Journal of Forensic Science and Medicine

ORIGINAL ARTICLE
Year
: 2016  |  Volume : 2  |  Issue : 1  |  Page : 1--7

Changes of Adenylate Cyclase and Guanylate Cyclase in the Frontal Cortex, Lenticula, Corpus Amygdaloideum, and Hippocampus in Morphine-dependent Rats


Shijun Hong, Dongxian Zhang, Lihua Li 
 School of Forensic Medicine, Kunming Medical University, Kunming, Yunnan, People's Republic of China

Correspondence Address:
Lihua Li
School of Forensic Medicine, Kunming Medical University, No. 1168 West Chun Rong Street, Cheng Gong, Kunming, Yunnan Province
People«SQ»s Republic of China

Abstract

To detect the changes of adenylate cyclase (AC) and guanylate cyclase (GC) in the four cerebral regions that are concerned with psychogenic dependence of morphine in rats, including the frontal cortex, lenticula, corpus amygdaloideum, and hippocampus. To discuss the relation between the expressions of AC and GC with the psychogenic dependence on morphine. Different periods of morphine-dependent rat models were established, and enzyme histochemistry was used to detect the variations of AC and GC in four cerebral regions. Compared with the control group, AC and GC in all the morphine-dependent groups increased. The data indicated that the amounts of AC and GC were significantly different between the morphine-dependent groups and the control group when tested at periods of 1 week, 2 weeks, 4 weeks, and 8 weeks (P ˂ 0.05 or P ˂ 0.01). There were significant differences when comparing the 1-week group with the 2-week, 4-week, and 8-week groups (P ˂ 0.05 or P ˂ 0.01). There were significant differences when comparing the 2-week dependent group with the 4-week dependent group or the 8-week dependent group (P ˂ 0.05 or P ˂ 0.01). The activities of AC and GC increased in four cerebral regions of morphine-dependent rats. The psychogenic dependence on morphine appears to be closely linked to the upgrade of AC and GC.



How to cite this article:
Hong S, Zhang D, Li L. Changes of Adenylate Cyclase and Guanylate Cyclase in the Frontal Cortex, Lenticula, Corpus Amygdaloideum, and Hippocampus in Morphine-dependent Rats.J Forensic Sci Med 2016;2:1-7


How to cite this URL:
Hong S, Zhang D, Li L. Changes of Adenylate Cyclase and Guanylate Cyclase in the Frontal Cortex, Lenticula, Corpus Amygdaloideum, and Hippocampus in Morphine-dependent Rats. J Forensic Sci Med [serial online] 2016 [cited 2021 Jan 28 ];2:1-7
Available from: https://www.jfsmonline.com/text.asp?2016/2/1/1/161631


Full Text

 Introduction



Previous results showed that the psychogenic symptoms of morphine dependence impacted on different cerebral regions, which included the frontal cortex, lenticula, corpus amygdaloideum, and hippocampus. Also, past studies have demonstrated a relationship between morphine dependence and the activity of adenylate cyclase (AC) (Nestler et al.) [1] and guanylate cyclase (GC) (Fang et al.). [2] Many researches had involved the changes of AC in the locus coeruleus (LC) (Nestler et al. [1] and Lane-Ladd et al.), [3] a region particularly related to the physiological dependence on morphine. Furthermore, the psychogenic dependence on morphine was more important and lasting. This research focused on the cerebral regions, including the frontal cortex, lenticula, corpus amygdaloideum, and hippocampus that concerned psychogenic dependence on morphine. Then, the mechanism of morphine-induced psychogenic dependence in rats was discussed.

 Materials and Methods



Animal model

Sixty healthy male and female Sprague Dawley(SD) rats that were 6-month old, weighing 260 ± 20 g were used in accordance with the Animal Experimental Ethical Committee of the Kunming Medical University's guidelines for the care and use of laboratory animals. The animals were divided into five groups (n = 12 per group). The groups were as follows: Morphine-dependent for 1 week, 2 weeks, 4 weeks, 8 weeks, and the control group. The rats of the control group were divided into four subgroups (n = 3) to match the four morphine-dependent groups. The rats were housed in a cage at 20°C with 40% humidity and under 12-h light, 12-h dark cycles, with free access to water and rat chow.

