The spinal cord as a major site for the antinociceptive action of nicotine in the rat

/veur0p/Iclrmae&gy Vol. 25, No. 9, pp. 1031-1036,1986 Printedin GreatBritain

0028-3908/86$3.00+ 0.04 PergamonJournalsLtd

THE SPINAL CORD AS A MAJOR SITE FOR THE ANTINOCICEPTIVE ACTION OF NICOTINE IN THE RAT M. D. ACETO, RUTH S. BAGLEY, W. L. DEWEY, T.-C. Fu and B. R. MARTIN* Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonweal~ University, MCV Station, Box 613 Richmond, Virginia 232984001, U.S.A. (Accepted I4 November 1985)

Summary-These studies were conducted to localize the antinociceptive action of nicotine witbin the CNS. Antinociceptive and biodispositional studies were carried out after the injection of [%]nicotine subcutaneously and intm~rebroven~~l~ly into the common carotid and vertebral arteries and into the subarachnoid space. The data indicated that [3HJnicotine was most potent when given into the subarachnoid space than by any of the other route of administration. Further, the disposition studies showed that [‘HInicotine was almost entirely contained in the thoracic and lumbar areas. These results are consistent with the hypothesis that the spinal cord is an important site for antinociception induced by nicotine. Key words: nicotine, ~tin~i~ption,

spinal cord, rH]-nicotine

Nicotine has been shown to produce antinociception in cats (Davis, Pollock and Stone, 1983), mice (Mattilla, Ahtie and Saamivara, 1968; Mansner, 1972; Phan, Doda, Bite and Gyorgy, 1973; Aceto, Martin, Tripathi, May and Jacobson, 1980; Tripathi, Martin and Aceto, 1982), rats (Phan et al., 1973; Sahley and Bemston, 1979; Tripathi er al., 1982; Martin, Tripathi, Aceto and May, 1983), dogs (Kamerling, Wettstein, Sloan, Su and Martin, 1982), rabbits and hamsters (Mattila et al., 1968). Antinociceptive activity in rats is not blocked by naloxone, which suggests that the mechanism for the action of nicotine is independent of opiate receptors and therefore distinct from that in mice (Sahley and Bernston, 1979; Tripathi et al., 1982). There is some controversy regarding the choline&c nature of the effects of nicotine in rats. Tripathi et al. (1982) have shown that mecamylamine, but not atropine or scopolamine, is an effective antagonist. However, Sahley and Bernston (1979) were able to block antinociception in the tail-flick test with both m~myl~ine and ~o~lamine. The inabi~ty of hexamethonium to antagonize systemically- (Tripathi er al., 1982) or intracerebroventricularly(Sahley and Bemston, 1979) administered nicotine suggests a central site of action. However, it has been shown that there is a poor correlation between levels of nicotine in whole brain and antino~i~ptive activity in rats after intravenous a~inistration (Tripathi et al., 1982). Studies were therefore conducted to determine whether a correlation exists between antinociceptive

*To whom correspondence should be addressed.

pksma, tissue levefs.

activity and levels of nicotine in specific areas of the brain or spinal cord in rats after intra~~broven~~lar and su~u~neous injection into the common carotid and vertebral arteries and into the subarachnoid space. METHODS

Animals and drugs Male Spragu~Dawley rats (200-460 g) were obtained from Flow Laboratories (Dublin, VA). They were housed individually in wire mesh cages and had free access to food and water. Nicotine was obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI) and converted to the ditartrate salt as described previously (Aceto, Martin, Uwaydah, May, Harris, Izazola-Conde, Dewey, Bradshaw and Vincek, 1979). rH]Nicotine ditartrate (4.7 Ci/mmol) was synthesized as described by Vincek, Martin, Aceto and Bowman (1980). The specific activity of t3H]nicotine was adjusted by the addition of nicotine so that each rat received appro~mately 5Oj.Ei of radioactivity. The drugs were dissolved in 0.9% saline for injection. The doses and tissue levels of nicotine are expressed as the free base. Routes

ofadministration

Un~~~etiz~ rats were used for the subcutaneous administration of nicotine. For all other routes of administration, the rats were anesthetized with methoxyflurane and then maintained under sodium pentobarbital (4s-50 mg/kg, i.p.) during surgery and antinociceptive testing. These anesthetic agents did not alter the times of the tail-flick response. Sub-

