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Effects of flunitrazepam on passive avoidance behaviour in mice subjected to immobilization stress or familiarized with the testing apparatus
91
Behavioural Bra#r Research, 22 (1986) 91-95 Elsevier BBR 00602
Effects of flunitrazepam on passive avoidance behaviour in mice subjected to immobilization stress or familiarized with the testing apparatus CLAUDIO CASTELLANO and F L A M I N I A PAVONE
Istituto di Psicobiologia e Psicofarmacologia, C.N.R., Roma (Italy) (Received 28 April 1986) (Revised version received 18 June 1986) (Accepted 23 June 1986)
Key words: Flunitrazepam - Passive avoidance - Endogenous opioid - Stress - Familiarization
The effects of flunitrazepam on passive avoidance behaviour were investigated in DBA/2 mice. In a first set of experiments retention performance impairment was observed in mice injected with the drug immediately but not 120 min after training. In a second set of experiments, immobilization stress enhanced, while familiarization with the apparatus decreased, the effects of flunitrazepam, suggesting involvement of emotional factors. All the effects observed were antagonized by naltrexone, showing involvement of opioid receptors.
A number of researches have demonstrated, in recent years, that benzodiazepines can produce some of their behavioural effects (anticonflict effect, appetite enhancement etc.) through a release of endogenous opioids within the brain s. Use and abuse of a 7-nitrobenzodiazepine, flunitrazepam, have recently been described, which are due to its properties of alleviating some of the symptoms typical of the abstinence syndrome (irritability, insomnia). Moreover, a number of data indicate that in animals and humans, some of the effects of this drug can be antagonized by administration ofopioid antagonists, or potentiated by opiate treatments 3,1 ~, Recent experiments, carried out with DBA/2 mice, tested in a passive avoidance condition, have shown that opioids impair memory in this strain, and that this effect is enhanced by immobilization stress, and reduced by familiarization with the apparatus 4,t2. These effects have been interpreted in terms of release ofopioids by stress,
and of attenuation of emotionality by familiarization. In the present research it has thus been decided to study the effects of flunitrazepam on memory processes, and the possible interference in these processes by immobilization stress and familiarization with the testing conditions. The naltrexone-reversibility of the effects observed has also been assessed. The subjects were naive male mice of the inbred strain DBA/2 (DBA) (River Lab., Como, Italy) weighing 23-25 g. The apparatus consisted of a 20 x 20 x 20 cm Lucite box with black walls and a grid floor. The front wall was placed at the edge of a table, A 12 cm long and 5 cm wide platform protruded from the front wall over this edge and was lit by a 40-W lamp positioned 50 cm above it. The box was in darkness. In the experiments involving immobilization stress, stress was produced by placing the animals for 15 rain in a snug-fit apparatus 2. During training each mouse was placed on the platform facing away from a
Correspondence: C. Castellano, Istituto di Psicobiologia e Psicofarmacologia, via Reno, 1-00198 Roma, Italy. 0166-4328/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
92 4 × 3 cm opening in the front wall. The time taken by the mouse to enter the box with all 4 feet (step-through latency) was recorded. When the animal had entered the box, the opening was closed by a hand-operated sliding door, and the mouse received a 50-Hz scrambled footshock of 0.7 mA through the grid floor, for 1 s. It was then returned to its home cage to await testing 24 h later. Test procedures were the same as training, except that no footshock was administered. Different groups of animals were injected intraperitoneally with flunitrazepam (0.0125, 0.025 and 0.05mg/kg) immediately after training. In addition, a group of mice was injected with flunitrazepam (0.05 mg/kg) 120min after training, and another group of mice did not receive footshock but were injected with flunitrazepam (0.05 mg/kg) immediately after training. The performances of all mice were compared with those of mice injected with flunitrazepam vehicle. The effects of flunitrazepam (0.0125, 0.025 or 0.05 mg/kg) were also investigated in pretrained mice, which were put into the apparatus for a familiarization period of 2 min for 2 consecutive days, and were trained in the apparatus on the third day4. Their performances were compared with those of pretrained mice injected with flunitrazepam