The groups of morphine-dependent rats were injected with morphine hydrochloride three times per day at 8:00 AM, 12:00 noon, and 16:00 PM for consecutive 5 days. The doses were as follows: 5 mg/kg on the 1 st day; 10 mg/kg on the 2 nd day; 20 mg/kg on the 3 rd day; 40 mg/kg on the 4 th day; and 50 mg/kg on the 5 th day. The control group was injected with the same dose of sodium chloride. Two rats were selected randomly from the four morphine-dependent groups and the control group, and were injected with 5 mg/kg naloxone hydrochloride to induce morphine withdrawal symptoms on the 6 th day. The degree of withdrawal symptoms was measured according to the methods in the literatures of Blasig et al., [4] Maldonade et al., [5] and Wei et al. [6] After confirming that the rats were dependent on morphine, the four dependent groups were injected with a dosage of 30 mg/kg of morphine hydrochloride (one time per day) for 1 week, 2 weeks, 4 weeks, and 8 weeks, respectively, to establish models of different dependent periods. The rats of each dependent group and the matched control group were euthanized with an overdose of pentobarbital, 3 h after the last time of injection. Then, the rats were perfused with 4% paraformaldehyde (PFA). Subsequently, the brains of the animals were removed. Blocks of the four specific cerebral regions were collected and fixed with 4% PFA for the enzyme histochemistry procedure. The staining procedure was performed on all the slices at the same time.

Enzymohistochemistry

The method of lead nitrate stain was used to detect AC and GC (Fujimoto et al.). [7] The total quantity of reaction liquids was 10 mL pH 7.4, containing 100 mmol/L tris-cis-butenedioic acid buffer solution (pH 7.4) 8 mL, adenylyl-imidodiphosphate (AMP-PNP) 2.6 mg, theocin 3.6 mg, 80 mmol/L magnesium sulfate 0.5 mL, L-tetramisole hydrochloride 6.02 mg, 20 mmol/L lead nitrate 1.0 mL, sodium fluoride 8.4 mg, dimethyl sulfoxide 0.5 ml, and sucrose 0.8 g. Then, the specimens were postfixed for 15 min in 2% PFA. Subsequently, these were washed three times with a cacodylic acid buffer solution that included with 8% sucrose, 5% volume/volume (V/V) dimethyl sulfoxide, pH 7.4, and 100 mmol/L. Then, 20 μm sections were cut by a cryostat. The sections were soaked in the reaction solution listed above in a water bath, which was placed inside a humid box for 60 min at a constant temperature of 37°C. The liquid was changed twice and the sections were soaked for 20 min each time. After the reaction, the sections were washed with distilled water, placed in a 1% ammonium sulfide solution for 2 min, and washed again with distilled water after the sections were colored. Then, the sections were covered with gelatin. The matched control group and all the four dependent groups underwent the same staining procedure, the negative control performed same procedure except for withdrawing the substrate. The detection of GC employed guanylyl-imidodiphosphate (GMP-PNP) as substrate; the other steps were the same as AC.

A high definition color pathological analytical system was performed to measure the gray scale of the sections. The data were analyzed by analysis of variance (ANOVA) following Fisher's protected least significant difference (PLSD) test, using SPSS software version 11.5 (Statistical Product and Service Solution, Chicago, U.S.A.). The criteria for significance were set at the 0.05 level.

