiety Anxiety-like behaviors were examined with a light/dark box test of anxiety based on our previous report (Ahmadi and Khaledi 2020). The apparatus consisted of a light chamber (30*30*24 cm) and a dark chamber (20*30*24 cm). The oors of the chambers were marked off in nine and six squares (10 × 10 cm), respectively. A white light source (6 W) lit the light chamber, whereas the dark chamber was illuminated by a dim red-light source (3 W), which both of them placed 40 cm above the oor of the chambers. The height of the dividing wall between the two chambers was 40 cm to prevent the emission of the light sources into the opposite chamber. A connecting door (10 × 10 cm) in the middle and at the oor level of the dividing wall permits voluntary transitions of the animals between the chambers. The oor and wall of both chambers were made of Plexiglass and cleaned between trials with an ethanol solution (10% v/v) and dried fully before the testing. Thirty min after the drug treatments at the morning session on days 1 and 8, the light/dark test was performed for each rat. The test started when each rat was placed in the center of the white area facing away from the middle door and was allowed for 5 min exploring the apparatus. Each animal was videotaped during the 5 min of exploration through a video camera xed above the apparatus (100 cm from the oor of the apparatus). Then, the videos were analyzed by a researcher blinded to experimental groups and different parameters including 1) lightbox time spent, 2) the number of squares crossing as an index of locomotor activity, and 3) the number of rearing behavior in the white chamber, as well as 4) the total crossing or transitions between the two chambers were evaluated (Bourin and Hascoet 2003;Costall et al. 1989;Ramos 2008).

Naloxone-precipitated withdrawal test
We assessed the induction of morphine dependence by measuring the behavioral expression of morphine withdrawal precipitated with naloxone on day 8 of the injections. In two independent groups of animals, naloxone hydrochloride (1 mg/kg, i.p.) was intraperitoneally injected 4 h after injection of saline or morphine at the morning session on day 8 (Yu et al. 2014). Each rat was immediately after the naloxone injection moved to a square clear Plexiglass chamber (40×40×40 cm high), and was videotaped for 15 mins with two video cameras (Sony, Japan). Two classes of signs, including graded and checked signs, were evaluated and scored as described elsewhere (Table 1) (Yu et al. 2014). The somatic signs of withdrawal were recorded as the number of events observed during 15 mins of the recorded experiment.

Brain dissections
In two experimental groups, two h after completion of the light/dark box test on day 8, each rat was decapitated, the whole brain was quickly removed from the skull, and the bilateral PFCs were immediately dissected on an ice-chilled sterile surface (Ahmadi et al. 2015a;Ahmadi et al. 2015b;Chiu et al. 2007).
Tissues were immediately frozen in liquid nitrogen and stored in a -80 freezer until using for molecular analysis.
Real-time polymerase chain reaction Total RNA was extracted by using a commercial kit according to the manufacturer's supplied protocol (High Pure miRNA isolation kit, Roche, Germany). The total RNAs were reverse transcribed to complementary DNA (cDNA) by using a two-step cDNA synthesis kit according to the manual provided by the manufacturer (First Strand cDNA Synthesis Kit, Thermo Fisher Scienti c, USA). Real-time PCR was performed using LightCycler 96 system thermal cycler (Roche, Germany). Each PCR reaction of 20 µl volume consisted of 10 µl YTA SYBR Green qPCR MasterMix (Yekta Tajhiz Azma Co., Tehran, Iran), 8 µl cDNA (4 ng/µl), 2 µl mix of the forward and reverse primers (5 µM). Triplicate technical repeats were determined for each biological sample. Thermal cycling was initiated with a pre-incubation step (95˚C for 30 Sec), followed by 40 cycles of two-step ampli cation (95˚C for 5 Sec and 60˚C for 30 Sec), followed by melting (95˚C for 5 Sec, 60˚C for 60 Sec, and 95˚C for 1 Sec), and nalized by cooling at 50˚C for 30 Sec.
We used the 2 -ΔΔCT method to assess relative gene expression (Livak and Schmittgen 2001). Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was selected as a housekeeping gene and was used for normalization. The sequences of PCR primers and the respective amplicon sizes have been summarized in Table 2.

Statistical analysis
We examined the normality of the data with the Shapiro-Wilk test and assessed equality of variance with the Brown-Forsythe test. All data passed normality and equal variance tests. A mixed between-within subject's ANOVA with two factors, including "the drug" with two levels (saline and morphine) and "days of testing" with two levels (days 1 and 8) was performed for analyzing anxiety-like behaviors. Post hoc Tukey's test was used for pairwise comparisons. An unpaired two-tailed Student t-test was used to analyze naloxone precipitated withdrawal symptoms and gene expression data. P < 0.05 was considered as a statistically signi cant level. Data analysis and plotting data were done using GraphPad Prism version 9.0. The corresponding author is responsible for data sharing upon reasonable request.