vehicle. When the naltrexone antagonism of the effects of flunitrazepam was investigated the dose of naltrexone (0.025 mg/kg) was selected on the basis of previous experiments and of preliminary work s. Finally, 3 groups of animals were immobilized for 15 min following immediate posttraining administration of saline + flunitrazepam vehicle, saline + flunitrazepam (0.025mg/kg) or naltrexone (0.025mg/kg)+ flunitrazepam (0.025 mg/kg) combinations and their performances were compared with those of corresponding non-immobilized groups. The duration of immobilization stress was selected on the basis of previous experiments 4. All groups consisted of 8 animals which were tested in the apparatus 24 h after training. Flunitrazepam (Darkene, Sigurta's, Milano) was diluted with 0.9~o NaC1. Naltrexone (HC1) (ENDO, Garden City, New York, NY) was dissolved in 0.9~o NaC1. Saline (0.9~,, NaC1) and flunitrazepam vehicle (at the concentration corre-
sponding to the highest dose of the drug) were used for control injections. All injections were given intraperitoneally (i.p.) in a volume of 4 ml/kg. The results were evaluated statistically by analysis of variance (ANOVA) (1- and 2-way) in which the mean step-through latencies of the groups on the test day were compared. Further analyses for individual between-groups comparisons were carried out with post hoc tests (Duncan multiple-range test). (1) Flunitrazepam administration immediately after training impaired retention performance of both non-pretrained and pretrained mice, but familiarization with the environment decreased this effect (see Table I). Moreover, (a) no significant difference was evident between the mean step-through latencies of mice injected with flunitrazepam (0.05 mg/kg) 120min after training (127.6 + 6.0) and those of their control group (129.2 + 2.8) (Fl,14 = 0.34, P > 0.05) and (b) the mean step-through latencies of the mice that had not received footshock on the training day but had been injected with flunitrazepam vehicle or flunitrazepam (0.05 mg/kg) did not differ from each other (mean step-through latencies: fluni-
TABLE I
Effects of flunitrazepam on passive avoidance behaviour #1 DBA/2 mice Mean step-through latencies ( _+ S.E.M.) on the test day of mice non-familiarized (A) and familiarized (B) with the apparatus. A N O V A (2-way) showed significant familiarization (Fi,56 = 35.957, P < 0.001) and flunitrazepam (F~,5~ - 136.281, P < 0.001) main effects, and a significant familiarization x flunitrazepam interaction (F3,5~ - 52.755, P < 0.001). The mean step-through latencies on the training day ranged between 9.1 + 1.1 and 10.7 _+ 1.3 s, for the nonfamiliarized groups, and 2.8 + 0.3 and 3.5 + 0.5 s for the familiarized groups.
Vehicle Flunitrazepam Flunitrazepam Flunitrazepam
mg/kg
A
0.0125 0.025 0.05
131.2 125.5 68.5 20,1
B +_ 5.5 + 5.8 _+ 5.0* +_ 3.3*
77.1 78.2 73.8 49.2
+ 2.3 + 2.5 _+ 2.0 _+ 2.6**
* P < 0.001 vs vehicle A (Duncan multiple-range test); ** P < 0.001 vs vehicle B (Duncan multiple-range test).
93 Two main points emerge from the present research (a) flunitrazepam exerted dose- and timedependent memory impairing effects on DBA mice; these effects were naltrexone reversible, suggesting the involvement of opioid receptors; (b) the effects of flunitrazepam were enhanced by immobilization stress, and decreased by familiarization with the passive avoidance apparatus. A number of experiments have recently shown that some of the behavioural effects of benzodiazepines can be counteracted by opioid antagonists, suggesting that they involve the release of endogenous opioids 5. Thus the present results extend to flunitrazepam, and in particular to its effects on memory, the observations carried out with other benzodiazepines on a variety of animal species tested in different experimental conditions. As far as the suggested link between opioids and benzodiazepines is concerned, it must be underlined that naltrexone-reversible impairing effects on memory have previously been observed, within the same range of doses of flunitrazepam used in the present research, in C57BL/6 (C57) mice 3. This result seems interesting since strainand dose-dependent behavioural effects have been observed in DBA and C57 mice following opioid treatment ~. In particular, as concerns
TABLE II
Antagonism by naltrexone of the effects offlunitrazepam (mean step-through latencies (± S.E.M.) on the test day) ANOVA (l-way) showed significant differences between groups (F3.2s = 166.97). The mean step-through latencies on the training day ranged between 9.2 ± 0.8 and 10.6 ± 0.9 s. Combinations of the substances were administered in a single treatment.
