Chou WB et al / Acta Pharmacol Sin 2002 Aug; 23 (8): 691-697

M₂ muscarinic receptor of spinal cord mediated increase of nNOS expression in locus coeruleus during morphine withdrawal

CHOU Wei-Bin, ZENG Yin-Ming¹, DUAN Shi-Ming, ZHOU Wen-Hua², GU Jun², YANG Guo-Dong²

Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou 221002;

²Ningbo Addiction Research and Treatment Center, Ningbo 315010, China

¹ Correspondence to Prof ZENG Yin-Ming. Phn 86-516-580-2018. Fax 86-516-569-9127. E-mail xmcfa@public.xz.Js.cn

Received 2001-12-04 Accepted 2002-05-13

KEY WORDS morphine; substance withdrawal syndrome; muscarinic receptors; nitric-oxide synthase; spinal cord; locus coeruleus

ABSTRACT

AIM: To investigate the effects of different muscarinic receptor (M) subtypes in the spinal cord on the scores of naloxone-precipitated morphine-withdrawal symptoms and the changes of nNOS expression in locus coeruleus (LC). METHODS: nNOS immunohistochemistry, intrathecal injection (it), and antisense oligonucleotides (AS-ONs) techniques were used. RESULTS: Intrathecal injection of M₂-antisense oligonucleotides (M₂-AS) decreased the scores of morphine withdrawal symptoms. M₁-AS attenuated morphine-withdrawal symptoms, but the effect was less than that of M₂-AS. The expression of nNOS positive neurons in the LC increased in morphine-dependent rats and increased to a greater extent during morphine withdrawal. Intrathecal injection of M₂-AS inhibited the increase of nNOS expression in LC during morphine-withdrawal, but there was no effect in case of M₁-AS. CONCLUSION: M₂ muscarinic receptor of spinal cord mediated the increase of nNOS expression in LC during morphine withdrawal.

INTRODUCTION

Many studies had demonstrated that nitric oxide (NO) contributed to the development of tolerance and physical dependence to morphine. Behaviour reports showed that the inhibitor of nitric oxide synthase (NOS) could effectively delay the development of morphine tolerance and physical dependence, and the donor of NO could improve the procedure of morphine tolerance^[1,2]. According to the traditional view, locus coeruleus (LC) is the main site mediating opioid withdrawal response, and the hyperactivity of LC neurons play an important role during opioid antagonist-precipitated morphine withdrawal^[3]. Chronic morphine exposure and naloxone-precipitated withdrawal upregulated neuronal nitric oxide synthase (nNOS) in the LC, and NOS inhibitors reduced the LC withdrawal response in voltammetric and electrophysiological experiments^[4,5]. It is suggested that the expression of nNOS in the neurons of LC showed selective activation during morphine withdrawal.

There are five subtypes of muscarinic receptor M₁-M₅ extensively distributing in the brain and spinal cord. It has been acknowledged that the subtypes of M₁ and M₂ are more important than the other subtypes in the central nervous system, which have many functions such as neural plasticity, acquisition of memory, or brain development. Because of the overlapped expression of muscarinic receptors and lack of sufficient selective muscarinic receptor antagonists, limited information is available concerning the exact function of muscarinic receptor subtypes. It is reported that cholinergic neurons of spinal cord participated in the draw-out of morphine withdrawal symptoms. Intrathecal injection of scopolamine could decrease the scores of morphine withdrawal symptoms, and more interestingly, decrease the expression of NOS mRNA in brainstem^[6]. So, it is suggested that the activity of muscarinic receptor of spinal cord was involved in the elicitation and development of physical dependence to morphine by influencing the activity of nNOS of LC. Then, which muscarinic receptor subtype of spinal cord mediates morphine withdrawal symptoms and whether the selective activation of nNOS in LC is modulated by muscarinic receptor of spinal cord are not clear. Antisense oligodeoxynucleotides (ODN) were capable of down-regulating the gene expression, and were used for assessment of gene function and for therapeutic purpose. However, functional efficacy of ODN required not only the selection of an appropriate target sequence, but also a sufficient concentration. Using cationic lipids LipofectinAMINE as carrier could enhance the cellular uptake and nuclear distribution of ODN^[7]. In the present study, we investigated the effects of different muscarinic receptor subtypes of spinal cord on the scores of naloxone-precipitated morphine-withdrawal symptoms and the changes of nNOS activity in LC.

