Time-dependent melatonin analgesia in mice: inhibition by opiate or benzodiazepine antagonism

European Journal of Pharmacology, 194 (1991) 25-30 © 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 001429999100175Y

25

EJP 51740

Time-dependent melatonin analgesia in mice: inhibition by opiate or benzodiazepine antagonism D i e g o A. G o l o m b e k , E s t e b a n Escolar, Leila J. Burin, M a r i a G. D e Brito S~nchez a n d D a n i e l P. C a r d i n a l i Departamento de Fisiologla, Facultad de Medicina, Universidad de Buenos Aires, CC 243, 1425 Buenos Aires, Argentina Received 16 August 1990, revised MS received 15 November 1990, accepted 4 December 1990

The aim of this study was to determine whether melatonin-induced analgesia in mice exhibits the time dependency known to occur for several other effects of the hormone, and to analyze to what extent the activity of melatonin can be inhibited by the opiate antagonist naloxone or the central-type benzodiazepine (BZP) antagonist Ro 15-1788. Analgesia was assessed with the hot plate procedure. There was a significant diurnal variation in the pain threshold, with an increase in latency during the dark phase of the daily photo period. Melatonin (20-40 mg/kg i.p.) exhibited maximal analgesic effects at late evening (20:00 h). The administration of naloxone or Ro 15-1788 at 20:00 h, although unable by themselves to modify pain threshold, blunted the analgesic response to melatonin. Significant increases in the latency of the hot plate response were found after diazepam injection, an effect blocked by Ro 15-1788 or naloxone. These results indicate that time-dependent melatonin analgesia is sensitive to opioid or central-type BZP antagonism. Melatonin; Analgesia; Benzodiazepine antagonism; Naloxone; (Time-dependent effects)

1. Introduction

Melatonin, a hormone secreted by the pineal gland, regulates seasonal reproduction (P6vet, 1987; Reiter, 1987; Arendt, 1988). In most species, including man, the circulating levels of melatonin display a circadian pattern, with maximal values occurring at night. The diurnal variations in melatonin encode a neuroendocrine time signal for seasonality (Goldman, 1983; Karsch et al., 1984; Tamarkin et al., 1985). Additionally, a number of behavioral effects of melatonin have been described, e.g. sleep promotion and activity depression in rats (Sugden, 1983; Mirmiran and P6vet, 1986), analgesic and anticonvulsant activities in mice and rats (Izumi et al., 1973; Albertson et al., 1981; Sugden, 1983), and entrainment of biological rhythms in rats (Redman et al., 1983). The physiologic activity of melatonin, as well as some of its pharmacological effects, depend on the time of hormone administration, with there being a late afternoon-early evening period of maximum sensitivity, and a second, short period of sensitivity just preceding lights on (Goldman, 1983;

Correspondence to: D. P. Cardinali, Departamento de Fisiologla, Facultad de Medicina, UBA, CC 243, 1425 Buenos Aires, Argentina.

Karsch et al., 1984; Tamarkin et al., 1985; Stetson et al., 1986; Reiter, 1987). Several observations point to a significant interaction between the pineal gland and opiate mechanisms in the brain (Lissoni et al., 1986; Esposti et al., 1988). Pinealectomy of rats (Kumar et al., 1982), or manipulation of the light-dark cycle in hamsters (Kumar et al., 1984), changes the levels and circadian rhythmicity of [MetS]enkephalin concentrations in hypothalamic areas. An effect of environmental lighting has also been described on fl-endorphin levels in the hypothalamus (Genazzani et al., 1987). In mice, removal of the pineal gland decreases the analgesic response to morphine while injection of melatonin (30-90 m g / k g ) brings about a naloxone-reversible analgesia (Lakin et al., 1981). The latter findings have not always been reproducible, and a non-specific action of the very high amounts of melatonin on analgesia, rather than an opiate-mediated effect of the compound, has been suggested (Sugden, 1983). Moreover, melatonin has hyperalgesic activity in mice (Takahashi et al., 1987). The present study was carried out to obtain additional information about the analgesic properties of melatonin in mice. Since a chronobiological examination of melatonin activity was not carried out in the above mentioned studies, we designed a series of experi-

26

ments to answer the following questions: (i) is there a diurnal rhythm of melatonin-induced analgesia in mice?; (ii) can this rhythm be disrupted by exposing the animals to continuous light?; (iii) can be opiate antagonist naloxone or the central-type benzodiazepine (BZP) antagonist Ro 15-1788 interfere with melatonin-induced analgesia? Our data argue in favor of a time-dependent, naloxone- and Ro 15-1788-sensitive effect of melatonin on pain perception.