Results
Morphine administration induced anxiolytic-like effects on day 1, which was signi cantly decreased on day 8 of the repeated injection The light/dark box test is based on an approach-avoidance con ict between exploration of novel environments and avoidance of a brightly lit open space. In this test, a higher time spent in the lightbox is Naloxone precipitated withdrawal expressions con rmed 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 con rm dependence to morphine in two independent experimental groups. The results of independent sample ttest revealed signi cant 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 signi cantly 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 signi cantly decreased [t (14) = 3.8, P < 0.01] but no signi cant 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).
Repeated injection of morphine had no effect on Erk1 gene expression but signi cantly increased the p38  (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 in uence 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.  (Fig. 7).

Discussion
Morphine is still the rst choice for controlling chronic pain in the clinic. Unfortunately, besides tolerance to the analgesic effect of morphine, anxiety has been described as an important comorbidity in patients suffering from chronic pain (Roeska et al. 2008). The present results revealed that morphine induces anxiolytic-like behaviors on day 1 of the repeated injection as revealed by a signi cant increase in lightbox time spent compared with the saline-treated rats. On day 1, consistent with the increased lightbox time spent, the locomotor activity of the animals in the lightbox signi cantly increased compared with the saline-treated control group. This signi cant increase in locomotion in the lightbox on day 1 discards the possibility of freezing behavior in the lightbox and further supports anxiolytic-like effects of morphine on day 1. However, rearing behavior in the lightbox and crossing between two chambers in morphine-treated animals showed a tendency to increase compared with the saline-treated animals but no signi cant alteration was detected on day 1. On contrary, repeated morphine injection induced anxietylike behaviors on day 8 of the treatment as revealed by signi cant decreases in lightbox time spent and locomotion in the light chamber compared with the saline-treated animals. However, on day 8, the number of crossing between light and dark chambers in the morphine-treated group was signi cantly higher than its respected number on day 1, which may also re ect an increase in anxiety or a decrease in anxiolyticlike effects after 8 days of repeated injection of morphine due to approach-avoidance con ict between exploration of both chambers. According to research, morphine shows anxiolytic-like properties during initial exposure, which is modulated by nitric oxide (NO) systems ( The involvement of TLR2 and TLR4 in the development of morphine tolerance and dependence has been previously reported (Eidson and Murphy 2013; Zhang et al. 2011b). However, there are not considerable reports on the involvement of TLR1 in morphine functions. The present results indicated downregulation of Tlr1 in the PFC after prolonged morphine injections for 8 days. However, no signi cant alteration was detected in the Tlr4 gene expression after repeated injection of morphine. The gene expression results in the present experiments also revealed signi cant increases in Il1, Tnfα, and Il6 expression in the PFC after repeated morphine treatment compared with saline-treated group. On contrary, Il1r and Tnfr were downregulated but Il6r was upregulated in the PFC in morphine-dependent rats. It has been shown that prolonged administration of morphine is followed by less effective opioid actions (Benyamin et al. 2008). Accumulating data shows that the ineffectiveness of morphine is results from adaptation within opioid receptor-expressing neurons in the reward pathways. However, this adaptation is not only restricted to neurons but also activation of glial cells by morphine, and subsequent production of proin ammatory cytokines is considered as an alternate process affecting morphine actions. Therefore, the role of glia and TLR4 in the development of morphine tolerance and addiction during the past two decades has been postulated and recently reviewed (Eidson and Murphy 2019; Wu and Li 2020). The present results may support the involvement of TLR1 in morphine actions in the PFC. Besides, activation of glia in the PFC after repeated morphine treatment in rats is proposed as revealed by the upregulation of proin ammatory cytokines. On the other hand, downregulation of Il1r and Tnfr may serve as homeostatic adaptations in response to the increased level of these cytokines due to repeated administration of morphine. The central immune signaling profoundly affects behavioral responses in the brain (Hutchinson et al. 2011).
Therefore, the increases in anxiety-like behaviors after repeated use of morphine may result, at least partly, from the altered cytokine levels and their contribution to neuroexitability in the PFC. In support of this suggestion, it has been reported that IL-1β can, directly and indirectly, increase NMDA receptor activity, which may also contribute to neuroexcitation (Hutchinson et al. 2011). However, we did not examine protein levels of these molecules and neurotransmitter levels, and further studies are needed to con rm this hypothesis.
A growing body of evidence shows that chronic morphine administration in uences different protein  . 2015). Therefore, chronic administration of morphine via a MAPK cascade may alter the expression of proin ammatory cytokines, which in turn via perturbation in neurotransmission and ring of the PFC outputs may affect anxiety-like behaviors.
There is some evidence that shows ERK is involved in the analgesic and hyperalgesic effects of acute morphine while p38, JNK, and CREB are involved in delayed hyperalgesia induced by the opioid (de Freitas et al. 2019). There is not enough report on the role of JNK3 in the PFC in behavioral alteration after morphine treatment. We have reported recently downregulation of Jnk3 in the PFC in a rat model of hepatic encephalopathy ). Since the role of neuroin ammation has been veri ed in hepatic encephalopathy, so the decreased Jnk3 gene expression in the PFC may re ect induction of neuroin ammation after repeated injection of morphine. Considering the increased expression of in ammatory cytokines in the present study, the downregulation of Jnk3 may play as a compensatory response to prevent further neuroexcitation induced by cytokines.
Activation of p38 MAPK in the spinal microglia mediates morphine antinociceptive tolerance (Cui et al. 2006), and its inhibition prevents the antinociceptive tolerance induced by the drug (Cui et al. 2008). p38 MAPK signaling pathway in the NAc is also involved in the induction of conditioned place preference (Hong et al. 2017). However, little is known about the role of p38 MAPK in the PFC in anxiety-like behaviors after prolonged treatment of morphine. It is possible that inhibition of cytokine signaling and MAPKs pathway especially p38 MAPK also be useful in the prevention of dependence to morphine and the anxiety-like behaviors after repeated use of the drug.
The present results also revealed that Creb1 and Nfkb expression in the PFC increased but Fos expression decreased after repeated injection of morphine. CREB is a target for opioid-regulated secondary messengers to alter gene expression (Bilecki and Przewłocki 2000), and thereby may regulate different adaptive responses, including emotional behavior and drug addiction (Valverde et al. 2004). Activation of the p38/NF-κB signaling pathway in the NAc plays a critical role in morphine conditioned place preference (Zhang et al. 2011a). The expression of both proin ammatory cytokine genes such as Il1 and Tnfα is dependent on the activation of NF-κB. Neuronal CREB has a selective role in anxiety-like behavior and the somatic expression of morphine withdrawal, without participating in the rewarding properties induced by morphine (Valverde et al. 2004). It is logical to propose that the increase in the gene expression of Nfkb is in line with the increased level of the above-mentioned cytokines as seen in the present experiments. However, there is no report on mir-133b expression in the PFC after repeated use of morphine. Considering the putative targets of mir-133b and mir-365 such as Il1r and Tnf family receptors based on TargetScan target gene prediction, we suggest that the increased level of mir-133b and mir-365, at least partly, be also involved in the downregulation of these receptors in the PFC. However, further experiments may require clari ng the exact effects of these miRNAs on the expression of the proposed targets, and also affecting other target genes cannot be excluded, which needs further experiments in the future.

