Psychopharmacology (1993) 1 t t : 62-68 Psychopharmacology © Springer-Verlag 1993

Low and high doses of midazolam differentially affect in rats conditioned to a heat stressor Justin A. Harris 1, lain S. McGregor 2, and R.F. Westbrook 1

School of Psychology, University of New South Wales, P.O. Box l, Kensington 2033, Australia 2 Department of Psychology, University of Sydney, Sydney, Australia

Received April 8, I992 / Final version September 9, 1992

hypoaigesia

Abstract. These experiments examined the effects of a benzodiazepine (midazolam) on rats' sensitivity-reactivity to the heated floor of a hot-plate apparatus. Rats were either previously exposed to the heated floor, or naive to the hot-plate apparatus, while control rats were familiar- ized with the apparatus in the absence of pain. A low dose (0.63 or 1.25 mg/kg) of midazolam attenuated the condi- tioned hypoalgesic response resulting from pre-exposure to a heated floor, but did not affect the hypoalgesic response elicited by exposure to a novel hot-plate appar- atus nor the "baseline" sensitivity-reactivity among con- trol rats. A high dose (2.5 mg/kg) of midazolam resulted in a naloxone-insensitive increase in both the conditioned and the novelty-induced hypoalgesia, and provoked a small, but naloxone-reversible increase in paw-lick lat- encies among control rats. The results were taken to mean that exposure to the heated floor results in hypoalgesic responses as a consequence of fear conditioning and the reinstatement of novelty. Midazolam was assumed to attenuate conditioned hypoalgesia by reducing fear but at the high dose to augment the hypoalgesic effects of novelty.

Key words: Midazo lam- Hypoalgesia Fear Novel ty- Hot-plate - Naloxone

A reduction in nociceptive sensitivity (hypoalgesia) can be elicited by a variety of stressors. Hypoalgesia can result from exposure to novel stimuli (Bardo and Hughes 1979; Foo and Westbrook 1991), nociceptive stimuli (Terman et al. 1984) and cues previously paired with nociceptive stimuli (Chance et al. 1978; Ross and Randich 1985). This last instance of hypoalgesia appears to depend upon classical conditioning processes, whereby an animal learns an association between environmental or predictive cues and the noxious stimulus (cf Ross 1985, 1986; Greeley and Westbrook 1990; Westbrook et al. 1991a).

Correspondence to: J.A, Harris

Chance (t 980) suggested that hypoalgesia is related to the arousal of fear. This was based largely upon evidence that rats made hyperemotional by septal or ventromedial hypothalamic lesions, displayed a diminished sensitivity to noxious stimulation (Chance et al. 1977, 1978). Further- more, both hyperemotionality and hypoalgesia were at- tenuated by treatment with the benzodiazepine diazepam.

This notion of a relationship between fear and hypo- algesia was taken up by Bolles and Fanselow (1980). They proposed a model of the organization of an animal's behaviour in response to both noxious stimulation and cues predicting such stimulation. Within the framework of this Perceptual-Defensive-Recuperative model, they ar- gue that cues which predict a noxious event activate a defensive motivational system that is linked to the arousal of fear. This defensive system mediates species-specific defensive responses such as freezing, as well as engaging endogenous antinociceptive mechanisms (Fanselow and Baackes 1982). In support of this theory, Fanselow and Helmstetter (1988) reported that conditioned freezing and hypoalgesia were reversed by pretreatment with several different benzodiazepines (chlordiazepoxide, diazepam and midazolam). This was said to represent evidence for a causal link between fear and hypoalgesia, on the basis of the reputed anxiolytic properties of these drugs.