2. Materials and methods

2.1. Animals Male Swiss Albino mice (20-30 g) were used in all experiments. Animals were fed with a rodent pellet diet and water ad-libitum, and were housed under a 12:12 l i g h t / d a r k regime (lights on at 08:00 h) at a room temperature of 20 + 2 ° C. Dim red lighting was provided in the experimental room to allow visualization and injection of mice during the dark period.

2.2. Drugs Melatonin (Sigma Chemical Co., St. Louis, MO, USA), naloxone (Endo Lab. Inc., USA) and Ro 15-1788 (a generous gift from H o f f m a n n La Roche, Basel, Switzerland) were dissolved in 1 : 4 ethanol-saline, with the total administration of ethanol never exceeding 20 ~tl. All animals were injected i.p. (100 #I per animal). Melatonin doses varied from 5 to 30 m g / k g , while naloxone and Ro 15-1788 were administered at doses of 10 and 20 m g / k g , respectively.

3. Results

Figure 1 shows the rhythm of analgesia in control mice treated with vehicle. There was a significant diurnal variation in the pain threshold, with a significant increase in latency in the hot plate test (FRT) during the dark phase of the daily photoperiod. Naive mice responded in the same way as the vehicle-treated animals (results not shown). Figure 2 summarizes a number of experiments carried out to assess the diurnal rhythmicity of melatonininduced analgesia. Data are expressed as the ratio between the time needed for F R T in melatonin-treated mice and that of vehicle-treated mice, assessed 30 min after injection. A m a x i m u m melatonin response was observed at 20:00 h. This time-dependent activity of melatonin was observed with 20 m g / k g of melatonin (fig. 2), as well as with 40 m g / k g (results not shown). No significant F R T increase was found with 5 m g / k g of melatonin (data not shown). The analgesic action of melatonin at 20:00 h was characterized further (fig. 3). A maximal effect of the hormone was found 30 min after injection of 20 m g / k g melatonin with significant activity also being observed 60 min after melatonin administration. M e l a t o n i n / control ratios in analgesic activity were 1.30 + 0.30, 1.44 + 0.08 and 1.27 + 0.03, after placing the animals on the hot plate for 15, 30 or 60 min, respectively. It is possible that a conditioned response of licking or jumping occurred when animals were placed on the hot plate for the second or third time during trial (i.e. 30 or 60 min after drug administration). In order to rule out this possibility, mice were placed on the hot plate only once, either 30 or 60 min after vehicle administra-

2.3. Evaluation of analgesia Pain thresholds were evaluated with the hot plate test, as described by Lakin et al. (1981). Mice were placed individually on the hot plate (50 + I ° C ) and their first response time (FRT) was recorded. F R T is defined as the time elapsed to obtain one of the following responses: licking the feet, jumping or rapidly stamping the feet, whichever occurred first. Analgesia is defined as a statistically significant difference in the F R T between control and treated mice. Mice were placed on the hot plate 15, 30 and 60 min after i.p. administration of the drugs or vehicle, unless stated otherwise. The F R T of non-treated animals was also recorded.

2.4. Statistical analysis Data were statistically analyzed with Student's t-test, a factorial analysis of variance, or a one or two-way A N O V A followed by Dunnett's t- or Tukey's tests.

20

15

',

iO

[

5

o8~o

,2'oo

' , oo

' ooo

o200

CLOCKTIME

Fig. 1. Diurnal rhythmicity of murine analgesia, as assessed with the hot plate test described in Methods. Animals were exposed to daily photoperiods of 12 h light, 12 h darkness (lights on from 08:00 to 20:00 h). Groups of 10 mice received i.p. injections of vehicle (1:4 ethanol : saline, 100 #1) 30 min before the hot plate test. Shown are the means + S.E. of the first response time (FRT), recorded as described in Methods. Asterisks designate significant differences (P < 0.05) compared to animals tested at 20:00 h, two-way ANOVA, Tukey's test.