1031

1032

M. D. ACETO et

arachnoid injections were performed in a manner similar to that described by Fu and Dewey (1981) for mice. Laminectomies were done at the high lumbar level (L,-L2) prior to insertion of the needle into the subarachnoid space. The needle was connected via PE tubing to a syringe that was clamped to a David Kopf 1272 Universal Holder (Tujunga, CA) which was, in turn, attached to a David Kopf 1460 Electrode Carrier mounted on a 1455 Electrode Angle Calibrator. The dose of the drug was in 10 ~1 volume and was administered slowly for 30 sec. The injection technique into the vertebral artery has been described and illustrated previously (Fu and Dewey, 1981). The right axillary artery was exposed by the removal of the overlying muscles. The PE-50 tubing was filled with nicotine (except the tip section which was filled with heparin, separated from the nicotine by a tiny air bubble), which was then inserted into the axillary artery up to the entrance of the vertebral artery. The level of the tubing was adjusted to face the opening of the vertebral artery. The right common carotid artery was also ligated in order to prevent the overflow of the solution of the drug from the subclavian artery into the common carotid artery during the injection. After each experiment was completed, the rat was sacrificed and the location of the tip of the PE tubing was verified. The success rate was approximately 75%. The volume of solution of the drug injected was held constant (45 ~1). For the injection into the common carotid, a 4-6 mm section of the right artery was exposed and cannulated with PE-50 tubing. The tubing was filled with nicotine in a manner identical to that described above. Nicotine was injected in 50 pl of saline. Intraventricuiar injections were made through a hole drilled into the skull 3 mm lateral to the midline and 3 mm caudal to the occipital suture line. The injection needle was placed 3 mm within the brain for injection into the ventricle. A volume of lo/*1 was injected. Measurement

ofantinociception

The tail-flick procedure @‘Amour and Smith, 1941; Dewey, Harris, Howes and Nuite, 1970) was used, which measures the time required for a rat to flick its tail away from a noxious stimulus (heat). A control response, which was always between 2 and 4 set, was determined in each anesthetized surgicailyprepared animal just prior to treatment with the drug. By moving the heat stimulus S-10 min proximal to the previous measurement, it was possible to make more than one measurement per animal in the timecourse studies. A maximal latency of 10 set was imposed so that injury to the tail was avoided. The percentage maximum possible effect (% MPE) was calculated as [(test-control)/(lO-control)] x 100. Measurement of [3H]nicotine in tissues Rats were given [3H]nicotine and all rats, other than those receiving subarachnoid injections, were decapitated at the selected intervals so that blood

al.

could be collected from the cervical wounds. For subarachnoid injections, the common carotid arteries of the rats were exposed so that they could be severed after administration of [3H]nicotine. This arrangement prevented blood from being contaminated with cerebrospinal fluid containing [3H]nicotine. These animals were decapitated after they were bled. Blood was centrifuged at 1OOOg for 20min in order to obtain plasma. Brains were dissected into those areas described by Glowinski and Iversen (1965). After isolation the spinal cords were dissected into cervical, thoracic and lumbar sections (Ohlsson, Fu, Jones, Martin and Dewey, 1982). Livers and gastrocnemius muscles were also removed for analysis. Areas of the brain and sections of spinal cord were homogenized in 4ml of 0.5 N HCI using a Polytron (Brinkman Instruments, Westbury, NY). Samples of liver and muscle were homogenized in 5 volumes of 0.5 N HCl. Total radioactivity in these tissues was determined by counting 100~~1 aliquots of homogenates and 200 ~1 of plasma directly in aqueous counting scintillant (Amersham Corp., Arlington Heights, IL). Heights, IL). The levels of [3H]nicotine in the tissue were determined using a solvent extraction technique that removes nicotine but not the metabolites (Hucker, Gillette and Brodie, 1960; Tsujimoto, Nakashima, Tanino, Dohi and Kurogochi, 1975; Mansner and Mattila, 1975; Tripathi et al., 1982). The procedure involves extraction of 13H]nicotine from homogenates of tissue and plasma with hexane under basic conditions. Radioactivity in each extract with hexane was quantified by liquid scintillation spectrometry. Both the selectivity and efficiency of this extraction procedure have been found to be > 90% (Tripathi et al., 1982; Martin et al., 1983). RESULTS