Other researchers have presented findings that speak against this as a general explanation of hypoalgesia. Maier (1990) examined a number of factors which influence the benzodiazepine sensitivity of post-shock hypoalgesia. He reported that diazepam did not affect the initial hypo- algesic response that occurs immediately after exposure to a small number of shocks. It appears from his research that diazepam was only effective in attenuating that por- tion of hypoalgesia which was dependent upon the pre- sence of environmental cues previously paired with the shock. However, Chance et al. (1979) reported that treat- ment with diazepam had no effect on hypoalgesia elicited by cues previously paired with shock. Further, Kinscheck et al. (1984) reported that chlordiazepoxide reduced con- ditioned hypoalgesia if given shortly before testing, but not if the drug had been chronically administered during training (exposure to environment-shock pairings). They

in terpre ted this to mean that the a t t enua t ion of hypo- algesia p roduced by ch lo rd iazepoxide was due to a gen- e ra l iza t ion decrement where learn ing failed to transfer to the drugged state. Fanse l0w and Helmste t te r (1988) have crit icised these studies, suggest ing tha t the benzodiazepine admin i s t r a t ion in each case m a y have been inadequate . F o r example , in reference to the findings of Kinscheck et al., Fanse low and Helmste t te r comment tha t the failure of ch to rd iazepox ide to reduce hypoa lges ia in the chronic admin i s t r a t ion group may have been due to the develop- ment of tolerance, render ing the d rug less effective in reducing fear. However , W e s t b r o o k et al. (1991a) r epor ted that midazo lam, given at a dose higher than tha t used by Fanse low and Helmste t te r (1988), d id no t a t t enua te condi- t ioned hypoalgesia . Indeed, this dose of m i d a z o l a m fur- ther reduced nocicept ive sensitivity, even though it was successful in b locking the acquis i t ion of cond i t ioned hypoalgesia .

The current series of exper iments was designed to provide a more deta i led inves t igat ion of the effects of a benzodiazep ine upon hypoa lges ia in the hot -p la te para - digm. The la tency for ra ts to ini t ia te paw-l icking when placed upon a hea ted f loor is affected by the t empera tu re of the floor and by p re t r ea tmen t with morph ine (Greeley and W e s t b r o o k 1990; W e s t b r o o k et al. 1991a,b). This suggests tha t la tency to paw-l ick represents a val id index of rats ' sensi t iv i ty / react iv i ty to a thermal noxious s t imu- lus. In add i t i on to these factors, the la tency for rats to paw-l ick is influenced by the h is tory of the rat 's experience in the test envi ronment . Rats that have been famil iar ized with a non-hea ted ho t -p la te a p p a r a t u s pr ior to testing, lick their paws with shor te r latencies than do rats de- pr ived of such fami l ia r iza t ion (Bardo and Hughes t979; F o o and W e s t b r o o k 1990). Fur ther , rats that have been previous ly exposed to the hea ted f loor lick with longer latencies when subsequent ly tested than do rats familiar- ized with a non-hea ted f loor but not pre-exposed to a hea ted f loor (Greeley and W e s t b r o o k 1990; W e s t b r o o k et al. 1991a). Therefore, it appears that appa ra tu s cues can elicit hypoa lges ia if they are novel or if they have been previous ly pa i red with a hea ted floor.

The purpose of the first three exper iments was to examine the effect of m i d a z o l a m at var ious doses on cond i t ioned and nove l ty - induced hypoalges ias as well as on "basel ine" noc icep t ion in rats s imply famil iar ized with the test envi ronment . In ano the r exper iment , the effects of a high dose of m i d a z o l a m in famil iar ized and non-famil i - ar ized rats were examined concurrent ly , so that a direct compar i son could be made between these two groups. A further exper iment examined whether the effects of mida- zo lam on the express ion of cond i t ioned hypoa lges ia are sensitive to t r ea tmen t with the benzodiazep ine an tagonis t flumazenil . Final ly , three exper iments were conduc ted to examine whether the appa ren t "hypoalges ia" induced by a high dose of m i d a z o l a m is sensitive to t rea tment with the op io id an tagon i s t naloxone.

Materials and methods

Subjects

The subjects in each experiment were experimentally naive, male Wistar rats, weighing between 280 and 430 g. Rats were obtained

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from the Specific Pathogen Free breeding unit at Little Bay, New South Wales. Throughout the experiment, rats were housed in plastic boxes (65 cm x 40 cm x 22 cm) kept in a colony room maintained on a 12 h light : dark cycle. There were eight rats in each box, with food and water made continuously available.