27 TABLE 1

1.75

Effect of prior exposure to the hot plate on the subsequent response of mice to the hot plate. Shown are the means + S.E. (n = 10 in each group). Pain thresholds were evaluated in the hot plate test (at 14:00 h), as described in Methods. Mice were injected i.p. with vehicle and placed individually on the hot plate, either once (at 30 ot 60 min of injecting vehicle, single exposure group) or after being exposed to the plate one (at 15 min) or two times (at 15 and 30 rain) (multiple exposure group). The first response time (FRT) was recorded as described in Methods. Student's t-test indicated the absence of significant differences as a function of prior exposure to the hot plate.

_o i .50

1.25 z z 1.00

J i.l.i ~E

~_

I 0.75

I

I

0800

1200

I

I

1600

2000

I 2400

I

Number of exposures

I

FRT (s)

0400

CLOCKTIME

Fig. 2. Diurnal rhythmicity of melatonin-induced murine analgesia, as assessed with the hot plate test described in Methods. Animals were exposed to daily photoperiods of 12 h light, 12 h darkness (lights on from 08:00 to 20:00 h). Groups of 10 mice received i.p. injections of 20 m g / k g of melatonin or vehicle (1:4 ethanol:saline, 100 pl), 30 min before the hot plate test. Shown are the means+S.E, of melatonin/vehicle ratio for the first response time (FRT), defined as described in Methods. Asterisks designate significant differences (P < 0.05) compared to animals tested at 20:00 h, two-way ANOVA, Tukey's test.

Single exposure Multiple exposures

cr

u_

Anal-

g e s i a w a s a s s e s s e d a t 1 2 : 0 0 o r 2 0 : 0 0 h, t h a t is, a t t h e maximum and nadir of the melatonin effects found in animals

under

in FRT

between

sent,

normal

lighting conditions.

Differences

t h e t w o t i m e i n t e r v a l s w e r e still p r e -

as revealed

by

a factorial

P < 0.0005). The results obtained significant differences in FRT

ANOVA

( F = 19.96,

indicate an absence of

between

melatonin-

10

5

0

-

V

M

V

M

Fig. 4. Effect of continuous (i.e. 1 week) exposure of mice to light (LL) on melatonin-induced analgesia, assessed at 12:00 to 20:00 h. Groups of 10 mice received i.p. injections of 20 m g / k g of melatonin (M) or vehicle (V) (1 : 4 ethanol : saline, 100 #1) 30 rrdn before the hot plate test. Shown are the means ___S.E. of FRT, computer as described in Methods.

and TABLE 2

16

Antagonism of melatonin-induced analgesia by naloxone or Ro 151788, assessed at 20:00 h. Animals received 20 m g / k g of melatonin or vehicle i.p. 15, 30 or 60 min before the hot plate test. Naloxone (10 mg/kg) was administered 5 min, and Ro 15-1788 (20 mg/kg) was administered i.p. 15 rain before melatonin or vehicle. Results are expressed as the means+S.E, of the first response time (FRT), recorded as described in Methods (n = 10 in each group).

o

u_

iLL)

light

schedule for 5 days to analyze the influence of continuous light on the analgesic activity of melatonin.

2000h

15

had previously experienced

of mice were placed under a continuous

12.4 + 1.4 13.9 + 0.4

1200h

q,

Groups

60 rain

12.2 _+1.8 13.4 _+2.1

20

tion. The results obtained indicated that FRT values did not differ significantly from those found in animals that t h e t e s t ( t a b l e 1).

30 rain

I0

Experimental group

control

8 0

t 15 MINUTES

I 30

t 45

t 60

AFTER MELATONIN ADMINISTRATION

Fig. 3. Melatonin-induced analgesia in mice, assessed late evening (20:00 h). Groups of 10 mice received i.p. injections of 20 m g / k g of melatonin or vehicle (1:4 ethanol:saline, 100 /.tl), 15, 30 or 60 min before the hot plate test. Shown are the means 4-S.E. of FRT, computed as described in Methods. Asterisks designate significant differences between melatonin and vehicle-injected control groups, P < 0.01, Student's t-test.