Antinociceptive activity Dose-response relationships and time-course studies were determined for nicotine after each route of administration. Except for the injections into the common carotid (400pg/kg) and vertebral (250pg/kg) arteries where the number was 3, all other doses had a number of 4-7. When given subcutaneously at a dose of 500 ,ugfkg, nicotine was completely inactive. At I mg/kg, the maximal increase in latency was 30% r4:13 at 2 min. At 1.5 mg/kg, the response time was increased to a maximum of 82% 2 12 at 2 min. The time course for this latter dose is illustrated in Fig. 1. When given by the intraventricular route, doses of 40 and 100 pg/kg were inactive. At 200 pg/kg, a maximum response of 17% was observed at 2 min. At 300 pg/kg, the maximum response was recorded as 100% at 2 min. The time course for this dose is also illustrated in Fig. 1. With injections into the common carotid artery, 4Ogg/kg did not significantly alter the latency. The maximal response obtained at 200 pg/kg was

Spinal antinociceptive action of nicotine

1033

IOOr

90-

8070 -

%O+I

I&l 50 I40s

3020 IO ooq

2

5

10

15

20

Time (mid

Fig. 1. Time course of antinociception induced by nicotine, given by different routes of administration: 4OOpg/kg carotid artery, IJ-0 e---e l.Smg/kg (SC), O-O 3OOMkg (i.c.v.), O-O 50 pg/kg vertebral artery, O-0 10 pg/kg subarachnoid space (L,-L2).

17% + 13 at 2 min. The largest dose tested, namely 400 gg/kg, produced a maximum response by 30 set and the effect was still present at 20 min as illustrated in Fig. 1. Twenty-five pg/kg injected into the vertebral artery was essentially inactive; maximum activity at this dose was 16% f 8 at 20 min. Doubling the dose resulted in maximum increase in latency of 72% + 20 by 30 sec. This effect gradually diminished tol7%+10byl5minasshowninFig.l.Adoseof 250 pg/kg increased the latency to 100% within 30 sec. Finally, subarachnoid injections at 5 pg/kg promptly increased the latency (41% f 25) within 30 set, and doses of 10, 25 and 4Opg/kg produced a maximum response of 100% within the same time interval. The time course for the 10 fig/kg dose is also illustrated in Fig. 1. Tissue levels of [3H]nicotine

The disposition of [‘HInicotine in areas of the brain and spinal cord was studied in an effort to identify the central sites most sensitive to nicotine. The objective of these experiments was to establish levels of rI-Ijnicotine in tissue after minimallyeffective, as well as effective doses, by each route of administration. Comparison of tissue levels after active and inactive doses should indicate which central sites are involved the effect of nicotine. Small and large doses of [3H]nicotine were administered by each route and the rats were decapitated at times which represented minimal or high antinociceptive activity, respectively. The concentrations of [“HInicotine in areas of the brain, sections of spinal cord and selected peripheral tissues are presented in Tables 1, 2 and 3. The disposition of [‘I-Qnicotine 2 min after subcutaneous injection was evenly distributed between all the areas of the brain for both large and small doses. There was a slight decrease in the levels in the spinal cord as the distance from the brain increased. In general, the levels in the spinal cord were approximately one-half of those in brain. Intraventricular administration of 200 fig rH]nicotine/kg, a dose