 Results



The withdrawal symptoms and the scores

The withdrawal symptoms were observed, which included shuddering like a wet dog, stretching, clearing the fur, swallowing, standing, jumping, and chattering the teeth. The results showed that the scores of the withdrawal symptoms of the dependent groups and the control group were significantly different (P ˂ 0.05 or P ˂ 0.01), which indicated that the morphine-dependent rat models were successfully established see [Figure 1].{Figure 1}

Results of AC

The results of AC showed the control group had weakly positive (+) staining, and there was a small quantity of dark brown granulation distributed within the cytoplasm in all the four regions. The 1-week group and the 2-week group had positive (++) staining and there was a major quantity of dark brown granulation distributed within the cytoplasm. The staining of the 4-week group was positive to strong positive (+++) and there was a great quantity of dark brown granulation distributed within the cytoplasm. The 8-week group had strongly positive (+++) staining. The negative control showed negative stain [see [Figure 2] [Figure 3] [Figure 4] [Figure 5] [Figure 6] [Figure 7].{Figure 2}{Figure 3}{Figure 4}{Figure 5}{Figure 6}{Figure 7}

The gray scales of AC in four regions were measured. The results of the same region in different dependent groups and control group were compared. There were significant differences between the control group and the 1-week, 2-week, 4-week, and 8-week dependent groups. There were significant differences when comparing the 1-week group with the 2-week, 4-week, and 8-week groups. The 2-week group was significantly different with the 8-week group. However, there was no significant difference when comparing the 2-week group with the 4-week group, and the 4-week group with the 8-week group [see [Table 1].{Table 1}

When comparing the four different regions in same group, there were significant differences between the regions except when comparing the lenticula with the frontal cortex and the corpus amygdaloideum. In the 1-week group, there was no significant difference when comparing the hippocampus with the frontal cortex and corpus amygdaloideum (P < 0.05 or P < 0.01). In the 2-week group, there was significant difference between the regions except when comparing the lenticula with the hippocampus and corpus amygdaloideum (P < 0.05 or P < 0.01). In the 4-week group, there was significant difference between the regions except when comparing the lenticula with the hippocampus, and the frontal cortex with the corpus amygdaloideum (P < 0.05 or P < 0.01). In the 8-week group, there was significant difference between the regions except when comparing the hippocampus with the corpus amygdaloideum (P < 0.05 or P < 0.01).

Results of GC

The results of GC were similar to the results of AC. The control group was weakly positive; the results ranged from weakly positive to strong positive in all the four regions from the 1-week group to the 8-week group [see [Figure 8] [Figure 9] [Figure 10] [Figure 11] [Figure 12] [Figure 13].{Figure 8}{Figure 9}{Figure 10}{Figure 11}{Figure 12}{Figure 13}

When comparing the gray scales of GC of the same region of different dependent groups, there were significant differences between the control group and all the four experimental groups (P < 0.05 or P < 0.01). There were significant differences when the 1-week group was compared with the 4-week group or the 8-week group (P < 0.05) as well as comparing the 2-week group with the 8-week group (P < 0.05). It was the same when the 4-week group was compared with the 8-week group (P < 0.05). There were no significant dereferences when comparing the 1-week group with the 2-week group, and the 2-week group with the 4-week group [see [Table 2].{Table 2}

Compared with the four regions in the same group, there were significant differences between the regions except when comparing the frontal cortex with the corpus amygdaloideum in the 1-week group (P < 0.05 or P < 0.01). There were significant differences between the four regions except when comparing the lenticula with the corpus amygdaloideum in the 2-week group (P < 0.05 or P < 0.01). There was significant difference in comparing the four regions in the 4-week group (P < 0.05 or P < 0.01). In the 8-week group, the corpus amygdaloideum was significantly different from the frontal cortex (P < 0.05 or P < 0.01).

 Discussions



AC

AC is an important enzyme related to morphine dependence, and the upgrade of the adenylyl cyclase/cyclic adenosine monophosphate-protein kinase A (AC/cAMP-PKA) system had an important contribution to the mechanism of morphine dependence (Nestler et al.). [1] The present results showed that the acute effect of an opioid could inhibit the function of AC by activating the binding G-protein opium receptor, thus degrading the cell's fundamental level of cAMP. The chronic effect of the opioid could upgrade the AC/cAMP system, which may have important effect on morphine dependence. Previous studies suggest that a compensatory increase of the activity of AC isoenzymes (AC superactivation) in response to chronic opioid receptor stimulation may contribute to cellular opioid tolerance (Williams et al.). [8]