Apparatus

The hot-plate apparatus consisted ofa Plexiglas cylinder (24 cm × 48 cm, diameter x height) with a copper floor fixed 12 cm above the base of the cylinder. The portion of the cylinder below the copper floor was perforated with holes (diameter 3 cm) to permit circulation of water beneath the copper floor. The cylinder stood in a water bath that could be maintained at a particular temperature ( + / -.- 0.5 °C) by a Haake D1 Open Bath Circulator. The laboratory also contain- ed plastic buckets (26 cm x 26 cm, diameter x height) with air holes drilled in the lid and side. These buckets served as chambers where rats were kept in isolation when brought to the laboratory.

Drugs

Midazolam (Hypnovel) was diluted with isotonic (0.9% w/v) saline. Flumazenil was suspended in a vehicle solution of distilled water to which was added two drops of Tween 80 per 10 ml. Midazolam and flumazenil were given as intraperitoneal injections, in a volume of 1.0 ml/kg. Naloxone hydrochloride was dissolved in isotonic (0.9% w/v) saline, and injected subcutaneously into the dorsal region of the neck in a volume of 1.0 ml/kg.

Procedure

Familiarization. Rats were handled for 3 days prior to the start of the experiment. Following this, on 4 consecutive days, pre-exposed and familiarized rats were transported to the laboratory where training and testing were to take place. Upon arrival, rats were kept in plastic buckets for 30 min before being placed in the hot-plate apparatus for 60 s. On these occasions, the water bath was maintained at an ambient temperature of 23°C. Non-familiarized rats were given equivalent handling experience in the colony room. Following this, rats were weighed and assigned to weight-matched groups.

Pre-exposure. On the day before testing, pre-exposed rats were transported to the laboratory and kept in plastic buckets for 30 min, before being placed on the heated floor for 30 s. The water bath was maintained at 54 °C, producing a floor temperature of approxim- ately 53.0 °C as measured by a digital thermometer (Amritsu).

Testing. On the next day, all rats in each experiment were trans- ported to the laboratory and kept in the buckets for 30 min. Rats were then placed upon the heated floor, while an observer used push-buttons connected to a microprocessor to record when the rat licked a front or back paw. Each rat was removed as soon as it responded or after a maximum of 60 s. The water bath was main- tained at 52 °C, producing a floor temperature between 50.0°C and 50.8 °C. The latency to first paw-lick (front or back) was recorded for every rat, and the mean of this latency was calculated for each group (cf Westbrook and Greeley 1990). Half the rats in each group were simultaneously rated by a second observer, who was unaware of the rat's group designation. The ratings made by each observer were compared in order to assess measurement reliability for latency to first paw-lick. These comparisons revealed a high degree of con- cordance between observers, with ratings rarely differing by more than 0.5 s.

Effect of midazotam at various doses. Twenty minutes before test, each rat was removed from its bucket, injected and returned to the bucket. Rats in groups SAL were injected with saline, while rats in groups MID-0.63, MID-1.25 and MID-2.5 received 0.63, 1.25 and 2.5 mg/kg of midazolam, respectively.

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Effects of midazotam in j~tmitiarized and non-familiarized rats. All rats were given an injection 20 rain before test. One familiarized and one non-familiarized group received 2.5 mg/kg midazolam, while the remaining rats received saline.

Effects of flumazenil and midazolam on conditioned hypoalgesia. Thirty minutes before being placed on the heated floor, three groups of rats were injected with 10 mg/kg flumazenil, while the remaining groups received an injection of the vehicle solution. Ten minutes later, all rats received a second injection. Two groups of rats were injected with saline (SAL), two groups received 0.63 mg/kg mid- azotam (MID-0.63) and two groups received 2.5 mg/kg midazolam (MID-2.5).

EfJect of naloxone on midazolam-induced "'hypoalgesia". Thirty minutes before exposure, rats received an injection of either saline (groups SAL/SAL and SAL/MID) or naloxone: pre-exposed rats in groups NAL-5.0/MID, NAL-1.25/MID and NAL-0.3/MID received 5.0, 1.25 or 0.3 mg/kg natoxone, respectively; familiarized and non- familiarized rats in groups NAL/SAL and NAL/MID received 5.0 mg/kg of naloxone. Ten minutes after this first injection, rats in groups SAL/SAL and NAL/SAL were given an injection of saline, while the remaining rats received 2.5 mg/kg midazolam (MID).