Conclusions
The present data revealed that prolonged morphine injection not only induces dependence on the drug but also decreases the anxiolytic effects of the drug on day 8 compared with day 1 of the injections. At gene expression level, alteration in proin ammatory cytokines including Il1, Il6, Tnfα, and their respective receptors, Tlr1, p38 and Jnk3 MAPKs, Creb, Nfkb, and Fos transcription factors as well as a few miRNAs including Let-7c1, mir-133b, and mir-365, were detected in the PFC after repeated morphine injection. It must be noticed that for many genes, transcript and protein levels do not correlate well (Tian et al. 2004), and therefore, examining protein levels of the examined receptors and downstream molecules is a limitation of our study and is suggested for future research. However, the alteration at the mRNA level has its own impact on the subsequent cellular and molecular processes in brain regions that may affect neural adaptations. Finally, considering the examined genes in the present experiments, it seems that alterations in the in ammatory signaling pathways either in neurons or in glia in the PFC after prolonged morphine treatment may affect a top-down neural system originating from the PFC projecting to the amygdala, which in turn affects emotional processing such as anxiety. However, future researches need to be conducted to examine this possibility.

Declarations
Zobeiri, Shiva Mohammadi Talvar, Kayvan Masoudi: Acquisition, analysis, interpretation of data, and Writing-Original Draft Preparation. All authors approved the nal version for publication.

Funding
This study was funded by Cognitive Sciences and Technologies Council of Iran (Contract No. D/100/11988).

Con icts of interests
The author(s) declare no potential con icts of interest concerning the research, authorship, and/or publication of this article.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.   Figure 1 Repeated injection of morphine induces anxiety-like behaviors. Two experimental groups (n = 8) received either saline (1 ml/kg) or morphine (10 mg/kg) twice a day for eight consecutive days. Anxiety-like behaviors in a light/dark box test, including lightbox time spent, locomotor activity, rearing, and crossing or transitions between the two light and dark chambers were assessed on days 1 and 8 of the injections. Each bar represents the mean ± SD of the speci ed behavior in each group on a respective day. Dots and triangles on the bars represent the individual data in each group. ** P < 0.01 and *** P < 0.001 compared with the speci ed group.

Figure 2
Repeated injection of morphine for eight days induced drug dependence. On day 8 of the repeated injection, naloxone precipitated withdrawal symptoms were assessed immediately after injection of naloxone (1 mg/kg, i.p.). Naloxone was injected 4 h after the injection of saline or morphine at the morning session. Each bar represents the mean ± SD of the speci ed sign of withdrawal in each group.
Circle dots and triangles on the bars represent the individual data in each group. *** P < 0.001 compared with the control group.     Effect of repeated morphine injection on the expression of Let7c1, mir-133b, and mir-365 in the PFC after induction of morphine dependence. Expression of three pre-miRNAs, including let-7c, mir-133b, and mir-365 were examined in the PFC on day 8 of the repeated injection. Each bar represents the mean ± SD of the gene expression data in each experimental group. Circle dots and diamond shapes on the bars represent the individual data in each group. *** P < 0.001 compared with the control group.