Pain, 22 (1985) 49-57 Elsevier

49

PAI 00739

Research Reports

A Study of Intracerebroventricular Self-Ad~nistration of Leucine or Met~onine

Enkephalin by Rats in Response to Intermittent Electric Shocks

Bassam Dib

L.uboratoire de Physioiogie, lJniversi3 Claude Bernard, Facukt? de Mhdecine Lyon - Sud B. P. 12, 69921 Oullins Cedex (France)

(Received 23 July 1984, accepted 26 November 1984)

Rats were taught to self-administer Leu-E (10, 25 and 100 pg/pl) or Met-E (0.5, 10 and 100 pg/$) through a cannula implanted in the lateral cerebral ventricle (i.c.v.). Their self-injection behaviour was studied before, during and after nocicep- tive stimulation. In the course of the control period of the experiment, the rats rapidly learned lever pressing for self-injection of enkephalin but they did not increase their self-administration of Leu-E or Met-E during the nociceptive electrical stimulation period. Also studied were the acute effect of i.c.v. enkephalin and morphine on tail-flick latency (set) and electrical threshold vocalization (mA). The analgesic effect of Leu-E and Met-E was of short duration (less than 2-6 min). The mean rise (i.e., analgesia) of the tail-flick threshold showed a significant difference after i.c.v. Met-E only. The acute i.c.v. effect of 20 or 30 hg of morphine induced a long-lasting analgesia, > 40 min.

These results show that Leu-E and Met-E are not rewarding during a nociceptive stimulus. This may be due to the short and inconstant analgesic action of i.c.v. enkephalins.

Intmduction

It has been suggested that methionine (H-Tyr-Gly-fly-Phe-Met-OH) and leucine (H-Tyr-Gly-Gly-Phe-Leu-OH) enkephalin are neurotransmitters involved in central pain mediation [ll]. The identification of both peptides had led to it being suggested

0304-3959/85/%03.30 0 1985 Elsevier Science Publishers B.V. (Biomedical Division)

50

that enkephalin receptors may be sites at which opiate drugs induce analgesia, and that the enkephalins themselves may be transmitters or modulators in brain systems of pain suppression or analgesia [12]. Consistently with these suggestions. central administration of enkephalin in animals produces morphine-like analgesia [3]. It has been demonstrated that laboratory rats will self-administer leucine- (Leu-E) and methionine-enkephalin (Met-E) at rates approximately 2-4 times as high as isotonic saline [3]. Recently we demonstrated that rats increased their intracerebroventricular (i.c.v.) self-administration of morphine in response to nociceptive stimulus [6]. This effect is specific for this opiate since under identical conditions the rats did not increase isotonic saline. We wished to find out whether, under the same conditions, the rats would increased their i.c.v. self-injection of enkephalins.

Methods

Surgery Surgery was performed on 64 Wistar albino rats weighing 280-300 g, under

surgical anaesthesia with 35 mg/kg i.p. pentobarbital. Ten minutes before anaesthesia the rats received 0.3 mg of atropine. A chronic guide cannula of stainless steel tubing, inner diameter 0.5 mm, was implanted stereotaxically in the right lateral ventricle. Stereotaxic coordinates were L = 2 mm, V = 3.5 mm, A = 6-6.5 mm.

Location of cannulae After a recovery period of at least 1 week, the correct positioning of the cannulae

was confirmed by the infusion of 3 ~1 of a solution containing 1 ng of human angiotensin II (Beckman). Given intraventricularly this octapeptide induces an immediate drinking response [7]. The angiotensin test was repeated after the comple- tion of the experiments. Nine rats showing no drinking response to angiotensin were eliminated. Histological examination of the brain confirmed that in these cases the cannulae had been placed outside the lateral ventricle.

Nociceptive thresholds The threshold for vocalization to radiant heat and electrical stimulation was

studied in 23 rats. Some rats were used in more than one experiment. Radiant heat and electrical stimulation thresholds were measured before and after injection of the drugs and during control. The noxious radiant heat threshold was measured by the tail-flick instrument described by D’Amour and Smith [4]. Nociceptive thresholds were measured as the latencies, or reaction times of movement of the tail, from above a source of noxious radiant heat. The intensity of noxious heat was adjusted to produce a baseline tail-flick of 8-10 sec. Tail-flick latency was tested every 2 or 5 min, and if tail-flick had not occurred by 30 set (cut-off) the heat was shut off in order to avoid tissue damage. The nociceptive electrical threshold (mA) was mea- sured in the Skinner box. An electrode was strapped at the base of the tail. The electrical nociceptive threshold was defined by the current between the tail and the grid of the Skinner box inducing vocalization.

51

Before and after the self-injection experiments radiant heat and electrical stimula- tion nociceptive thresholds were measured twice. The results are expressed as a mean of the successive measurements.