Results

The effect o f midazolam at various doses

The results of the first three experiments are presented together in Fig. 1. The mean latencies to first paw-lick on second (52°C) exposure to the heated floor (plate temperature = 50.8°C) for each group (n = 8) of pre- exposed rats are presented in the left-hand panel of Fig. 1. It is clear that the different doses of midazolam had very different effects on response latency. Because of the un- expected nature of this finding, the planned contrasts were abandoned in favour of a set of post-hoc, orthogonal contrasts. With alpha set at 0.05, and 3, 28 df, the critical F value for three contrasts is 5.9 (Rodger 1967). Rats given the highest dose (2.5 mg/kg) of midazolam took signific- antly longer to initiate paw-licking than did rats in the remaining three groups (F = 19.0). In contrast, rats given the two lower doses of midazolam licked with significantly shorter latencies than did saline-treated rats (F = 6.5). Therefore, it appears that these two doses of the drug were able to attenuate the expression of conditioned hypoal- gesia.

The mean latencies to first paw-tick (plate temperature = 50.8 °C) for each group of familiarized rats (n = 6) are

presented in the central panel of Fig. 1. For the post-hoc, orthogonal contrasts used to analyse this set of results, the critical F value is 6.1, with alpha set at 0.05 and 1, 20 df (Rodger 1967). Rats given the highest dose of midazotam licked their paws with significantly longer latencies than did rats in the remaining three groups (F = 7.1). There was no significant difference in response latencies between saline-treated rats and rats given the two lower doses of midazolam (F < 1), nor between groups MID-0.63 and MID-1.25 (F < 1). Therefore, it appears that a large dose of midazolam (2.5 mg/kg) produces a slight increase in the latency to paw-lick for rats familiarized with a non-heated floor but not pre-exposed to a heated floor. In contrast to the reduction in response latency in pre-exposed rats, lower doses of the drug (1.25 and 0.63 mg/kg) did not have

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Fig. 1. Mean Iatencies to first paw-lick: for rats pre-exposed to a heated floor (left); for rats familiarized with the apparatus but not pre-exposed to a heated floor (centre); and for non-familiarized rats (right). Prior to testing, rats were injected with either saline (SAL) or midazolam (MID) at a dose of 0.63, t.25 or 2.5 mg/kg. Vertical bars represent standard errors of the mean. * Indicates group signific- antly different from remaining groups (P < 0.05); 4~ indicates groups significantly different from group SAL (P < 0.05)

any detectable effect here. However, this may have been due to a floor effect in the current experiment.

The mean latency to first paw-lick (plate temperature = 50.1 °C) for each group of non-familiarized rats (n = 8)

are presented in the right-hand panel of Fig. 1. For the post-hoc, orthogonal contrasts used to analyse this set of results, the critical F value is 5.9, with alpha set at 0.05 and 1, 28 df (Rodger 1967). Rats injected with the high dose (2.5 mg/kg) of midazolam took significantly longer to initiate paw-licking than did rats in the remaining groups (F = 7.4). There was no reliable difference between rats given saline and those given low doses of midazolam (0.63 and 1.25 mg/kg), nor between groups MID-0.63 and MID-1.25 (Fs < 1). Therefore, the results of this experi- ment are similar to those of pre-exposed rats, in that latency for rats to respond on the heated floor is increased by a high dose of midazolam but unaffected by lower doses. In this case, it is unlikely that floor effects are present.

Taken together, the current experiments confirm that administration of a large dose (2.5 mg/kg) of midazolam results in an increase in the latency for rats to respond when placed upon a heated floor. This effect occurs in rats with no previous experience of the apparatus or proced- ure, in rats familiarized with the apparatus but with no prior experience of the heated floor, and in rats familiar- ized with the apparatus and pre-exposed to the heated floor. Therefore, the increase in latency to paw-lick ap- pears to occur despite marked differences in the rat's prior experience with the test environment. There is less gen- erality for the effects of low doses of midazolam (0.63 and 1.25 mg/kg). At these doses, midazolam reduces response latency in rats pre-exposed to the heated floor, but has

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Fig. 2. Mean latencies to first paw-lick for familiarized and non- familiarized rats, administered either saline or midazolam (2.5 mg/kg), Vertical bars represent standard errors of the mean. Non- familiarized rats (*) significantly different from familiarized rats (P < 0.05); midazolam-treated rats (4~) significantly different from saline-treated rats (P < 0.05)

no effect in non-pre-exposed rats (familiarized and non- familiarized).