mg/kg Saline Naltrexone 0.025 Saline Naltrexone 0.025
mg/kg Vehicle Vehicle Flunitrazepam Flunitrazepam
0.5 0.5
132.3 130.0 21.5 124.6
± + ± +
4.6 5.0 2.3* 4.0
* P < 0.001 vs controls (Duncan multiple-range test).
trazepam vehicle: 8.0 _+ 3.4 s; flunitrazepam" 7.3 + 2.5 s) showing that the effects of the drug were not attributable to non-specific proactive pharmacological actions acting more than 24 h. (2) Naltrexone co-administration antagonized the retention performance impairment exerted by flunitrazepam (see Table II). (3) A significant enhancement of the effects of flunitrazepam was observed in mice subjected to a 15-min immobilization stress; this effect was antagonized by naltrexone administration (see Table III). TABLE llI
Effects of immobilization stress on flunitrazepam-injected mice and their antagonism by naltrexone (Mean step-through latencies (+_S.E.M.) on the test day) Separate ANOVAs (2-way) were carried out in order te analyze. (1) The effects of±mobilization stress on flunitrazepam-injected mice (groups A.B.D.E.) and (2) the antagonism of these effects by naltrexone (groups B.C.E.F.). The first ANOVA showed significant s t r e s s (F1,28 = 28.73, P < 0.001 and Flunitrazepam (F1,2s = 680.43, P < 0.001) main effects, and a significant stress × flunitrazepam interaction (F~.2s = 70.09, P < 0.001). The second ANOVA showed significant main effects for both immobilization s t r e s s ( F l , z s = 30.85, P < 0 . 0 0 1 ) and naltrexone on fiunitrazepam-injected mice (F~.2s = 368.20, P < 0.001) and a significant stress × drug treatment interaction (F 1,2s = 24.51, P < 0.001). The mean step-through latencies on the training day ranged between 8.7 + 0.8 and 10.2 + 0.7 s. Combinations of the substances were administered in a single treatment.
Min A. B. C. D. E. F.
Non-immob. Non-immob. Non-immob, Immob. Immob. Immob.
15 15 15
mg/kg Saline Saline Naltrexone Saline Saline Naltrexone
* P < 0.001 vs controls (Duncan multiple range test).
0.025
0.025
mg/kg Vehicle Flunitrazepam Flunitrazepam Vehicle Flunitrazepam Flunitrazepam
0.025 0.025 0.025 0.025
123.3 61.7 126.6 134.0 13.7 123.2
+ + ± ± ± ±
4.6 3.5* 5.3 4.0 3.0* 4.7
94 memory processes, previous investigations carried out in a patterns discrimination test have shown that post-trial peripheral (i.p.) administrations of/~-receptor agonists (heroin) improve memory in the latter strain, while in the former strain the effects are dose-dependent. In addition, recent experiments, carried out in passive avoidance conditions, have shown that post-trial central (i.c.v.) administrations of the b-receptor agonist (D-Ala2-D-LeuS)-enkephalin result in memory impairment in DBA mice, and in dosedependent effects in C57 mice 1. Finally, it seems interesting to underline that pre-trial i.p. injections of morphine have been shown to improve active avoidance responding in C57 but not in DBA mice, and that co-administration of chlordiazepoxide (which by itself improved avoidance behaviour in both strains of mice) resulted, in those experiments, in favourable effects in the latter strain only 6. Further investigations will indeed be necessary in this field to fully elucidate the behavioural effects of opioids and benzodiazepines, administered alone or in combination, depending on mouse strain, drug, dose and experimental conditions. As concerns, in particular, flunitrazepam, and related compounds, the pharmacogenetic approach based on C57 and DBA mice might be useful to clarify their mechanisms of action and in particular the neural structures, and the receptor populations, involved in the observed effects. A number of points emerge, in the present research, from the familiarization experiments. As far as the pretrained mice are concerned, the control groups showed retention scores lower than those of the non-familiarized subjects. This effect has previously been interpreted by considering the retention test as an approach-avoidance conflict for the animals, pre-training resolving this conflict in favour of 'approach' after initial avoidance behaviour 13. Mainly, this set of experiments shows a lower efficacy of flunitrazepam in DBA mice familiarized with the apparatus, in comparison with the non-experienced groups, as it has already been observed in the same strain following opioid treatment 4"12. This result has in particular been interpreted on
