Effects of electric footshock on barviturate sensitivity, nociception and body temperature in mice

European Journal of Pharmacology, 89 (1983) 119-123

119

Elsevier Biomedical Press

E F F E C T S OF ELECTRIC F O O T S H O C K ON BARBITURATE SENSITIVITY, N O C I C E P T I O N AND BODY T E M P E R A T U R E IN MICE JOHN CARMODY

School of Physiology and Pharmacology, University of New South Wales, Kensington (Sydney), NSW 2033, Australia Received 16 November 1982, revised MS received 18 January 1983, accepted 3 February 1983

J. CARMODY, Effects of electric footshock on barbiturate sensitivity, nociception and body temperature in mice, European J. Pharmacol. 89 (1983) 119-123. In female (Balb/c) mice, electric footshock increases the animals' sensitivity to pentobarbitone; this action is reversed by naloxone. It also induces relative analgesia (hotplate test) which is also reversed by a low dose of naloxone. The stress also increases rectal temperature and naloxone blocks this effect as well. Footshock

Barbiturate sensitivity

Analgesia

Opioids

1. Introduction

With the discovery, in recent years, of the 'endogenous opioids', the enkephalins and endorphins, there has been considerable interest in the physiological roles of these compounds and the conditions which alter their secretion. The stress of electric footshock has been reported (Madden et al., 1977) to increase the levels of these opioids in rat brain, with parallel changes in nociception. The stress of various handling procedures has been reported (Bl~isig et al., 1978) to increase brain levels of endorphins in rats. Direct measurements of the opioids have not been made in mice, after stress, but the involvement of these substances has been inferred by the use of naloxone (considered to be a specific opiate antagonist (Jaffe and Martin, 1975)) or animals made tolerant to morphine (Bl~isig and Herz, 1976). Chesher and Chan (1977) used the latter technique and implicated these compounds in footshock analgesia in mice; Willow et al. (1980) used naloxone and suggested an opioid basis for the decreased nociception and increased barbiturate sensitivity which they observed in mice following swimming. However, as Fields and Basbaum (1978) ob0014-2999/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press

served, pain research which uses behavioural criteria is often controversial because of 'the lack of an adequate behavioural paradigm for pain'. In addition, results from rats, for example, cannot be simply extrapolated to refer to mice (Rossier et al., 1977) and even strain differences can be important in the use of mice (Catz and Yaffe, 1967; Vessell, 1968; Alleva et al., 1978; Lush and Lovell, 1978). Accordingly, the experiments reported in this paper attempted (i) to confirm the previous observations of the influence of footshock on nociception, but with a different strain of mouse and a different nociceptive test (the classic h o t p l a t e / footflick test of Woolfe and McDonald, 1944); and (ii) to check if the stress of footshock, as with swimming, increases the animals' sensitivity to hypnotic barbiturate. In addition, the influences of these procedures on rectal temperature were examined as well as the possible involvement of opioids in any of the responses. The results have been reported briefly in abstract form (Carmody, 1980). 2. Materials and methods

Virgin female mice of B a l b / c strain were used. They were all in good health, had received no

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drugs and had not previously been exposed to the experimental situation. This is necessary as the animals show habituation to the stress and so multiple experiments may not validly be done on a single animal. They had been allowed food and water ad libitum up to the time of the experiments. Their mean weight was 23 g and there were no differences between the test and control groups in this respect. For the footshock, animals were put individually into a smooth-walled perspex box (measuring 22 x 22 x 21 cm) the floor of which was a series of parallel metal bars (3 mm diameter, 9 mm apart). The shocks of 0.3-0.5 mA nominal strength were administered (10 s on - 10 s off) by a device (R.S. Hales Instruments, Sydney) which delivered the current only to those bars on which the animal was standing at any moment. The experimental design was as follows: measurement of rectal temperature (using a lubricated Yellow Springs Instruments probe); measurement of the time for the appearance of a characteristic fast flick of a hindlimb after the animal was placed on the copper hotplate (Woolfe and McDonald, 1944; Carmody et al., 1979); footshock session (10 min) followed immediately by a further measurement of rectal temperature and footflick latency. When pentobarbitone (Pentobarbitone sodium, Chemical Operations, Abbott Australasia Pty Ltd.) was administered this was immediately after the measurement of the post-stress nociception. It was given in a dose of 50 m g / k g body weight, dissolved in 0.9% saline, 0.1 m l / 1 0 g body weight. Each mouse was then placed in a plastic holding box (30 x 30 × 11 cm, one mouse per box) and checked at 30 s intervals for loss of righting reflex; when this was unequivocally lost the mouse was placed on its back in a large, thermally insulated chamber (88 × 59 x 88 cm) which was maintained at 30-31°C. It remained there until the righting reflex had returned. The timing of these procedures was such that the second measurement of nociception was usually begun within 15 s of the end of the shock period and the pentobarbitone administrated within 60 s of the end of this period. When naloxone ('Narcan', Endo Laboratories) was used it was administered (i.p., 100/~g/kg in 0.9% saline,

0.1 m l / 1 0 g body weight) 30 min before the beginning of the experimental sequence described above; in addition, rectal temperature was measured before the naloxone was given. All experiments were performed in an air-conditioned laboratory held at 21-23°C, and were done at approximately the same time each day to avoid circadian influences.