Effects of midazolam in familiarized and non-familiarized rats

The mean latencies to first paw-lick for each group (n = 8) during exposure to the heated floor (plate temperature = 50.1 °C) are presented in Fig. 2. To analyse the data,

three planned non-orthogonal contrasts were constructed in accordance with the Bonferroni technique (cf Miller, 1966). With alpha set at 0.05, the critical F value (1,28 dr) is 6.5. As predicted, rats that were familiarized with the experimental procedure were faster to initiate paw-licking than were non-familiarized rats (F = 28.3). Therefore, in agreement with previous research (Bardo and Hughes 1979; Foo and Westbrook 1990), it appears that rats are hypoalgesic when tested in a novel environment. Further, rats given midazolam took longer to lick their paws than did saline-treated rats (F = 12.7). However, the inter- action between these two factors was not significant (F = 2.9). Therefore, there was no reliable difference for the effect of midazotam between familiarized and non- familiarized rats.

Effects of flumazenil and midazolam on conditioned hypoalgesia

The mean latencies to first paw-lick for each group (n = 8) during second exposure to the heated floor (plate temperature = 50.3 °C) are presented in Fig. 3. The data were analysed using planned, orthogonal contrasts, with alpha set at 0.05. The critical F value (1,42 df) is 4.t (Hays 1972). Vehicle-treated rats that received the high dose of

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Fig. 3, Mean latencies to first paw-lick for pre-exposed rats treated with either 10 mg/kg ftumazenil or vehicle, and injected with either saline (SAL) or midazolam (MID) at a dose of either 0.63 or 2.5 mg/kg. Vertical bars represent standard errors of the mean. * indicates group significantly different from other groups (P < 0.05); ~ indicates group significantly different from remaining groups (P < 0.05)

midazolam (MID-2.5) took significantly longer to paw- lick than those in the other five groups (F = 43.3). In addition, vehicle-treated rats given the low dose of midaz- olam (MID-0.63) responded faster than the other four groups of rats (F = 4.8). These results confirm the findings made earlier, that the latency for rats to paw-lick on second exposure to a heated floor is increased by a high dose but attenuated by a low dose of midazotam. These effects of midazolam appear to have been blocked by the antagonist flumazenil. The three flumazenil-treated groups showed response latencies that were similar to those of vehicle-treated rats given saline (SAL) (F < 1). Further, among the three flumazenil-treated, there was no reliable difference between rats given saline and those given midazolam (F < t), nor between the two midazolam groups (F < 1). Therefore, it appears that the attenuation and the increase in response latency produced by the different doses of midazolam are both mediated by the drug's action at benzodiazepine receptors.

Effect of naloxone on midazolam-induced "hypoaIgesia"

The mean latencies to respond (plate temperature = 50.0 °C) for each group of pre-exposed rats (n = 8) are

presented in the left hand panel of Fig. 4. The results were analysed using a set of post-hoc, orthogonal contrasts. With alpha at 0.05, and 3, 35 df, the critical F value is 5.8 (Rodger 1967). Rats given midazolam (groups SAL/MID, NAL-5.0/MID, NAL-1.25/MID and NAL-0.3/MID) were slower to lick their paws than were rats in group SAL/ SAL (F = 9.13). Therefore, as demonstrated previously, injection of 2.5 mg/kg midazolam before second exposure to a heated floor significantly increases latency to respond.