the basis of the hypothesis that opioid administration might result in memory impairment due to attenuation of emotionality 9 and that a lower effect can be evident in experienced animals since their emotional level is lower in comparison with that of non-experienced subjects 4'1°. Thus, according to this previous hypothesis, it can be suggested that the memory impairment observed in the present research following the administration of the 7-nitrobenzodiazepine flunitrazepam might be due to its anti-emotional properties. Finally, in the present research, a by itself ineffective immobilization stress enhanced the effects exerted by flunitrazepam. In addition this effect was antagonized by naltrexone. As far as this point is concerned, a number of previous experiments have shown that stress can affect memory processes in DBA mice, and that its effect can be ascribed, at least in part, to release of endogenous opioids under stressful conditions 2. Naloxone-reversible enhancement of the effects of opioids on memory following a short period of immobilization, suggesting the involvement of opioid receptors, has moreover been recently demonstrated in DBA mice 4"1e. Thus it can be hypothesized that opioid receptors might be involved in the enhancement of the effects of flunitrazepam by immobilization stress observed in the present study. It must finally be underlined that recent experiments 7 have shown that administration of chlordiazepoxide (CDP) prior to exposure to inescapable shock prevents the shuttleescape deficit typically observed following this type of stress. It must be pointed out that these results were obtained with rats tested in active avoidance conditions. Thus further experiments are now in progress in which CDP, and also other benzodiazepines, will be administered to C57 and DBA mice tested in our experimental conditions, in order to compare their effects with those of flunitrazepam observed in the present research, and possibly better clarify the already obtained results.
We thank E N D O Pharmaceuticals for the gift of naltrexone.
95
1 Castellano, C., A pharmacogenetic approach to opiates and memory. In J.L. McGaugh (Ed.), Contemporary Psychology: Biological Processes and Theoretical Issues, Elsevier, Amsterdam, 1985, pp. 45-67. 2 Castellano, C. and Puglisi-Allegra, S., Strain-dependent modulation of memory by stress in mice, Behav. Neural Biol., 38 (1983) 133-138. 3 Castellano, C., Filibeek, U. and Pavone, F., Naltrexonereversible effects of flunitrazepam on locomotor activity and passive avoidance behavior in mice, Eur. J. Pharmacol., 104 (1984) 111-116. 4 Castellano, C., Pavone, F. and Puglisi-Allegra, S., Morphine and memory in DBA/2 mice: effects of stress and of prior experience, Behav. Brain Res., 11 (1984) 3-10. 5 Cooper, S.J., Benzodiazepine-opiate antagonist interactions in relation to anxiety and appetite, Trends Pharmacol. Sci., 4 (1983) 456-458. 6 Cuomo, V., Cortese, I. and Racagni, G., Effects of morphine and chlordiazepoxide on avoidance behaviour in two inbred strains of mice, Neuropharmacology, 20 (1981) 301-304. 7 Drugan, R.C., Ryan, S.M., Minor, T.R. and Maier, S.F.,
8
9
10
11
12
13
Librium prevents the analgesia and shuttle-box deficit typically observed following inescapable shock, Pharmacol. Biochem. Behav., 21 (1984) 749-754. Gallagher, M., Naloxone enhancement of memory processes: effects of other opiate antagonists, Behav. Neural Biol., 35 (1982) 375-382. Gallagher, M. and Kapp, B.S., Manipulation of opiate activity in the amygdala alters memory processes, Life Sci., 23 (1978). Haycock, J.W., Van Buskirk, R. and Gold, P.E., Effects on retention of posttraining amphetamine injections in mice: interaction with pretraining experience, Psychopharmacology, 54 (1977) 21-24. Nicolai, P., Di Carlo, M., Alfani, F., Cerbo, R., Smeriglio, M., Borgo, S., Cipriani, E., Tarquini, S. and Malizia, E., La nostra esperienza sull'intossicazione acuta da flunitrazepam, Riv. Tossicol. Sperim. Clin., 3 (1981) 47-59. Pavone, F. and Castellano, C., Effects of tifluadom on passive avoidance behaviour in DBA/2 mice, Behav. Brain Res., 15 (1985) 177-181. Rigter, H., Vasopressin and memory: the influence of prior experience with the training situation, Behav. Neural Biol., 34 (1982) 337-351.