3. Results

The results are summarised in the tables. From table 1A it can be seen that the stress of footshock p r o l o n g e d the footflick latency by 57%. If the reciprocal of this latency be taken as an index of pain sensitivity (Carmody et al., 1979) one can say that pain perception was reduced by 36% by the stress. The (relatively small) dose of naloxone totally abolished the analgesic effect in a separate group of mice (table 1B). The stress of footshock, as with swimming (Willow et al., 1980), increased the animals' sensitivity to pentobarbitone. Table 2A shows that the stressed mice were asleep 27% sooner than those which had not been subjected to the electric shock. Further, the footshock stress increased the duration of the sleep induced by this dose of pento-

TABLE 1 Effect of footshock on nociception in mice. These are four separate groups of mice. Values are mean ± S.E.M. a p < 0.01. The hotplate temperatures of these two sets of experiments are different because of a fault in the thermal control system. This does not vitiate the results. The response latencies of part A are longer because the temperature is 2.4°C lower than in part B, but these experiments clearly show a prolongation of the latency by the stress procedure. Part B shows that this effect of stress is abolished by a very low dose of naloxone. Footflick latency (s)

Pain index

n

0.048 0.030

24 24

(,4) Normal mice, 53.6°C hotplate Pre-stress Post-stress

20.9 ± 1.55 a 32.9 ± 3.60 a

(B) Animals treated with naloxone (100 t t g / k g i.p.), 30 rain prior to testing, 56°C hotplate. Pre-stress Post-stress

9.3 + 1.43 8.4 + 1. i 2

0.107 0.119

25 25

121 TABLE 2 Effect of footshock on sensitivity to pentobarbitone (50 m g / k g i.p.). These are four separate groups of mice. The mice in part A of this table did not receive i.p. saline 30 min before the beginning of the stress. Carmody et al. (1979) showed that the injection procedure (i.e. of the vehicle alone) was without effect. Values are mean + S.E.M. Results of significance testing (unpaired, two-tailed t-tests) indicated by the symbols, as follows: a significant difference, P < 0.001; b no significant difference, 0.3 > P > 0.2; c significant difference, P < 0.02; d no significant difference, 0.2 > P > 0.1; e no significant difference, P = 0.2; r no significant difference, 0.3 > P > 0.2. Time to sleep

n

(rain) (.4) Normal mice Unstressed animals Stressed animals

6.9 + 0.62 a,b 4.7 + 0.26 a

Sleeping time

n

(rain) 13 24

33.7 _+6.24 ¢,a 55.3 _+4.86 c

13 24

(B) Animals treated with naloxone (100 p g / k g i.p.), 30 rain prior to stress Unstressed animals 8.1+0.73 b.e 17 24.8+2.66 d.f 17 Stressed animals 7.0+0.49 e 25 29.1 + 2.51 f 25

barbitone, by 55% (in fact, although it was not reported by WiUow et at. (1980), the stress of a three-rain swim also leads to a shorter time-to-sleep in mice; in experiments with Balb/c females, the present author found that time-to-sleep (pentobarbitone 50 mg/kg) was reduced by 44% from

TABLE 3 Effect of footshock on rectal temperature and the influence of naloxone. Rectal temperature

n

(°C) (,4) Normal animals Pre-stress Post-stress

36.4 + 0.20 a 37.3 + 0.25 a

33 33

(B) Animals treated with naloxone (100 p g / k g i.p.), 30 rain prior to stress Pre-naloxone 37.4 + 0.30 b 25 Pre-stress 38.0-1- 0.28 b 25 Post-stress 37.9 + 0.23 b 25 a The post-stress temperature is significantly higher: two-tailed, paired t-test, P < 0.001 b The temperatures are not significantly different in these three circumstances: P > 0.2 (F-ratio) in a one-way analysis of variance for repeated measurements.

11.3 to 6.3 rain). Thus footshock stress (like swimstress) increases the animals' sensitivity to this drug: by 55% if the sleeping time is taken as the index and by 36% if the reciprocal of the time-tosleep is used. Table 2B shows that the small dose of naloxone completely abolished this effect of stress and returned the sleeping times and timesto-sleep (in different groups of animals) to normal values. In addition, there was a small, but significant, rise in rectal temperature (approximately 2.5%) as a result of the footshock stress (table 3); this was abolished by naloxone. This dose of naloxone did not have a significant effect on rectal temperature in unstressed animals (P > 0.2).

