Evidence for the involvement of a descending ... - Science Direct

Brain Research, 478 (1989) 293-300 Elsevier

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Evidence for the involvement of a descending cholinergic pathway in systemic morphine analgesia Chen-Yu Chiang and Min Zhuo Shanghai Institute of Physiology, Academia Sinica, Shanghai (People'sRepublic of China) (Accepted i2 July 1988) Key words:Morphine; Atropine; Phentolamine; Methysergide; Analgesia; Tail-flick; Hot-plate; Rat

The analgesic effect of morphine sulfate (3-5 mg/kg, i.p.) was assessed by both tail-flick and hot-plate tests in unanesthetized restrained rats. Intrathecal administration of atropine sulfate (10/~g) in the lumbar region of the spinal cord powerfully reduced the analgesia induced by systemic administration of morphine. This action did not result from the diffusion of atropine from its administration site to more rostral sites in the central nervous system. In spinal rats, atropine failed to reverse morphine analgesia, thus strongly suggesting that either a cholinergic descending pathway or a spinal local cholinergic circuit activated by an unknown descending pathway may be involved in the systemic morphine analgesia. In addition, the involvement of the a-adrenergic descending pathway in morphine analgesia is confirmed, whereas that of the serotonergic descending pathway is less prominent. INTRODUCTION Our previous work indicated that a non-serotonergic descending pathway originating in nucleus raphe magnus (NRM) and adjoining structures may mediate in part the analgesia produced by systemic administration of morphine 8'9. NRM and the surrounding region are generally accepted to be composed of heterogeneous populations of neurons a, including in addition to the serotonergic raphe-spinal neurons also descending fibres containing enkephalin, substance p6,22, thyrotropin-releasing hormone (TH-RH) 25, cholecystokinin 34 and acetylcholine 6'26. Possibly due to the technical difficulties, there have been few studies concerning the function of the peptidergic descending system 52. However, the enhancement of morphine analgesia by cholinomimetics has been reported clinically in the early 1940's 11'44, and then confirmed later in animal experiments 4'23'33. Analgesic effects of anticholinesterase and cholinergic agonists have been demonstrated in man ~, monkey 4°, rat and mouse 4'12'18'21'29'45'46. It has been reported recently that acetylcholine ar carbachol ad-

ministered intracerebroventricularly (i.c.v.) 39, intrathecally (i.t.) 51 or locally into particular brainstem nuclei 7,2a'as can produce pronounced analgesia that is reversible by atropine. In addition, recent anatomical studies have demonstrated that cholinergic muscarinic bindings are concentrated in the substantia gelatinosa of the dorsal horn45'48; that acetylcholinesterase and choline acetyltransferase are distributed superficially in the dorsal horn as wel12"a7"43; and that there are several cholinergic descending pathways 6,26.27. Electrophysiological studies have shown that iontophoretic administration of muscarinic agonists onto spinal neurons resulted in neuronal depression 36 and application of acetylcholine facilitated evoked inhibitory postsynaptic potentials 53. On the basis of these findings, the present study aimed to investigate the possible role of cholinergic descending pathways in mediating systemic morphine analgesia, by examining the effects of i.t. administration of cholinergic antagonist and comparing them to those produced by serotonergic and a-adrenergic antagonists. A preliminary report has been published elsewhere l°.

Correspondence and present address: C.Y. Chiaag, Faculty of Dentistry, University of Toronto, 124 Edward St., Toronto, Ontario M5G IG6, Canada. 0006-8993/89/$03.50 © 1989Elsevier Scie~lcePublishers B.V. (Biomedical Division)

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Shanghai bred Wistar male rats weighing 220-400 g were housed in an environmental controlled room and supplied with Wayne Rodent Block food and tap water ad libitum. No special care was taken after surgery except for routine antibiotic injections for the 3 consecutive postoperative days.

Prophylactic antibiotic injections were administered postoperatively. In order to inject drugs, a segment of 30-gauge stainless steel tube was introduced into the guide cannula so that just 0.5 mm protruded from it into the ventricle. The other end of that tube was attached to a PE 10 polyethylene tubule. Upon completion of the experiment, cannula placement was determined by dye injection (10#! of 2% Pontamine sky blue dissolved in saline).