Vehicle Melatonin Melatonin plus naloxone Melatonin plus Ro 15-1788 Naloxone Ro 15-1788

FRT (s) 15 min

30 min

60 min

10.6 4- 0.5 19.2+2.1 a

13.9 4- 0.7 17.2-t-1.8 b

15.3 + 0.8 21.4+2.1 a

12.5 + 0.8

12.5 + 1.2

15.2 + 0.9

11.4+1.3 11.9+0.5 12.6+0.8

12.6+0.8 14.9+0.5 11.4+1.3

15.34-0.9 16.9-t-0.4 12.6+0.8

a p < 0.05, two-way ANOVA, Dunnett's t-test; b p < 0.05, one-way ANOVA, Dunnett's t-test; significant differences compared to vehicle-injected controls.

28 TABLE 3 Antagonism of diazepam-induced analgesia by naloxone or Ro 151788, assessed at 20:00 h. Animals received 5 mg/kg of diazepam or vehicle i.p. 15, 30 or 60 min before the hot plate test. Naloxone (5 mg/kg) or Ro 15-1788 (10 mg/kg) was administered i.p. 5 or 15 min before diazepam, respectively. Results are expressed as the means_+ S.E. of the first response time (FRT), recorded as described in Methods (n = 10 in each group). Experimental group

FRT (s) 15 min

30min

60min

Vehicle Diazepam Diazepam+ naloxone Diazepam plus Ro 15-1788

8.7+0.4 14.4_+2.2 a

8.3_+0.6 15.9_+2.1a

10.9_+1.2 15.4_+1.9 b

10.9_+1.1

12.5_+0.8

14.2_+0.9

8.4_+0.7

11.2_+0.9

11.3_+0.9

p < 0.05, two-way ANOVA, Dunnett's t-test; b p < 0.05, one-way ANOVA, Dunnett's t-test; significant differences compared to vehicle-injected controls. a

vehicle-treated mice when tested at 12:00 or 20:00 h, or a factorial main effect of the treatment (F =0.123, P > 0.73) (fig. 4). The effect of naloxone or Ro 15-1788 on melatonin analgesia is summarized in table 2. When assessed at 20:00 h, both naloxone and Ro 15-1788 blunted the analgesic response to melatonin. Neither drug exerted effects by itself. In an independent experiment, a significant induction of analgesia was found 15-60 rain after diazepam injection, an effect blocked by Ro 151788 (at all tested intervals) or naloxone (at 15 and 30 min after injection) (table 3).

4. Discussion

Our data further demonstrate the existence of a diurnal rhythm in the latency to respond to a pain stimulus in mice, with maximal latencies being found at night (Frederikson et al., 1977; Lakin et al., 1981; Oliverio et al., 1982; Kavaliers and Hirst, 1983). Injection of the pineal hormone melatonin in pharmacological amounts (20-40 m g / k g ) caused analgesia, an effect which peaked late in the evening. These data, which constitute the first description of a time-dependent effect of melatonin on analgesia, generally agree with the circadian variation in melatonin activity reported for other CNS functions, e.g. inhibition of hypophysialgonadal axis in hamsters (Tamarkin et al., 1985; Stetson et al., 1986; Reiter, 1987) and rats (Lang et al., 1983), entrainment of circadian rhythm of locomoter activity in rats (Redman et al., 1983) and effect of melatonin on 2-deoxyglucose uptake (Cassone et al., 1988) or electrical activity of hypothalamic suprachiasmatic nuclei in rats (Shibata et al., 1989; Stehle et al., 1989). In addition, exposure of mice to continuous light, a situation