MATERIALS AND METHODS

Animals and reagents Male adult Sprague-Dawley rats, weighing 200-250 g, were obtained from Shanghai Experimental Animal Center (Grade II, Certificate No 005). Morphine was purchased from Shenyang Pharmaceutical Factory, China. nNOS antibody was product of Santa Cruz (USA). Biotinylated goat anti-rabbit IgG, avidin-biotin peroxidase complex (ABC), and 3,3'-diaminobenzidine (DAB) were purchased from Vector (USA). LipofectinAMINE was purchased from Gibco (USA). Other chemicals were of analytical grade.

Intrathecal cannulation Rats were anesthetized with sodium pentobarbital (50 mg/kg, ip). A polyethylene catheter (inner diameter of 0.28 mm and outer diameter of 0.61 mm) filled with saline, with a parafilm knot in heat-sealed side, was inserted via an incision of the atlanto-occiptal membrane and advanced 5.5-6.0 cm caudally to leave its tip in the thoracolumbar level. The catheter was fixed to muscle. Correct catheter placement was confirmed by injection of 15 ¦ÌL of 2 % lidocaine into the subarachnoid space 20 min after recovery from anesthesia. The catheter was judged to be intrathecal if paralysis and dragging of the hind legs occurred within 30 s of this injection. Animals with negative lidocaine test and neurologic damage after catheter implantation were excluded from the study. The rats were housed individually after surgery and allowed 5-6 d recoveries before testing.

Animal model and experimental groups Experiments were carried out in intrathecal cannulated rats. To set up dependence model (bid, 5 d), the dose of morphine was 10 mg/kg at the first day and increased by 10 mg/kg each day. An equal volume of saline was administered in the control group. On d 6, 4 h after injection of morphine 50 mg/kg, morphine withdrawal syndrome was precipitated by injection of naloxone (4 mg/kg, ip). M₁-AS 2 nmol, M₂-AS 2 nmol (dissolved in 20 ¦ÌL of 20 % LipofectinAMINE), and 20 ¦ÌL of 20 % LipofectinAMINE were intrathecally injected at 24 h before naloxone-precipitated withdrawal. So we got five groups: control group, dependence group, withdrawal group, M₁-AS group, and M₂-AS group.

nNOS immunohistochemistry One hour after injection of naloxone, all rats for immunohistochemistry were deeply anesthetized with sodium pentobarbital (50 mg/kg, ip) and underwent sternotomy and transcardial aortic needle cannulation, then perfused with saline 100 mL, followed by 400 mL of 4 % ice-cold paraformaldehyde in phosphate buffer (PB) 0.1 mol/L. The brainstem was removed quickly, postfixed for 3 h in the same solution of paraformaldehyde at room temperature, then immersed in 30 % sucrose in PB overnight at 4 ¡æ. Frozen series sections (25 mm) were cut and collected in PB. Tissue sections were washed in phosphate-buffered saline (PBS) and incubated in PBS containing 5 % normal goat serum and 0.3 % Triton X-100 at room temperature (25±2 ¡æ) for 30 min, and then in rabbit anti-nNOS serum diluted at 1: 800 in PBS/Triton X-100 at 4 ¡æ for 48 h. After several washes in PBS, the sections were incubated with biotinylated goat anti-rabbit IgG (1:200) at 37 ¡æ for 1 h and in avidin-biotin-peroxidase complex (1:100) at 37 ¡æ for 2 h. Finally, the sections were reacted with DAB for 5-10 min.

Synthesis of ODN and selection of sequence targets The sequences of ODN against M₁ and M₂ were the initiation of translation sites of rat M₁ mRNA and M₂ mRNA. M₁ antisense: 5'-TGT GCA TCC CTG TGT GCT-3'; M₂ antisense: 5'-TAT TCA TTT TGT GTT CAG-3'. ODN was synthesized by Sangon Co Ltd, Shanghai, and modified with phosphorothioate^[8,9].