which produced 17% of the maximum possible effect, resulted in very large concentrations of [‘HInicotine in all areas of the brain. There was an even distribution throughout the brain, with the exception of a larger concentration in hypothalamus. Levels in the spinal cord were only a small fraction of those in supraspinal structures. The intraventricular administration of 300 pg/kg of [3H]nicotine resulted in a similar distribution pattern but with somewhat higher levels than those after 200 pg/kg. [‘HINicotine was also injected via the common carotid and vertebral arteries, which supply the rostral and caudal structures of the brain, respectively. The right common carotid artery bifurcates forming the internal and external carotid arteries, and the thalamus, corpus striatum, cortex, etc. are supplied by the internal carotid artery. The cervical branch of the vertebral artery serves the spinal cord and the meninges, before the vertebral arteries form the basilar arteries. Under normal physiological conditions, the basilar arteries provide the sole arterial blood supply to the medulla, pons, midbrain cerebellum, posterior thalamus and occipital lobe. The distribution of both doses of [‘HInicotine after infusion via the common carotid artery showed that hypothalamus and hippocampus were perfused perferentially with somewhat lesser amounts present in the other rostra1 brain structures and cervical spinal cord. The caudal brain structures, with the exception of the midbrain, contained low levels of [3H]nicotine. Conversely, when [‘HInicotine was injected into the vertebral artery, it was found predominantly in cauda1 structures of the brain and the cervical spinal cord as expected. All of the rostra1 brain areas, with the exception of hippocampus in the group given 50 pg/kg, contained small concentrations relative to the caudal areas. The subarachnoid injection of [3H]nicotine showed clearly that at the time of antinociception, [3H]nicotine was localized almost entirely at the site of injection. Only trace quantities of [‘HInicotine

172&S 144+3 183*11 188 f 14 162k3 l88+6 229 * 13

459 f 12 399+65 568 + 82 568 jf 97 525 + 58 592 St 88 814 & 155

subcutaneous (2) 5OtP.b I,sM)c

10,100 k 3,170 13,200 + 140 36,300 * 4,110 14,400 + 2,230 14,800 -+ 2,800 IS,000It 4,260 13,800 k 5,070

(2) 3OOc 741 & 403 354 + 104 1,330 & 452 320 & 51 451 + 163 %I + 294 545 I 123

173 f 18 131 220 104*12

117+ 18 45 i 3 142 + IO

(2)

1,5w

428 _+137 93+12 518 rt 84

Su~u~neous Wb 228 + 94 42 it 8 312?~115

237 + 70 99+9 357?99

191 rt 39 lSf2 159 rt 16

374 & 140 35 i 5 321 f 42

11*3 7k2 237 + 124

1,300 58 336 10 590 688 188

(0.5) 25” 26+4 318+41 2,250 + 391

Subarachnoid 5b 15&6 235+78 528+71

12* 1 Ii *4 862 +_140

Route of administration (min after drug) Common carotid artery (0.5) Intraventricular (2) Vertebral artery (0.5) 3tw 50’ 2GQb 2oOb 4w 25s

“Doses as ngjkg; binactive dose; ‘effective or active dose

Liver Muscle Plasma

Tissue

4,929 f 3,750 + 650 + 70 k 1,580 + 1,840 + 414 +

1 rto 0 6&I

Su~~chnoid

6rt2 2+0 2553

(0.5) 25’

tissue or ~1 of plasma and are presented

3,030 f 64 681 -f: 166 1,601 + 279 234k64 229+41 388+139 177+43 32+15 96f52

Table 3. Levels of [WJnicotine in peripheral tissues. The results are expressed as picograms/mg as means +_SE of 5 animals

569 + 79 365+42 196+29

binactive dose; ‘effective or active dose.