Behavioural Bra#r Research, 22 (1986) 91-95 Elsevier BBR 00602
Effects of flunitrazepam on passive avoidance behaviour in mice subjected to immobilization stress or familiarized with the testing apparatus CLAUDIO CASTELLANO and F L A M I N I A PAVONE
Istituto di Psicobiologia e Psicofarmacologia, C.N.R., Roma (Italy) (Received 28 April 1986) (Revised version received 18 June 1986) (Accepted 23 June 1986)
Key words: Flunitrazepam - Passive avoidance - Endogenous opioid - Stress - Familiarization
The effects of flunitrazepam on passive avoidance behaviour were investigated in DBA/2 mice. In a first set of experiments retention performance impairment was observed in mice injected with the drug immediately but not 120 min after training. In a second set of experiments, immobilization stress enhanced, while familiarization with the apparatus decreased, the effects of flunitrazepam, suggesting involvement of emotional factors. All the effects observed were antagonized by naltrexone, showing involvement of opioid receptors.
A number of researches have demonstrated, in recent years, that benzodiazepines can produce some of their behavioural effects (anticonflict effect, appetite enhancement etc.) through a release of endogenous opioids within the brain s. Use and abuse of a 7-nitrobenzodiazepine, flunitrazepam, have recently been described, which are due to its properties of alleviating some of the symptoms typical of the abstinence syndrome (irritability, insomnia). Moreover, a number of data indicate that in animals and humans, some of the effects of this drug can be antagonized by administration ofopioid antagonists, or potentiated by opiate treatments 3,1 ~, Recent experiments, carried out with DBA/2 mice, tested in a passive avoidance condition, have shown that opioids impair memory in this strain, and that this effect is enhanced by immobilization stress, and reduced by familiarization with the apparatus 4,t2. These effects have been interpreted in terms of release ofopioids by stress,
and of attenuation of emotionality by familiarization. In the present research it has thus been decided to study the effects of flunitrazepam on memory processes, and the possible interference in these processes by immobilization stress and familiarization with the testing conditions. The naltrexone-reversibility of the effects observed has also been assessed. The subjects were naive male mice of the inbred strain DBA/2 (DBA) (River Lab., Como, Italy) weighing 23-25 g. The apparatus consisted of a 20 x 20 x 20 cm Lucite box with black walls and a grid floor. The front wall was placed at the edge of a table, A 12 cm long and 5 cm wide platform protruded from the front wall over this edge and was lit by a 40-W lamp positioned 50 cm above it. The box was in darkness. In the experiments involving immobilization stress, stress was produced by placing the animals for 15 rain in a snug-fit apparatus 2. During training each mouse was placed on the platform facing away from a
Correspondence: C. Castellano, Istituto di Psicobiologia e Psicofarmacologia, via Reno, 1-00198 Roma, Italy. 0166-4328/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
92 4 × 3 cm opening in the front wall. The time taken by the mouse to enter the box with all 4 feet (step-through latency) was recorded. When the animal had entered the box, the opening was closed by a hand-operated sliding door, and the mouse received a 50-Hz scrambled footshock of 0.7 mA through the grid floor, for 1 s. It was then returned to its home cage to await testing 24 h later. Test procedures were the same as training, except that no footshock was administered. Different groups of animals were injected intraperitoneally with flunitrazepam (0.0125, 