Other researches indicated that brain region-selective changes in AC signal transduction are critical, and both these biochemical changes and the behavioral effects are prevented by facilitating endocytosis of the μ-opioid peptide (MOP) receptor (He and Whistler). [9] In a prior study, we found that the upgrade of AC activity not only occurred in the LC, but also in the periaqueductal gray and the substantia nigra, cerebral regions that were related to the physiological symptoms of morphine dependence in rats (Hong et al.). [10]

The data of the present study suggested that the increase in AC activity existed in all the four cerebral regions that were detected, which indicates the increase in AC activity was closely correlated with the morphine psychogenic dependence in rats. The activity of AC went higher following the progress in morphine dependence. The upregulation of cAMP could induce psychogenic dependence on morphine. The enzyme changes in the four regions altered the morphine-dependent rats' ability to learn and memorize. Other studies also indicated that the activity of AC subtype was increased. Hao et al. [11] found that AC VIII messenger RNA (mRNA) was increased in the ventral tegmental region as well as the nucleus accumbens septi, periaqueductal gray, corpus amygdaloideum, and the hippocampus CA1 area. That result indicated that the expression of the AC gene may play a crucial role in morphine dependence. Hao et al. [11] and Kim, et al. [12] considered AC type 5 (AC5) as an important component of μ and d opioid receptor signal transduction mechanisms in vivo as did Kim et al. [12] These researches provided further support for the importance of the cAMP pathway as a critical mediator of opioid action. Also, the spinal AC and PKA pathway through intracellular mitogen-activated protein kinase (MAPK) may be contributory to the cellular mechanisms of morphine-induced apoptosis (Lim et al.). [13]

Our results indicated that increase of AC in the four regions related to psychogenic dependent of morphine. This change may influence the rat's ability to memorize. And in the long term, the animal relapsed and returned to drug-seeking behavior.

GC

GC is also an important enzyme in the mechanism of morphine dependence but the results of past studies of GC had differences (Fang et al., [2] Sullivan et al., [14] and Yang et al.). [15] The present results suggest that nitric oxide served as substrate for GC and that the nitric oxide/nitric oxide synthase-guanylate cyclase-cyclic guanosine monophosphate (NO/NOS-GC-cGMP) messages transmit system might have an important role in the procedure of morphine dependence. Researchers like Zhang, Zhang et al. [16] had proved that the pathway of NO-cGMP had a crucial role in the progress on morphine dependence. But other researches like that of Fang et al. [2] proved that NO/NOS might have another pathway other than that of cGMP. Sullivan et al. [14] suggest that soluble GC (sGC) played an intermediary role in the genesis of LC neuronal hyperactivity and the behavioral signs of morphine withdrawal. Our prior results show that the increase in GC activity existed in the LC, the periaqueductal gray, and the substantia nigra. It also indicated that GC may play a role in the physiological dependence of morphine as showed by Hong et al. [10]

Furthermore, other studies found that sildenafil significantly increased the morphine-induced antinociception that was produced by the drugs alone or combined due to a local action. Pretreatment of the paws with 1H-[1,2,4]-oxadiazolo[4,3-a] quinoxalin-1-one (ODQ, a GC inhibitor) blocked the effect of the combination. The results suggest that opioid receptors, NO, and cyclic GMP are relevant in the combination-induced antinociception (Mixcoatl-Zecuatl et al.). [17] At the spinal level, morphine could produce antiedematogenic and the NO-cGMP pathway seems to be an important mediator in this effect (Brock and Tonussi.). [18]

The results of the present research showed that GC increased in the four regions that relate to the psychogenic dependence of morphine, which indicated that the changes of GC might have a close correlation with the psychogenic dependence; such changes seem to happen mostly in the hippocampus and the corpus amygdaloideum.

To summrize, the increase in AC and GC activities may have effect by changing the function of AC/cAMP-PKA and NO/NOS-GC-cGMP pathways in morphine-dependent rats. These changes could alter the capacities of learning and memory through the message-transmitting pathway, leading to relapse of the rat and further drug-seeking behavior.

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