Scores of morphine withdrawal response After the last injection of morphine, the rats were placed in a plexiglass observation chamber to allow for acclimatization to the environment. Following naloxone precipitated withdrawal, response was obtained according to morphine withdrawal syndrome. Two classes of withdrawal signs were measured: counted signs and observed signs. The counted signs were made every 15 min for 1 h. During each period of 15 min, the number of bouts of wet dog shakes, teeth chatting, and irritability (jumping and attack) were counted and a withdrawal score for each sign was assigned as follows: 0=no occurrence; 1=1 occurrence; 2>2 occurrences. Abnormal position was observed over periods of 2 min, with two points being given for the presence during each period. The scores of four periods showing the signs were added. Diarrhea was evaluated and given a score based on the severity of the response observed (0=absent, 4=mild and moderate, 8=severe). The score of salivation was based on the severity of the response observed (0=absent, 1=mild and moderate, 2=severe). The withdrawal score of body weight loss was determined by the weight difference (¦¤W ) before and 60 min after administration of the naloxone, which was assigned as follows: 0=no change; 1=¦¤W<2 %; 5=¦¤W<4 %; 10=¦¤W<6 %; 15=¦¤W<8 %; 20=¦¤W>8 %.

Quantification For each animal, five LC sections were analyzed. The sections of interest initially selected for counting were those which had the largest number of nNOS positive neurons by visual inspection at ×40 magnification. The number and the average optical density of nNOS positive cell bodies were analyzed by pathological image analysis system PAS-5000.

Statistical analysis The data were presented by mean±SD and compared with one-way ANOVA.

RESULTS

Effects of intrathecal injection of M₁-AS and M₂-AS on the response scores of morphine withdrawal symptoms Intrathecal injection of M₂-AS could decrease the total scores of morphine withdrawal symptoms (P<0.01), but the effects of injection of M₁-AS were less than that of M₂-AS. Pretreatment of rats with M₂-AS (it) attenuated the expression of withdrawal symptoms as reflected by low scores. However, not all of the withdrawal signs were influenced by the treatment. For example, M₂-AS attenuated irritability, diarrhea, and weight loss (P<0.01), but had no effect on other withdrawal symptoms (P>0.05). M₁-AS only attenuated wet dog shakes (P<0.01) (Tab 1).

Tab 1. Effects of intrathecal injection of M₁ -AS and M₂-AS on the response scores of morphine withdrawal symptoms. n=6. Mean±SD. ^aP >0.05, ^bP<0.01 vs withdrawal group.

Effects of intrathecal injection of M₁-AS and M₂-AS on the number of nNOS positive neurons in LC during morphine withdrawal nNOS positive neurons in the LC increased in morphine-dependent (223.5 % from control, P<0.01) and in naloxone-precipitated withdrawal (243.2 % from control, P<0.01) rats. Intrathecal injection of M₂-AS inhibited the increase of nNOS-immunoreactive (IR) neurons in the LC during morphine-withdrawal (P<0.01), but there was no effect in case of M₁-AS group (P>0.05) (Fig 1, 3).

Fig 1. Effects of intrathecal injection of M₁ and M₂ antisense oligonucleotides on the number of nNOS positive neurons in LC during morphine withdrawal. n=6. Mean±SD. ^cP < 0.01 vs control. ^dP > 0.05, ^fP < 0.01 vs withdrawal group.

Fig 2. Effects of intrathecal injection of M₁ and M₂ antisense oligonucleotides on the average optical density of the nNOS positive cells in LC during morphine withdrawal. n=6. Mean±SD. ^cP < 0.01 vs control. ^dP > 0.05, ^fP < 0.01 vs withdrawal group.

Fig 3. Effects of intrathecal injection of M₁ and M₂ antisense oligonucleotides on the expression of nNOS positive neurons in the LC during morphine withdrawal. A: Control group; B: Dependence group; C: Withdrawal group; D: M₁-AS group; E: M₂-AS group. ×120.