Drug injection Before the experiment, the dose concentrations of met- and leu-enkephalin were

tested in 8 rats in order to obtain a reasonable estimate of the liminal dose. We chose 200 pg of enkephalin. In all the experiments on the acute effect of drugs, morphine (20 or 30 pg) was injected after enkephalin. Rats were sometimes used for more than one experiment, and so received 2 injections.

Skinner box For intraventricular self-administration each rat, with the cannula correctly

placed, was tested in a plastic cylinder 0.35 m high and 0.30 m in diameter. The plastic cage was equipped with a lever 37 mm long, 5 mm broad and 20 mm above the cage floor.

Set-administration A stainless steel injection needle, outer diameter 0.4 mm, was inserted in the

lateral ventricle via the guide cannula. The needle was connected by polyethylene tubing to an infusion pump. This infusion tube and the wire for nociceptive stimulation were wound in a spiral enabling the rat to move freely in the Skinner box. Details of this technique have been given elsewhere [S]. Each lever press delivered 1 ,ul fluid within 15 set to the ventricle and was recorded. Responses during the 15 see infusion intervals were of no consequence and went unrecorded. During the week before the first experiment all rats were placed in the Skinner box for 2 days to learn to use the lever for self-administration of Leu-E or Met-E. During each experiment the rat could self-administer 0.5, 10, 100 pg/pl of Met-E or 10, 25, 100 pg/pl of Leu-E. Each experiment lasted 60 min and was divided into 4 parts: exploration, 15 min; control, 15 min; nociceptive stimulation, 15 min; control, 15 min. Immediately on entering the Skinner box the rat had access to the lever. The nociceptive stimulus consisted of electrical pulses of 5 set duration, applied every 5 set, between the base of the tail and the grid of the Skinner box floor. Threshold level for vocalization was measured, and the current then adjusted to between 0.4 and 0.8 mA below the threshold, depending of the rat. Two series of experiments were carried out using the above protocol. In the first series, the rats had access to Met-E by self-administration; one experiment was carried out on each rat (n = 18). In the second series, the rats had access to Leu-E by self-administration; one experiment was carried out on each rat (n = 21). Some rats were used in more than one experiment, the experiments being separated by a period of 1 week.

Rest&s

(I) Acute effects of i.c.v. Met-E or Lw-E injection on nociceptive electrical threshold

CmA) Fig. 1A shows the mean electrical threshold for vocalization. The electrical

52

threshold remained unchanged after the i.c.v. injection of 200 pg of Met-E or Leu-E. However, in the same rats the electrical threshold rose after the i.c.v. injection of 30

pg of morphine. This rise was statistically significant from 5 min morphine injection till the end of the experiment (Student’s t test, P < 0.001).

(2) Acute effects of i.c.v. fluid injection on tail-flick latency (set) Fig. 1B shows mean tail-flick latency. The rise of tail-flick latency threshold was

significant immediately after 200 pg of Met-E. Compared with that of the preinjec- tion period this rise was obvious between the 1st and 7th min (Student’s t test,

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Fig. 1. A: mean f S.E.M. of vocalization threshold before and after i.c.v. injection of met- or leu-enkepha- lin or morphine. Asterisks indicate statistical levels of significance: Student’s t test, ** P < 0.02,

*** P i 0.01, **** P < 0.001. n indicates the numbers of rats. One experiment per rat. B: mean f S.E.M.

of tail-flick latencies before and after i.c.v. injection of leu-enkephalin and morphine (see A). C:

mean f S.E.M. of tail-flick latencies before and after i.c.v. injection of met- or leu-enkephahn or NaCl (see

A).

53

P < 0.001). After i.c.v. injection of 200 ,ug of Leu-E we noted partial analgesia, and also tail-flick latency rising little above baseline levels and lasting for 1 min only. When compared with the preinjection tail-flick threshold this rise did not show any significance. Partial analgesia was also observed in other groups of rats after the i.c.v. injection of 200 pg of Leu-E (Fig. 1C). Sixty minutes after Leu-E injection, 20 pg of morphine was administered i.c.v., and this immediately induced a rise in the latency threshold. Compared with the preinjection period (50th min) this rise was significant from the 75th min till the end of the experiment (Student’s t test, P < 0.001).

(3) Intracerebroventricular self-injection behaviour (a) Met-E injection. The mean results of the 3 series of experiments at different

doses of Met-E (0.5, 10 and 100 pg) are shown in Fig. 2A and Table I. In the first, second and third series of experiments, the rats could self-administer 0.5, 10 or 100

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Fig. 2. A: group mean of mean response ratesf S.E.M. in 60 min experiments under conditions of self-injection of met-enkephalin: in the first experiment 0.5 pg/pl per injection, in the second 10 pg/pl per injection, in the third 100 ng/pl per injection. In both control periods at all doses, during nociceptive stimulation, the rats did not significantly increase i.c.v. self-injection of met-enkephalin. B: group mean of mean response rates* S.E.M. in 60 min experiments under conditions of self-injection of leu-enkephalin: in the first experiment 10 pg/pl per injection, in the second 20 pg/ul per injection, and in the third experiment 100 pg/cl per injection. The rats did not increase self-injection during the nociceptive period. For mean lever-pressing during the pre- and post-nociceptive control as compared with the nociceptive period Student’s t test shows a significant difference (*P < 0.05, *** P-c 0.01, ****P < 0.001). n indicates the numbers of rats. One experiment per rat.