4. Discussion

These results indicate that the stresses of swimming and footshock have in common, inter alia, the facts that each (i) reduces sensitivity to pain, at least of the cutaneous, thermal kind, and (ii) increases sensitivity to a hypnotic barbiturate as well. This finding of hypoanalgesia (a 36% reduction) is consistent with results from other laboratories where other nociceptive tests and other genera (or strains) have been used: Chesher and Chan (1977) reported reductions in nociception of 23-56% (abdominal constriction test) with much the same intensity of shock stress; with an apparently more severe degree of shock, Madden et al. (1977) also found (in rats) reduced nociception by approximately 50% but it is difficult to quantify their result. They also found an approximately 60% increase in whole-brain levels of 'opioids' (their test used [3H]naloxone binding) while Rossier et al. (1977; 1978), used even greater stress and found increases in levels of fl-endorphin of up to five-fold in plasma but decreases in the hypothalarnic levels of fl-endorphin (Rossier et at., 1977) and leucine-enkephalin (Rossier et al., 1978). Fratta et at. (1977) found no changes in levels of methionine-enkephalin in various parts of the brain after 'considerable' footshock stress. However, the fact that naloxone totally eliminated the effects on nociception and drug sensitivity in the present experiments strongly sug-

122 gests that the e n d o g e n o u s o p i o i d s are involved in these responses. F u r t h e r , the dose used (100 # g / k g ) is a very low one - C h e s h e r a n d C h a n (1977) used 1 m g / k g a n d l0 m g / k g is c o m m o n l y used - so its p o t e n t action suggests a p a r t i c u l a r specificity. It is p r o b a b l e that these e n d o g e n o u s o p i o i d s p l a y some role in the h y p n o t i c a c t i o n of b a r b i t u r a t e . Ftirst et al. (1977) showed that naloxone t r e a t m e n t o f n o r m a l rats r e d u c e s their sensitivity to b a r b i t u r a t e , so the stress effects could be c o n s i d e r e d an a u g m e n t a t i o n of this p h e n o m e n o n . T h e i r dosage of n a l o x o n e was ten times that of the p r e s e n t e x p e r i m e n t s ; the results of table 2 show a similar trend, n o t quite reaching significant levels, suggesting that o p i o i d p r o d u c t i o n , at relevant sites, is low in n o r m a l animals. T h e p r e s e n t results also indicate that these stress effects, i n c l u d i n g the stress h y p e r t h e r m i a , have a fairly r a p i d onset. Bl~isig et al. (1978) r e p o r t e d that, in rats, the h y p e r t h e r m i a o c c u r r e d within 15 m i n of their stress p r o c e d u r e : from the p r e s e n t work, the onset, in mice, w o u l d seem to be even sooner. These a u t h o r s also c o n s i d e r e d that the n e u r o n e s involved in the t h e r m a l response were ' n o t very sensitive to n a l o x o n e ' . T h e present result, seen with 1% of their dose, w o u l d argue against that view. T h e r e is also a puzzle in reconciling the result of Lin a n d Su (1979) w h o f o u n d .that injection of f l - e n d o r p h i n into the c e r e b r o s p i n a l fluid of r a b b i t s p r o d u c e d falls (5% m a x i m u m ) in rectal t e m p e r a t u r e which were p a r t i a l l y a b o l i s h e d b y adm i n i s t r a t i o n of n a l o x o n e (into the C.S.F.) in dosages c o m p a r a b l e to those Of the present experiments. Like Lotti et al. (1965), these a u t h o r s f o u n d t h a t the a d m i n i s t r a t i o n of m o r p h i n e i n t o the b r a i n induced hypothermia. It is relevant to p o i n t out, though, that a temp e r a t u r e change in a p a r t i c u l a r direction is n o t intrinsic to the stressful state: a three-rain swim in w a t e r at r o o m t e m p e r a t u r e reduces b o d y t e m p e r a ture b y a b o u t 15% in mice. It c a n be clearly stated, though, that like swimming, the f o o t s h o c k releases n a l o x o n e - b l o c k a b l e m a t e r i a l which can affect b r a i n systems s e p a r a t e f r o m those involved with pain, as well as the p a i n systems themselves. This m a t e r i a l p r e s u m a b l y serves some" s y n a p t i c function, either acting as a t r a n s m i t t e r it[elf or otherwise m o d u l a t i n g s y n a p t i c efficacy.

Acknowledgements I thank the Department of Psychology, University of Sydney, for the loan of the stimulating device, and Abbott Australasia Pry, Ltd. for a gift of pentobarbitone.

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