Effects of intrathecal injection of M₁-AS and M₂-AS on the average optical density of the nNOS positive cells in LC during morphine withdrawal The expression of nNOS IR in the LC were more intense in morphine-dependent and morphine-withdrawal rats than in control animals (P<0.01). Pretreatment of rats with M₂-AS (it) decreased the average optical density during morphine withdrawal (P<0.01), but M₁-AS had no effect on the average optical density in the LC (P>0.05, from withdrawal) (Fig 2, 3)

DISCUSSION

Spinal pathways are important for the expression withdrawal signs following both local and systemic administration of naloxone in morphine dependent rats. Marshall et al^[10] reported that spinal cholinergic pathways played a role in the expression of spinal mediated withdrawal symptoms such as mean arterial pressure (MAP). Pretreatment of dependent rats with intrathecal injection of atropine reduced the pressor and several behavioural responses elicited by naloxone. Further more, intrathecal injection of M₁ selective antagonist pirenzepine and M₂ selective antagonist methoc-tramine could attenuate morphine withdrawal symp-
toms^[11]. As described above, the activation of spinal muscarinic receptor participated morphine withdrawal symptoms. The present study was consistent with their results. We also found that M₂ activation played a more important role than M₁ at the spinal cord level. Behaviour results showed that inhibiting M₂ gene expression could decrease the total and several response scores of morphine withdrawal symptoms. In contrast, inhibiting M₁ gene expression had no obvious effect during morphine withdrawal.

Previous studies have suggested that increased glutamatergic afferent neurons input to the LC is mainly responsible for its hyperactivity during opioid with- drawal^[12]. There are many reports showing that antagonists of N-methyl-D-aspartate (NMDA) excitatory amino acid (EAA) receptors or inhibitors of EAA release modify tolerance and attenuate or prevent precipitated withdrawal syndrome in morphine-dependent animals^[13]. Stimulation of NMDA receptor is the main synthesis pathway for the high expression of nNOS in the LC during morphine dependence and withdrawal. Influx of Ca²⁺ during depolarization of the postsynaptic cell activates a calmodulin-dependent enzyme nNOS, which generates NO. NO acts as a retrograde messenger by diffusing back to the presynaptic terminal where it activates the heme-bearing enzyme guanylate cyclase. The resultant increased level of cGMP then causes enhancement of the release of the transmitter, strengthening the synaptic signal. Consistent with results from our immunohistochemistry studies, morphine dependence and withdrawal induce high expression in both number and intensity of nNOS immunoreactive neurons in the LC.

One proposed hypothesis suggested that the behavioural symptoms of morphine withdrawal be partially attributed to a message wind-up phenomenon from spinal cord to brainstem. In morphine dependent rats, systemic injection of naloxone does provoke enhanced neuronal firing in dorsal root ganglion (DRG)/dorsal horn at spinal cord level, which can be inputted to the nucleus paragigantocellularis (PGi) and the LC by ascending glutamatergic afferent neurons^[14]. Therefore, the enhanced firing in LC neurons underlied the hyperactivity of LC and the expression of withdrawal symptoms during morphine withdrawal. In the present study, intrathecal injection of M₂-AS inhibited the increase of nNOS expression in both number and intensity in LC during morphine-withdrawal, but there was no effect in M₁-AS group. The results suggested that inhibiting M₂ gene expression could decrease the firing input of spinal ascending glutamatergic afferent neurons, and activating M₂ receptors of spinal cord could lead to the hyperactivity of LC during morphine withdrawal.

Zhou et al reported that pretreatment with scopolamine decreased the expression of NOS mRNA in spinal cord^[6]. Previous behavioral studies showed that the manner of inhibiting morphine withdrawal symptoms with intrathecal injection of NMDA receptor antagonist was similar with muscarinic receptor antagonist^[15,16]. It is possible, therefore, that some relationship exists between NMDA/NO/cGMP pathway and cholinergic system in spinal cord during morphine withdrawal. Spinal cholinergic neurons enhance glutamatergic tone to supraspinal regions. In both cases, muscarinic receptor-mediated morphine-withdrawal responses appear to be influenced by a local NO generating system. The most likely scenario is that during withdrawal there is an enhanced release of acetylcholine, which acts upon muscarinic receptor which may located on glutamatergic neurons. The release of glutamate, in turn, activates NMDA receptors on presynaptic terminal where it activates NMDA/NO/cGMP pathway^[2]. Certain symptoms, such as wet dog shakes and diarrhea are most likely expressed in response to withdrawal-evoked enhanced autonomic nerve discharge, whereas other behaviour symptoms may be due to activation of ascending spinal pathways, including those to the LC. So, we considered that downregu-lation of M₂ subtype gene expression indirectly inhibited the activation of glutamatergic neurons in spinal cord, then blocked the firing input ascending to LC which could decrease the expression of nNOS in the LC. Further studies will be required to elucidate the precise mechanisms of communications between spinal muscarinic receptor and supraspinal regions during morphine withdrawal.