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pg of Met-E each time bar-pressing activated the infusion pump. In the first and third experiments, during the exploration and control period, the rats self-injected Met-E. Bar-pressing did not increase Met-E self-administration during nociceptive stimulation or the post-control periods. In the second series of experiments, the animals did not activate the infusion pump much.

In each experiment, cumulative Met-E intake over the whole experiment did not modify the nociceptive electrical threshold or tail-flick latency (Table I).

(b) La-E self-injection. The mean results of the 3 series of experiments at different doses of Leu-E (10, 25 and 100 pg) are shown in Fig. 2B and Table I. In these series of experiments the rats could self-administer 10, 25 or 100 pg of Leu-E each time bar-pressing activated the infusion pump. In all 3 series of experiments, the rats did not activate the infusion pump during the pre- and post-control periods except in the third series of experiments where the rats activated the infusion pump. Bar-pressing did not increase Leu-E self-injection during nociceptive stimulation. When mean lever-pressing during the nociceptive period and mean lever-pressing during the post-nociceptive control period are compared Student’s t test shows a significant difference (P < 0.001).

Discussion

The results of the present experiment on rats do not show an increase of i.c.v. self-injection of Met-E or Leu-E. This confirms the previous observations of Dib and Duclaux [6], when rats self-administered morphine or isotonic saline intracerebrally in the absence of a nociceptive stimulation period. In the control period before the nociceptive stimulation experiment, the rats pressed the lever for self-injection of enkephalin, morphine or isotonic saline in exactly the same way. That we did not find any difference between the enkephalin, and morphine or isotonic saline injec- tions during the first control period may be due to the short time (15 min) used by us. Belluzzi and Stein [2] demonstrated that when the rats had access to the lever over several days they showed a preference first for Leu-E, then for morphine Met-E and lastly for isotonic saline, as is shown by the number of bar-presses.

In the present experiments, the rats did not increase self-injection of Met-E or Leu-E during nociceptive stimulus as they did in the pre-nociceptive control periods. Self-injection of Leu-E during nociceptive stimulation was significantly different from the post-nociceptive control period. The difference is due to the absence of lever-pressing behaviour in the second control period. In the precontrol period rats pressed the lever for enkephalin self-injection. This behaviour is probably due to euphoria or the rapid reward of enkephalin [2]. In fact, the present findings do not reproduce those observed by Dib and Duclaux [6]. These authors have recently demonstrated that rats significantly increase i.c.v. self-injection of morphine in response to a nociceptive stimulation. From the present experiment it may be concluded that enkephalins are not rewarding during a nociceptive stimulation period. This may be due to the short and inconstant analgesic action of i.c.v. enkephahn. This hypothesis seems likely when we compare the effect of Leu-E or

56

Met-E and morphine on tail-flick latency and electrical threshold for vocalization. Both substances (Met-E and Leu-E) had a short-duration analgesic effect. The

rise (i.e., analgesia) in tail-flick latency threshold was significant after Met-E injection only. A non-significant partial analgesia was observed from 1st to 2nd min after Leu-E injection. In this experiment, contrary to enkephalin, morphine induced a long analgesia > 40 min in tail-flick latency (set) and in the nociceptive electrical threshold (mA) test. The moderate analgesia (2-6 min) observed in the present results confirms previous findings [3,9,13]. In our experiment, Met-E appears to be more potent than Leu-E for the same doses. Met-E and Leu-E were less potent than morphine. The analgesia caused by i.c.v. injection of 20 or 30 pg of morphine lasted substantially longer than 1 h [8]. The strong analgesia induced by i.c.v. injection of morphine may be compared with the effect of /3-lipotropin hormone [9]. The shorter duration of the action of these endogenous peptides may be attributed, at least in part, to their relatively rapid degradation by brain enzymes [10,14].

In conclusion, these experiments demonstrate that the rats did not increase i.c.v. self-administration of enkephalins during nociceptive stimulation. The enkephalin reinforcement after each bar-press appears insufficient to alleviate pain sensation. This reduction in behavioural response after self-injection of enkephalin is probably due to rapid degradation due to brain enzymes [ll]. Further research will be necessary to elucidate the precise role of these endogenous peptides (Met-E and Leu-E).

Acknowledgements

I would like to thank Florette Godinot for her technical assistance. The present work received the support of the C.N.R.S. (Centre National de la

Recherche Scientifique, L.A. 180).

References

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3 Belluzzi, J.D., Grant, N., Garsky, V., Sarantakis, D., Wise, C.D. and Stein, L., Analgesia induced in

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