0.025 and 0.05mg/kg) immediately after training. In addition, a group of mice was injected with flunitrazepam (0.05 mg/kg) 120min after training, and another group of mice did not receive footshock but were injected with flunitrazepam (0.05 mg/kg) immediately after training. The performances of all mice were compared with those of mice injected with flunitrazepam vehicle. The effects of flunitrazepam (0.0125, 0.025 or 0.05 mg/kg) were also investigated in pretrained mice, which were put into the apparatus for a familiarization period of 2 min for 2 consecutive days, and were trained in the apparatus on the third day4. Their performances were compared with those of pretrained mice injected with flunitrazepam vehicle. When the naltrexone antagonism of the effects of flunitrazepam was investigated the dose of naltrexone (0.025 mg/kg) was selected on the basis of previous experiments and of preliminary work s. Finally, 3 groups of animals were immobilized for 15 min following immediate posttraining administration of saline + flunitrazepam vehicle, saline + flunitrazepam (0.025mg/kg) or naltrexone (0.025mg/kg)+ flunitrazepam (0.025 mg/kg) combinations and their performances were compared with those of corresponding non-immobilized groups. The duration of immobilization stress was selected on the basis of previous experiments 4. All groups consisted of 8 animals which were tested in the apparatus 24 h after training. Flunitrazepam (Darkene, Sigurta's, Milano) was diluted with 0.9~o NaC1. Naltrexone (HC1) (ENDO, Garden City, New York, NY) was dissolved in 0.9~o NaC1. Saline (0.9~,, NaC1) and flunitrazepam vehicle (at the concentration corre-
sponding to the highest dose of the drug) were used for control injections. All injections were given intraperitoneally (i.p.) in a volume of 4 ml/kg. The results were evaluated statistically by analysis of variance (ANOVA) (1- and 2-way) in which the mean step-through latencies of the groups on the test day were compared. Further analyses for individual between-groups comparisons were carried out with post hoc tests (Duncan multiple-range test). (1) Flunitrazepam administration immediately after training impaired retention performance of both non-pretrained and pretrained mice, but familiarization with the environment decreased this effect (see Table I). Moreover, (a) no significant difference was evident between the mean step-through latencies of mice injected with flunitrazepam (0.05 mg/kg) 120min after training (127.6 + 6.0) and those of their control group (129.2 + 2.8) (Fl,14 = 0.34, P > 0.05) and (b) the mean step-through latencies of the mice that had not received footshock on the training day but had been injected with flunitrazepam vehicle or flunitrazepam (0.05 mg/kg) did not differ from each other (mean step-through latencies: fluni-
TABLE I
Effects of flunitrazepam on passive avoidance behaviour #1 DBA/2 mice Mean step-through latencies ( _+ S.E.M.) on the test day of mice non-familiarized (A) and familiarized (B) with the apparatus. A N O V A (2-way) showed significant familiarization (Fi,56 = 35.957, P < 0.001) and flunitrazepam (F~,5~ - 136.281, P < 0.001) main effects, and a significant familiarization x flunitrazepam interaction (F3,5~ - 52.755, P < 0.001). The mean step-through latencies on the training day ranged between 9.1 + 1.1 and 10.7 _+ 1.3 s, for the nonfamiliarized groups, and 2.8 + 0.3 and 3.5 + 0.5 s for the familiarized groups.
Vehicle Flunitrazepam Flunitrazepam Flunitrazepam
mg/kg
A
0.0125 0.025 0.05
131.2 125.5 68.5 20,1
B +_ 5.5 + 5.8 _+ 5.0* +_ 3.3*
77.1 78.2 73.8 49.2
+ 2.3 + 2.5 _+ 2.0 _+ 2.6**
* P < 0.001 vs vehicle A (Duncan multiple-range test); ** P < 0.001 vs vehicle B (Duncan multiple-range test).