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Fig. 4. Mean latencies to first paw-lick: for rats pre-exposed to a heated floor (left); for rats familiarized with the apparatus but not pre-exposed to a heated floor (centre); and for non-familiarized rats (riyht). Prior to testing, rats received two injections: either saline (SAL) or naloxone (NAL), followed by either saline or 2.5 mg/kg midazolam (MID). Naloxone was administered to pre-exposed rats at a dose of 0.3, 1.25 or 5 mg/kg, and administered to familiarized and non-familiarized rats at 5 mg/kg. Vertical bars represent stand- ard errors of the mean. * Indicates groups significantly different from remaining groups (P < 0.05)

The latencies to paw-lick for rats given naloxone with midazolam were similar to those of rats given midazolam alone (F = 2.58). There were no significant differences between the three groups of rats given naloxone (Fs < 1). Therefore, it appears that all three doses of naloxone failed to affect midazolam-enhanced hypoalgesia, suggesting that midazolam's effect is independent of activity in opioid systems.

The mean tatencies to first paw-lick (plate temperature = 50.8 °C) for each group of familiarized rats (n = 8) are

presented in the central panel of Fig. 4. For the planned, orthogonal contrasts used to analyse these results, the F critical value is 4.2, with alpha at 0.05 and 1, 28 df(Hays 1972). Rats in group SAL/MID were slower to initiate paw-licking than were rats in the other three groups (F = 5.5). Therefore, as demonstrated earlier, it appears that midazolam produced a primary increase in response latency in familiarized rats not pre-exposed to a heated floor. Furthermore, this effect of midazolam appears to have been reversed by pretreatment with naloxone, be- cause rats in group NAL/MID licked with similar lat- encies to those of rats in groups SAL/SAL and NAL/SAL (F < 1). There was no significant difference between group SAL/SAL and group NAL/SAL (F < 1), indicating that naloxone, on its own, did not affect response latencies. Therefore, it appears that opioid mechanisms do mediate the slight increase in paw-lick latencies induced by mid- azolam in rats familiarized with the apparatus but not pre-exposed to a heated floor.

The mean latencies to first paw-lick (plate temperature = 50.4 °C) for each group of non-familiarized rats (n = 8)

are presented in the right hand panel of Fig. 4. For the

post-hoc, orthogonat contrasts used to analyse these res- ults, the F critical value is 5.9, with alpha at 0.05 and 3, 28 df (Rodger 1967). Rats given midazolam before test (groups SAL/MID and NAL/MID) were slower to initiate paw-licking than were rats in groups SAL/SAL and NAL- /SAL (F = 19.0). Therefore, as previously demonstrated, pretreatment with a high dose of midazolam increased response latency in rats not familiarized with the appar- atus. This effect of midazolam was not reversed by nalox- one, because rats in group NAL/MID licked with similar latencies to those of rats in group SAL/MID (F < 1). Further, there was no significant difference between groups SAL/SAL and NAL/SAL (F < 1), indicating that naloxone, on its own, did not affect response latencies. Therefore, it appears that both novelty-induced hypo- algesia, displayed by rats tested in an unfamiliar environ- ment, and the enhancement of this hypoalgesia by a high dose of midazolam are not mediated by opioid mech- anisms.

General discussion

The current series of experiments has demonstrated that the effects of midazolam on nociceptive sensitivity differ greatly, depending upon the dose administered and the history of the rat's experience in the test environment. Specifically, for rats previously exposed to a heated floor, low doses (0.63 and 1.25 mg/kg) of midazolam decreased while a high dose (2.5 mg/kg) increased the latency to paw-lick in response to a heated floor (Fig. 1). Further- more, both the decrease and increase in paw-lick latency produced by midazolam were completely reversed by the competitive antagonist flumazenit (Fig. 3). This suggests that both effects of rnidazolam are mediated by the drug's action at the benzodiazepine receptor (cf Haefely 1990). In contrast, in rats tested without prior experience of the heated floor, low doses of midazolam had no effect on paw-lick latency while the large dose (2.5 mg/kg) increased latency to respond (Fig. 1).