356 & 131 235+68 178&40

Woses as ggbg;

380 t_ 50 293rt.44 288+.35

118?~:3 9453 85&10

subcutaneous (2) 500@ 1.5tlF

Route of administration (min after drug) Common Intraventricmar (2) carotid artery (0.5) Vertebral artery (0.5) 200” 3w 200s 4Ow 25b MC

Cervical Thorncic Lumbar

TisrUC

3,090 +- 1,210 I.310 rt 630 306f118 93 * 19 t ,070 f 433 620 A 182 173 &45

Vertebral artery (0.5) 25b 5w

tissue, and are presented as means i SE of

1,INl f 270 643rfr91 10,700 rt 2,590 3,420 & 1,130 4,660 -+ 1,840 8,000~ 3,100 4,610 rt 1,510

Table 2. Levels of [‘H]nicotine in spinal cord. The results are expressed as picograms/mg 5 animals

14,4tXl* 3,580 11,500+2,130 62,200 & 23,900 16,500 + 5,360 36,200 & 7,710 23,200 f 1,900 35,800 * 10,400

Inlravent~cular 2tw

‘Doses as &g/kg; ‘inactive dose; ‘effective or active dose.

Cerebelhrm Medulla Hypothalamus Striatum Midbrain Hippocampus Cortex

Tissue

tissue, and are presented as means + SE of 5 animals

Route of administration (min after drug) Common carotid artery (OS) 2Oob 460”

Table 1. Levels of [‘HI nicotine in brain. The results are expressed as picograms/mg

351 3+1 2+1 251 3&l 3_+1 2+0

Su~~hnoid I! 212 12+2 12_+2 lo+ 1 Ilk2 1312 IOk2

(OS) 25’

Spinal antinociceptive action of nicotine

structures. This finding strongly suggests the thoracic and/or lumbar sections as sites of the antinociceptive action. Linear regression analysis revealed a reasonably good correlation (r = 0.77) between the levels of [3H]nicotine in the thoracic region to the percentage of maximum possible effect after each route of administration, considering the variability in these two systems and the inclusion of 0% and 100% effects. A possible aberrant finding was the low levels of nicotine in the thoracic region after 400 pg/kg injected into the common carotid artery, a dose that produced 92% of the maximum possible effect. Omission of these data from the linear regression analysis would result in a correlation of r = 0.85. The correlation between the levels of nicotine in tissue and percentage of maximum possible effect for all routes of administration were as follows: cervical (0.38), lumbar (0.45), cortex (0.33), hippocampus (0.30), midbrain (- 0.07), corpus striatum (0.18), hypothalamus (0.27), medulla (0.08) and cerebellum (0.17). The levels of rH]nicotine were also measured in plasma, liver and muscle in order to provide a general assessment of the concentration of [3H]nicotine in central compared to peripheral tissues. It is clear that after intraventricular administration, the major portion of the [‘HInicotine was in central tissue. Intra-arterial injections resulted in relatively small concentrations of [‘HInicotine in liver and muscle, particularly in the case of vertebral arteries. It was also quite evident that little nicotine had escaped from the spinal cord after subarachnoid injections. were found in supraspinal

DISCUSSION

Taken collectively, the results suggest that an important site of action for antinociception induced by nicotine is the spinal cord. Firstly, nicotine was more potent when injected directly into the subarachnoid space than by any other route of administration. Significantly, the most marked difference in potency was between the intraventricular and subarachnoid routes; nicotine was at least 40 times more potent. Secondly, nicotine had the fastest onset of action after subarachnoid injection than by any other route of administration. The rapid onset of action after arterial injection may be due to the direct blood supply from the vertebral artery to the spinal cord. Also, injection into the common carotid artery may have led to a partial reversal of the pressure gradient in the Circle of Willis, which could have resulted in a greater than normal flow to the spinal cord and a resultant faster onset of action. Thirdly, the disposition studies demonstrated that [3H]nicotine was contained almost entirely in the thoracic and lumbar sections during antinociception, after subarachnoid injection. The correlation between the levels in spinal cord and antinociceptive activity after all routes of administration suggests that the thoracic region, rather than the lumbar (r = 0.85), is important with