93 Two main points emerge from the present research (a) flunitrazepam exerted dose- and timedependent memory impairing effects on DBA mice; these effects were naltrexone reversible, suggesting the involvement of opioid receptors; (b) the effects of flunitrazepam were enhanced by immobilization stress, and decreased by familiarization with the passive avoidance apparatus. A number of experiments have recently shown that some of the behavioural effects of benzodiazepines can be counteracted by opioid antagonists, suggesting that they involve the release of endogenous opioids 5. Thus the present results extend to flunitrazepam, and in particular to its effects on memory, the observations carried out with other benzodiazepines on a variety of animal species tested in different experimental conditions. As far as the suggested link between opioids and benzodiazepines is concerned, it must be underlined that naltrexone-reversible impairing effects on memory have previously been observed, within the same range of doses of flunitrazepam used in the present research, in C57BL/6 (C57) mice 3. This result seems interesting since strainand dose-dependent behavioural effects have been observed in DBA and C57 mice following opioid treatment ~. In particular, as concerns
TABLE II
Antagonism by naltrexone of the effects offlunitrazepam (mean step-through latencies (± S.E.M.) on the test day) ANOVA (l-way) showed significant differences between groups (F3.2s = 166.97). The mean step-through latencies on the training day ranged between 9.2 ± 0.8 and 10.6 ± 0.9 s. Combinations of the substances were administered in a single treatment.
mg/kg Saline Naltrexone 0.025 Saline Naltrexone 0.025
mg/kg Vehicle Vehicle Flunitrazepam Flunitrazepam
0.5 0.5
132.3 130.0 21.5 124.6
± + ± +
4.6 5.0 2.3* 4.0
* P < 0.001 vs controls (Duncan multiple-range test).
trazepam vehicle: 8.0 _+ 3.4 s; flunitrazepam" 7.3 + 2.5 s) showing that the effects of the drug were not attributable to non-specific proactive pharmacological actions acting more than 24 h. (2) Naltrexone co-administration antagonized the retention performance impairment exerted by flunitrazepam (see Table II). (3) A significant enhancement of the effects of flunitrazepam was observed in mice subjected to a 15-min immobilization stress; this effect was antagonized by naltrexone administration (see Table III). TABLE llI
Effects of immobilization stress on flunitrazepam-injected mice and their antagonism by naltrexone (Mean step-through latencies (+_S.E.M.) on the test day) Separate ANOVAs (2-way) were carried out in order te analyze. (1) The effects of±mobilization stress on flunitrazepam-injected mice (groups A.B.D.E.) and (2) the antagonism of these effects by naltrexone (groups B.C.E.F.). The first ANOVA showed significant s t r e s s (F1,28 = 28.73, P < 0.001 and Flunitrazepam (F1,2s = 680.43, P < 0.001) main effects, and a significant stress × flunitrazepam interaction (F~.2s = 70.09, P < 0.001). The second ANOVA showed significant main effects for both immobilization s t r e s s ( F l , z s = 30.85, P < 0 . 0 0 1 ) and naltrexone on fiunitrazepam-injected mice (F~.2s = 368.20, P < 0.001) and a significant stress × drug treatment interaction (F 1,2s = 24.51, P < 0.001). The mean step-through latencies on the training day ranged between 8.7 + 0.8 and 10.2 + 0.7 s. Combinations of the substances were administered in a single treatment.
Min A. B. C. D. E. F.
Non-immob. Non-immob. Non-immob, Immob. Immob. Immob.
15 15 15
mg/kg Saline Saline Naltrexone Saline Saline Naltrexone
* P < 0.001 vs controls (Duncan multiple range test).