It has been demonstrated previously that pre-exposure to a 54°C floor produces an increase in the latency to initiate paw-licking, when rats are subsequently tested on a heated floor (Greeley and Westbrook 1990; Westbrook et al. 1991a). If response latency is taken as an index of nociceptive sensitivity/reactivity, then the increase in lat- ency to respond indicates that rats are hypoalgesic during their second exposure to the heated floor. Therefore, it appears that the hypoalgesia which accrues as a result of pre-exposure to a heated floor is attenuated by low doses of midazotam, and that these doses have no effect on baseline response latencies in rats not pre-exposed to a heated floor. This is consistent with reports by Fanselow and Helmstetter (1988) that treatment with a variety of benzodiazepines (including midazolam) can attenuate hy- poalgesia elicited in rats tested in an environment in which they had been previously exposed to shock. Kinscheck et at. (1984) have argued that the attenuation of condi- tioned hypoalgesia by benzodiazepines is due to a drug- induced generaIization decrement, However, the demon- stration that a high dose of midazolam enhances condi- tioned hypoalgesia rules out the possibility that a gen-

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eralization decrement might be operating in the current experiments. There have been numerous demonstrations that benzodiazepines inhibit conditioned fear responses (e.g. Tenen 1967; Miczek 1973; Davis 1979; Pascoe et al.). Therefore, one interpretation of the current findings is that midazolam attenuates hypoalgesia by inhibiting con- ditioned fear. This would support the proposal by Bolles and Fanselow (1980) that hypoalgesia occurs as a result of the arousal of fear.

The notion that fear mediates hypoalgesia cannot be used to explain the hypoalgesic response elicited in rats when tested in an unfamiliar environment (Fig. 2). This "novelty-induced" hypoalgesia was not reduced by mid- azolam at any of the doses tested (Fig. 1). If it is to be assumed that fear is attenuated by midazolam at the doses used, then it must be concluded that this hypoalgesia is not a product of the arousal of fear. One alternative explanation is that novelty-induced hypoalgesia reflects a "distraction" effect (Bardo and Hughes 1979) whereby the presence of novel stimuli interferes with attention to the noxious heat, and presumably that rats habituate to these stimuli during familiarization.

The current series of experiments has demonstrated that a relatively high dose of midazolam produces an increase in response latency in rats tested on a heated floor. The slight increase induced by midazolam in famil- iarized rats that were not pre-exposed to a heated floor most likely reflects a primary effect of the drug, due to the absence of novelty or conditioned effects. Furthermore, it was demonstrated that this increase is reversed by pre- treatment with naloxone (Fig. 4), which suggests that the effect is mediated by endogenous opioids. The increase may simply be due to motoric incapacitation induced by the high dose of the drug (cf Lau et al. 1990). This would be consistent with the demonstration that naloxone re- verses the locomotor depressant effect of benzodiazepines (Nowakowska and Chodera 1990). While this possibility cannot be ruled out, it is also possible that the increase is due to a direct antinociceptive action of midazolam. There have been numerous demonstrations of antinociceptive effects associated with benzodiazepines (e.g. Walsh et al. 1986). Furthermore, this antinociceptive effect appears to be mediated at the spinal cord: intrathecal injection of midazolam induces hypoalgesia (Niv et al. 1988; Zambotti et al. 1991); and systemic injection of midazolam dose- dependently depresses evoked responses in A-delta fibres in the dorsal horn (Clavier et al. 1992). Therefore, the current demonstration that naloxone can reverse mid- azolam-induced reduction in nociceptive sensitivity/reac- tivity would implicate the involvement of opioid systems at the spinal level. This possibility could be investigated further by assessing the effect of intrathecal injection of various opioid antagonists on midazolam-induced hy- poalgesia.

It was also demonstrated that a high dose of mid- azolam produces a marked increase in response latency in rats which had been pre-exposed to a heated floor and in rats that had not been familiarized with the apparatus or heated floor. However, these drug-induced reductions in nociceptive sensitivity were not affected by naloxone (Fig. 4). This means that the midazolam-induced hypoal- gesia in non-familiarized or pre-exposed rats cannot be