1035

regard to the antinociceptive activity of nicotine. Although distribution data per se does not prove that nicotine acts on the spinal cord, it is consistent with the hypothesis. While it has been clearly demonstrated that administration of morphine into the spinal cord inhibits the tail-flick response (Yaksh, 1981; Fu and Dewey, 1981), morphine undoubtedly acts at the spinal and supraspinal levels (Yaksh and Rudy, 1978; Yaksh, 1981). A similar dual action of nicotine remains to be established. however, there is some evidence suggesting that nicotine does not act supraspinally. In fact, one of the major reasons for initiating these experiments was that. the time course of antinociception induced by nicotine did not correlate well with the time course of the levels of nicotine in whole brain and areas of the brain (Martin et al., 1983). More importantly, nicotine was found to be relatively inactive when given intraventricularly. A dose of 200 pg/kg produced only a 17% change in the reaction latency whereas peripheral administration of a dose only 7 times greater (1.5 mg/kg s.c.) produced a change of 82% in the latency of the response. Sahley and Bemston (1979) also reported a low potency for nicotine by the intraventricular route of administration. Furthermore, the disposition studies showed that intraventricular administration of [3H]nicotine (200 pg/kg) resulted in high levels of [‘HInicotine in all areas of the brain; yet there was very little antinociceptive activity. Administration of active doses of [‘HInicotine by all other routes resulted in concentrations in areas of the brain far less than those following an inactive dose, administered by the intracerebral route. While the data discussed thus far collectively argues strongly against supraspinal mechanism, it is difficult to rule out such a possibility. The very low levels of [31-IJnicotine in the thoracic area after injections into the common carotid artery may be artifactual. If not, then cervical or supraspinal mechanisms must be responsible for the effect of nicotine. The possibility that large concentrations of nicotine in supraspinal structures could contribute to the spinal actions has not been eliminated. The mechanism whereby nicotine produces antinociception in rats has not been elucidated but it has been shown that the effect of nicotine on the tail-flick response was not blocked by naloxone or atropine but that it was blocked by mecamylamine. Muscarinic and nicotinic cholinergic receptors in the spinal cord have been demonstrated (Headley, Lodge and Biscoe, 1975; Kayaalp and Neff, 1980). It seems reasonable to conclude that nicotine is stimulating the nicotinic cholinergic receptor in the spinal cord to block the tail-flick response. Nicotinic cholinergic innervation of Renshaw cells results in neuronal inhibition via the recurrent collateral, as well as inhibition of motor neurons. Stimulation of the Renshaw cell by nicotine or a similar inhibitory system could result in blocking either the perception or the reaction to painful thermal stimuli.

1036

M. D. ACETOet al.

Acknowledgements-This research was supported by Grant DA-02384 and DA-01647 from the National Institute on Drug Abuse.

Mansner R. (1972) Relation between some central effects of nicotine and its brain levels in the mouse. Annls. Med.

REFERENCES

Mansner R. and Mattila M. J. (1975) Nicotine-induced tremore and antidiuresis and brain nicotine levels in the rat. Med. Biol. 53: 169-176. Martin B. R., Tripathi H. L., Aceto M. D. and May E. L. (1983) Relationship of the biodisposition of the stereoisomers of nicotine in the central nervous system to their pharmacological actions. J. Pharmac. exp. Ther. 226:

Aceto M. D., Martin B. R., Uwaydah I. M., May E. L., Harris L. S., Izazola-Conde C., Dewey W. L., Bradshaw T. J. and Vincek W. C. (1979) Optically pure (+)-nicotine from (&)-nicotine and biological comparisons with (-)-nicotine. J. mednl Chem. 22: 174-177. Aceto M. D., martin B. R., Tripathi H. L., May E. L. and Jacobson A. E. (1980) Antinociceutive effect of onticallvpure stereoisomers ofnicotine. Pharmacologist 22‘: p. 30% D’Amour F. E. and Smith D. L. (1941) A method for determining loss pain sensation. J. Pharmac. exp. Ther. 72: 7&79.