Intrathecal cannulation surgery

Spinal cord transection

Rats were anesthetized with Nembutal (55 mg/kg, i.p.) and placed in prone position. Chronic catheterization of the spinal subarachnoid space was performed according to the procedure developed by Yaksh and Rudy 49. Briefly, a length of saline-filled PE 10 tubing was inserted for a distance of 8.5 cm caudal to a midline cutting in the atlanto-occipital membrane to terminate in the lumbar enlargement of the spinal cord in one group of animals, and 4.0 cm caudal to the midline cutting to terminate in the T3-4 segments in another group. The other end of the catheter was tied to a short pin, anchored at the protuberance of the occipital bone and externalized at the top of the head. The pin together with the catheter were anchored with acrylic cement to the skull. The rostral end of the catheter was plugged with a removable 30-gauge stylette. At the end of the surgical procedure, aureomycin ointment was topically applied to the skin wound. Animals were allowed at least 4 days to recover before being used in experimental testing. Upon completion of the experiment, catheter placement was confirmed by Pontamine sky blue dye injection.

Surgery was carried out under Nembutal anesthesia. An incision was made at the mid-thoracic region. A dorsal laminectomy at the T7-8 segments was performed. The dura mater was incised and reflected. The spinal cord was transected with a scalpel following topical application of 2% Xylocaine solution which eliminated any motor responses elicited during the transection. Then, an intrathecal cannulation was made as described above. Special care was taken postoperatively to help restore auto-urination and defecation of the animal. Those animals that did not recover were rapidly euthanized.

MATERIALS AND METHODS

Animals

Surgery for i.c. v. cannula implantation Under Nembutal anesthesia, the head of the animal was placed in a stereotaxic instrument and a midline incision on the scalp was made. A burr hole (1.0 mm in diameter) was drilled through the skull at 2.5 mm lateral to the saggital suture and 2.0 mm caudal to the coronal suture. A 26-gauge needle cut to a length of 3.5 mm and inserted into the lateral ventricle served as a guide tube for later i.c.v, injections. The flow of cerebrospinai fluid out of the guide tube confirmed correct placement. The guide tube was fixed to the skull with dental cement and its opening plugged with a segment of stainless steel stylette.

Drug administrations Morphine sulfate (Qing Hai Pharmaceutical Plant), atropine sulfate (Shanghai No. 10 Pharmaceutical Plant), phentolamine hydrochloride (CibaGeigy) and methysergide maleate (Sandoz) were all freshly prepared with 0.9% sodium chloride solution before use. Dosages are expressed in terms of the salt. For both Lc.v. and i.t. administrations, a volume of 10 .ul of drug solution was delivered via a 50/zl Hamilton syringe driven by an electric microdriver with a speed of 5!:!/min and then followed with a 10/~1 saline flush to clean the i.c.v, or i.t. catheter of the drug.

Tail-flick (TF) response latency measurement Animals were wrapped in a long cotton towel which was clipped over the head of the animal by a clamp, but left a slit for the PE 10 tubing catheter to be introduced. The tail of the animal protruded from" the towel and was blackened with black ink on the ventral surface. Four spots were selected on that surface at intervals of 1 cm. The distal spot was located at about 3 cm from the tip of the tail. On testing, the tail was gently placed on a wooden board with a 0.5

295 mm width slit, through which passed.intense light of a radiant heat algesiometer. The radiant heat caused the tail to flick away from the slit. The reaction time of the TF response was detected by a photoelectric cell and displayed automatically on a digital timer. The TF response latency varied greatly between individual animals. To minimize these individual differences, the voltage of the algesiometer was adjusted for each animal at the beginning of an experiment in order to obtain a stable baseline TF response latency of around 3 s. During the experiment, the TF response latency elicited from one of the 4 spots on the tail selected at random was measured. One trial comprised two measurements of TF response latency. The average TF response latency value from 3 consecutive trials before drug or saline administration was taken as the baseline TF response latency. Then the trials were repeated at 5 min intervals during the following 2 h. A TF latency of twice the baseline latency was taken as the cut-off time of illumination to avoid skin damage. The analgesic effect was estimated as the analgesic index (A.I.) according to the following formula: A.I. =

postdrug latency-predrug latency

corresponding time of any two curves was analysed by the two-tailed Student's t-test. RESULTS

L Reversal of systemic morphine analgesic effect by intrathecal injection of atropine A. TF testing. Three groups of rats, 5 rats in each group, Two groups received morphine sulfate (3 mg/kg, i.p.), and the third group that served as a control was given sterile normal saline of an equal volume (see arrow 1 in Fig. 1A). Half an hour later, one of the morphine-treated groups received an i.t. injection of the cholinergic antagonist, atropine sulfate (10 pg) and another was given i.t. saline. The third

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