known to disrupt circadian rhythmicity, also blunted the time dependency of melatonin-induced analgesia. It must be noted that the doses of melatonin needed to obtain analgesia in mice are somewhat greater than those required for neuroendocrine effects. Although there are no data available on the levels of melatonin in murine brain after systemic administration of the hormone, unpublished data from our laboratory indicate that, in rats, the levels of melatonin (assayed by high pressure liquid chromatography) reached in the hypothalamus 20 min after i.p. administration of 5 or 10 m g / k g were 3.1 + 0.5 and 6.8 _+ 0.9 n g / m g medial basal hypothalamus (H.E. Chuluyan, B.I. Kanterewicz, D.P. Cardinali, unpublished data). The site and mechanism of action of melatonin to induce analgesia remain to be defined. One obvious candidate is the central opioid system, since pinealectomy or melatonin injection causes modifications in brain [MetS]enkephalin content ( K u m a r et al., 1982; 1984). Melatonin administration reduces blood fl-endorphin levels and impairs the LH-releasing effect of the opioid receptor antagonist naloxone in humans (Lissoni et al., 1986; Esposti et al., 1988). Another opioid antagonist, naltrexone, counteracts the immuno-enhancing activity of melatonin in mice (Maestroni et al., 1989). Conflicting reports, however, have been published about the efficacy of naloxone to block the analgesic effect of melatonin. Although an effective inhibition of melatonin action by naloxone was initially observed (Lakin et al., 1981), a subsequent study could not confirm this effect, which suggests that the hormone has a non-specific effect when given at high concentrations (Sugden, 1983). Our present results support the existence of a naloxone-sensitive effect of melatonin on murine analgesia, since opioid antagonism fully prevented the melatonin effect. The proposed link between melatonin and the central serotonergic system (AntonTay et al., 1971; Gaffori and Van Ree, 1985), and the role that serotonergic neurons play in controlling opioid-mediated endogenous analgesia (Le Bars et al., 1986), suggest that the mechanisms are linked, a hypothesis that deserves to be explored further. Results obtained from rats indicate that central synapses employing -f-aminobutyric acid (GABA) as an inhibitory transmitter can be a target for melatonin. Pinealectomy disrupts circadian rhythmicity of brain G A B A and BZP binding (Acuha-Castroviejo et al., 1986a,b; Kennaway et al., 1988), with low doses of melatonin counteracting the modifications in BZP and G A B A binding. Additionally, melatonin administration accelerates turnover rate of G A B A in the brain (Rosenstein and Cardinali, 1986), augments G A B A synthesis, and potentiates G A B A postsynaptic activity on G A B A type A receptor sites (Rosenstein et al., 1989). The activity of melatonin to induce these effects was greater

29 in t h e e v e n i n g t h a n in t h e m o r n i n g . O u r p r e s e n t results p r o v i d e f u r t h e r s u p p o r t for a l i n k b e t w e e n m e l a t o n i n a n d c e n t r a l G A B A e r g i c r e c e p t o r s , since t h e e f f e c t o f m e l a t o n i n to p r o m o t e m u r i n e a n a l g e s i a c o u l d b e prev e n t e d b y t h e c e n t r a l - t y p e B Z P a n t a g o n i s t R o 15-1788. In our study, the central-type BZP agonist diazepam i n d u c e d an i n c r e a s e in l a t e n c y a f t e r h o t p l a t e a n a l g e s i a in m i c e , a n d this effect, as in the c a s e o f m e l a t o n i n , was significantly impaired by naloxone. However, diazepam produced sedation, amnesia and motor weakness, and t h e s e effects c o u l d c o n t r i b u t e to t h e effects o b s e r v e d . It m u s t b e n o t e d t h a t a l t h o u g h t h e r e is e x p e r i m e n t a l evid e n c e in v i t r o i n d i c a t i n g t h a t n a l o x o n e b l o c k s G A B A p o s t s y n a p t i c a c t i v i t y ( D i n g l e d i n e et al., 1978), the levels o f n a l o x o n e r e a c h e d in t h e b r a i n at the d o s e s u s e d in t h e p r e s e n t s t u d y are p r e s u m a b l y m u c h l o w e r t h a n t h o s e u s e d in vitro.

Acknowledgements These studies were supported by grants from the Consejo Nacional de Investigaciones Cientificas y Trcnicas, Argentina (PID 211/89), Fundacirn Antorchas, Buenos Aires, Argentina (Project No. 11083/1) and Volkswagen-Stiftung, Hannover, FRG (Project 1/65 721).

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