linked to the naloxone-reversible hypoalgesia induced by the drug in familiarized rats. Therefore, it is unlikely that midazolam-induced hypoalgesia in non-familiarized or pre-exposed rats could be due to novelty-induced or conditioned hypoalgesic responses summing with a pri- mary antinociceptive effect or motoric incapacitation in- duced by the drug. Consequently, the high dose of midaz- olam must be modulating some aspect of the rat's re- sponse to novel or aversively-conditioned environmental cues, in order to produce the enhanced hypoalgesia. While it may be possible to conclude that midazolam dose- specifically increases the rat's hypoalgesic response to novelty, the situation is more complicated in pre-exposed rats due to the fact that low doses of midazotam have the opposite efl~ct. It is difficult to explain how a single dose- dependent effect of midazolam could produce both at- tenuation and enhancement of conditioned hypoalgesia. One possible explanation is that the high dose of midaz- olam directly enhances some aspect of the conditioned hypoalgesic response which is distinct from a benzodiaze- pine-reversible hypoalgesia also present in we-exposed rats. In this case, it becomes possible (and parsimonious) to link the effect of the high dose of midazolam in non- familiarized rats with the effect in pre-exposed rats. Pre- exposure to a heated floor could be viewed as having dual consequences for a rat's subsequent reaction to that envir- onment: the rat becomes frightened, producing a fear- dependent hypoalgesia; and the rat responds to the envir- onment as if it were novel. The notion that aversive conditioning reinstates the novelty value of an environ- ment has been comprehensively argued by Gray (1982), who described in detail the similarity between rats' re- sponses to novelty and their responses to aversively condi- tioned stimuli. In the current experiments, evidence for a link between the rat's response to a novel environment and that to an aversively conditioned environment comes from the observation made during testing that pre-exposed and non-familiarized rats displayed very similar behaviours (rearing, jumping and generally very active) when treated with the high dose of midazolam. A similar effect has been reported by Rushton and Steinberg (1966), who found that rats treated with chlordiazepoxide (25 mg/kg) displayed an initial increase in the spontaneous activity evoked by an unfamiliar environment. Therefore, low doses of midaz- olam attenuate a fear-related hypoalgesic response, but have no effect on novelty-induced hypoalgesia. However, at the high dose, midazolam augments the novelty-in- duced hypoalgesic response, producing a profound hy- poalgesia in both pre-exposed and non-familiarized rats. While there have been demonstrations that benzodiaze- pines attenuate responses to novelty (e.g. File 1980), the effects of these drugs on rats' responses to novel environ- ments are generally mixed and contradictory (see Gray t 982, ch. 2, for a review). If the above account is correct, then novelty-induced hypoalgesia should be observed in pre-exposed rats tested with a low dose of midazolam. Such an effect may explain why these doses of midazolam failed to completely remove all hypoalgesia in pre-exposed rats (compare with familiarized rats, Fig. 1). Unfortu- nately, a direct comparison between pre-exposed rats and non-familiarized rats cannot be made due to differences in floor temperature on test.

68

In conclusion, the current series of experiments has shown that hypoalgesia resulting from pre-exposure to a heated floor is a t tenuated by the receptor-specific act ion of midazolam at low doses that do not affect nociceptive sensitivity in rats not pre-exposed to a heated floor. This is consistent with other reports that benzodiazepines reduce condi t ioned hypoalgesia (Fanselow and Helmstetter 1988), and supports the a rgument that hypoalgesia is linked to the arousal of fear (Bolles and Fanselow 1980). However, the current experiments failed to demonstra te a similar effect of midazolam on hypoalgesia in rats tested in an unfamiliar environment . Therefore, it would appear that some instances of hypoalgesia cannot be explained in terms of an association with benzodiazepine-sensit ive mo- t ivational arousal. Finally, the current experiments dem- onstrated that a high dose of midazolam increases the latency of rats to paw-lick on a heated floor, and that this hypoalgesia occurs despite differences in rats' prior experi- ences in the test environment . However, differences in naloxone-reversibil i ty indicate that midazolam's effect on pre-exposed and non-familiarized rats is distinct from that in familiarized rats that were not pre-exposed to a heated floor.

Acknowledgements. The authors wish to thank Kate Trimble at Roche Products Pry. Ltd. for the generous gift of flumazenil. The authors are also grateful to Rebecca McKetin, Anthony Good and Anne Swinbourne for their assistance with experimentation and care of the animals, and to Hayley Foo for comments on the manuscript. This research was supported by an Australian Research Council grant to R. F. Westbrook and postgraduate scholarship to J. A. Harris. The procedures used do not cause any tissue damage and have been approved by the Animal Care and Ethics committee at the University of New South Wales.

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