Davis L., Pollock L. J. and Stone T. T. (1932) Visceral pain. Surg. Gynecol. Obst. 55: 418427. Dewey W. L., Harris L. S., Howes J. F. and Nuite J. A. (1970) The effect of various neurohumoral modulators on the activity of morphine and the narcotic antagonists in tail-flick and phenylquinone tests. J. Pharmac. exp. ther. 175: 435-442.

Fu T. C. and Dewey W. L. (1981) A new technique for the in vivo bioassay of opiates and other drugs on mouse spinal cord. Eur. J. Pharmac. 74: 239-242. Glowinski J. and Iversen L. L. (1965) Regional studies of catecholamines in the rat brain-I. The disposition of ‘H-norepinephrine, 3H-dopamine, and ‘H-DOPA in various regions of the brain. J. Neurochem. 13: 655-669. Headley P. M., Lodge D. and Biscoe T. J. (1975) Acetylcholine receptors on Renshaw cell of the rat. Eur. J. Pharmac. 30: 252-259.

Hucker H. B., Gillette J. R. and Brodie B. B. (1960) Enzymatic pathway for the formation of cotinine, a major metabolite of nicotine in rabbit liver. J. Pharmac. exp. Ther. 129: 94100.

Kamerling S. G., Wettstein J. G., Sloan J. W., Su T.-P. and Martin W. R. (1982) Interaction between nicotine and endogenous opioid mechanism in the unanesthetized dog. Pharmac. Biochem. Behav. 17: 733-740. Kayaalp S. 0. and Neff N. H. (1980) Regional distribution of cholinergic muscarinic receptors in spinal cord. Brain Res. 196: 426436.

exp. Biol. Fenn. 50: 205-212.

157-163.

Mattila M. J., Ahtie L. and Saarnivara L. (1968) The analgesic and sedative effects of nicotine in white mice, rabbits and golden hamsters. An&s. med. exp. Biol. Fenn. 46: 7&84.

Ohlsson A. E., Fu T.-C., Jones D., Martin B. R. and Dewey W. L. (1982) Distribution of radioactivity in the spinal cord after intracerebroventricular and intravenous injection of radiolabelled opioid peptides in mice. J. Pharmac. exp. Ther. 221: 362-367.

Phan D. V., Doda M., Bite A. and Gyorgy L. (1973) Antinociceptive activity of nicotine. Acta physiol. hung. 44: 85-93.

Sahley T. L. and Bemtson G. G. (1979) Antinociceptive effects of central and systemic administration of nicotine in the rat. Psychopharmacology 65: 279-283. Tripathi H. L., Martin B. R. and Aceto M. D. (1982) Nicotine-induced antinociception in rats and mice: Correlation with nicotine brain levels. J. Pharmac. exp. Ther. 221: 91-96.

Tsujimoto A., Nakashima T., Tanino S., Dohi T. and Kuroaochi Y. (1975) Tissue distribution of I’Hl-nicotine in dogs and rhesus ‘monkeys. Toxic. appl. ihaimac. 32: 21-31.

Vincek W. C., Martin B. R., Aceto M. D. and Bowman E. R. (1980) Synthesis and preliminary binding studies of 4,4-ditritio-( -)-nicotine of high specific activity. J. mednl Chem. 23: 960-962.

Yaksh T. L. (1981) Spinal opiate analgesia: Characteristic and principles of action. pain 11: 293-346. Yaksh T. L. and Rudy T. A. (1978) Narcotic analgesia: CNS sites and mechanism of action or revealed by intracerebral injection techniques. Pain 4: 299-359.