In conclusion, our results suggested that M₂ muscarinic receptor of spinal cord mediated increase of nNOS expression in LC during morphine withdrawal.

REFERENCES

• 1 Bhargava HN, Thorat SN. Evidence for a role of nitric oxide of the central nervous system in morphine abstinence syndrome. Pharmacology 1996; 52: 86-91.
• 2 Babey AM, Kolesnikov Y, Cheng J, Inturrisi CE, Trifilletti RR, Pasternak GW, et al. Nitric oxide and opioid tolerance. Neuropharmacology 1994; 33: 1463-70.
• 3 Maldonado R, Stinus L, Gold LH, Koob GF. Role of different brain structures in the expression of the physical morphine withdrawal syndrome. J Pharmacol Exp Ther 1992; 261: 669-77.
• 4 Cuellar B, Fernandez AP, Lizasoain I, Moro MA, Lorenzo P, Bentura ML, et al. Up-regulatioon of neuronal NO synthase immunoreactivity in opiate dependence and withdrawal. Psychopharmacology 2000; 148: 66-73.
• 5 Hall S, Milne B, Jhamandas K. Nitric oxide synthase inhibitors attenuate acute and chronic morphine withdrawal response in the rat locus coeruleus: an in vivo voltammetric study. Brain Res 1996; 739: 182-91.
• 6 Zhou WH, Xie XH, Liu HF, Yang GD. The expression of nitric oxide synthase mRNA in spinal cord and brainstem during morphine dependence and withdrawal in rats. Chin Pharmacol Bull 2000; 16: 99-102.
• 7 Akhta S, Agrawal S. In vitro studies with antisense oligonucleotides. TIPS 1997; 18: 12-8.
• 8 Klett CPR, Bonner TI. Identification and characterization of the rat m₁ muscarinic receptor promoter. J Neurochem 1999; 72: 900-9.
• 9 Gocayne J, Robinson DA, Fitzgerald MG, Chung FZ. Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family. Proc Natl Acad Sci USA 1987; 84: 8290-300.
• 10 Marshall DC, Buccafusco JJ. Spinal cholinergic neurons and the expression of morphine withdrawal symptoms in the rat. J Neurosci 1987; 7: 621-8.
• 11 Yang GD, Zhou WH, Zhang FQ, Zhang YH, Liu HF, Zhu B, et al. Experimental study of selective muscarinic receptor antagonists on attenuation of morphine tolerance and dependence in rats. Natl Med China 1997; 77: 130-3.
• 12 Aghajanian GK, Kogan JH, Moghaddam B. Opiate withdrawal increases glutamate and aspartate efflux in the locus coeruleus: an in vivo microdialysis study. Brain Res 1994; 636: 126-30.
• 13 Trujillo KA, Akil H. Inhibition of morphine tolerance and dependence by the NMDA receptor antagonists MK-801. Science 1991; 251: 85-7.
• 14 Wang B, Luo F, Han JS. Central areas and post-receptor mechanisms underlying opioid physical dependence. Chin J Neurosci 1999; 15: 63-8.
• 15 Holland LN, Shuster LC, Buccafusco JJ. Role of spinal and supraspinal muscarinic receptors in the expression of morphine withdrawal symptoms in the rat. Neuropharmacology 1993; 32: 1387-95.
• 16 Buccafusco JJ, Terry AV Jr, Shuster L. Spinal NMDA receptor — nitric oxide mediation of the expression of morphine withdrawal symptoms in the rat. Brain Res 1995; 679: 189-99.