0.025
0.025
mg/kg Vehicle Flunitrazepam Flunitrazepam Vehicle Flunitrazepam Flunitrazepam
0.025 0.025 0.025 0.025
123.3 61.7 126.6 134.0 13.7 123.2
+ + ± ± ± ±
4.6 3.5* 5.3 4.0 3.0* 4.7
94 memory processes, previous investigations carried out in a patterns discrimination test have shown that post-trial peripheral (i.p.) administrations of/~-receptor agonists (heroin) improve memory in the latter strain, while in the former strain the effects are dose-dependent. In addition, recent experiments, carried out in passive avoidance conditions, have shown that post-trial central (i.c.v.) administrations of the b-receptor agonist (D-Ala2-D-LeuS)-enkephalin result in memory impairment in DBA mice, and in dosedependent effects in C57 mice 1. Finally, it seems interesting to underline that pre-trial i.p. injections of morphine have been shown to improve active avoidance responding in C57 but not in DBA mice, and that co-administration of chlordiazepoxide (which by itself improved avoidance behaviour in both strains of mice) resulted, in those experiments, in favourable effects in the latter strain only 6. Further investigations will indeed be necessary in this field to fully elucidate the behavioural effects of opioids and benzodiazepines, administered alone or in combination, depending on mouse strain, drug, dose and experimental conditions. As concerns, in particular, flunitrazepam, and related compounds, the pharmacogenetic approach based on C57 and DBA mice might be useful to clarify their mechanisms of action and in particular the neural structures, and the receptor populations, involved in the observed effects. A number of points emerge, in the present research, from the familiarization experiments. As far as the pretrained mice are concerned, the control groups showed retention scores lower than those of the non-familiarized subjects. This effect has previously been interpreted by considering the retention test as an approach-avoidance conflict for the animals, pre-training resolving this conflict in favour of 'approach' after initial avoidance behaviour 13. Mainly, this set of experiments shows a lower efficacy of flunitrazepam in DBA mice familiarized with the apparatus, in comparison with the non-experienced groups, as it has already been observed in the same strain following opioid treatment 4"12. This result has in particular been interpreted on
the basis of the hypothesis that opioid administration might result in memory impairment due to attenuation of emotionality 9 and that a lower effect can be evident in experienced animals since their emotional level is lower in comparison with that of non-experienced subjects 4'1°. Thus, according to this previous hypothesis, it can be suggested that the memory impairment observed in the present research following the administration of the 7-nitrobenzodiazepine flunitrazepam might be due to its anti-emotional properties. Finally, in the present research, a by itself ineffective immobilization stress enhanced the effects exerted by flunitrazepam. In addition this effect was antagonized by naltrexone. As far as this point is concerned, a number of previous experiments have shown that stress can affect memory processes in DBA mice, and that its effect can be ascribed, at least in part, to release of endogenous opioids under stressful conditions 2. Naloxone-reversible enhancement of the effects of opioids on memory following a short period of immobilization, suggesting the involvement of opioid receptors, has moreover been recently demonstrated in DBA mice 4"1e. Thus it can be hypothesized that opioid receptors might be involved in the enhancement of the effects of flunitrazepam by immobilization stress observed in the present study. It must finally be underlined that recent experiments 7 have shown that administration of chlordiazepoxide (CDP) prior to exposure to inescapable shock prevents the shuttleescape deficit typically observed following this type of stress. It must be pointed out that these results were obtained with rats tested in active avoidance conditions. Thus further experiments are now in progress in which CDP, and also other benzodiazepines, will be administered to C57 and DBA mice tested in our experimental conditions, in order to compare their effects with those of flunitrazepam observed in the present research, and possibly better clarify the already obtained results.
We thank E N D O Pharmaceuticals for the gift of naltrexone.
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1 Castellano, C., A pharmacogenetic approach to opiates and memory. In J.L. McGaugh (Ed.), Contemporary Psychology: Biological Processes and Theoretical Issues, Elsevier, Amsterdam, 1985, pp. 45-67. 2 Castellano, C. and Puglisi-Allegra, S., Strain-dependent modulation of memory by stress in mice, Behav. Neural Biol., 38 (1983) 133-138. 3 Castellano, C., Filibeek, U. and Pavone, F., Naltrexonereversible effects of flunitrazepam on locomotor activity and passive avoidance behavior in mice, Eur. J. Pharmacol., 104 (1984) 111-116. 4 Castellano, C., Pavone, F. and Puglisi-Allegra, S., Morphine and memory in DBA/2 mice: effects of stress and of prior experience, Behav. Brain Res., 11 (1984) 3-10. 5 Cooper, S.J., Benzodiazepine-opiate antagonist interactions in relation to anxiety and appetite, Trends Pharmacol. Sci., 4 (1983) 456-458. 6 Cuomo, V., Cortese, I. and Racagni, G., Effects of morphine and chlordiazepoxide on avoidance behaviour in two inbred strains of mice, Neuropharmacology, 20 (1981) 301-304. 7 Drugan, R.C., Ryan, S.M., Minor, T.R. and Maier, S.F.,
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