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Anxiolytic profiles of alprazolam and ethanol in the elevated plus-maze test and the early acquisition of shuttlebox avoidance
Pharmacological Research. Vol. 29, No. I. 1994
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ANXIOLYTIC PROFILES OF ALPRAZOLAM AND ETHANOL IN THE ELEVATED PLUS-MAZE TEST AND THE EARLY ACQUISITION OF SHUTTLEBOX AVOIDANCE M. PRUNELL*,
R. M. ESCORIHUELAt, A. FERNANDEZ-TERUELt, J. F. NfiREZt and A. TOBERAt
“Laboratorio de Fisiologia Animal, Facultad de Biologia, Universidad de la Laguna, Tenerife, Spain and I‘Unitat de Psicologia Mbdica, Dept. de Farmacologia i Psiquiatria, Facultat de Medicina, Universidad Autbnoma de Barcelona, 08193-Bellaterra, Barcelona, Spain Received in final form
27 July 1993
SUMMARY Rats pretreated either with the anxiolytic triazolobenzodiazepine alprazolam (0.75 and 1.75 mg kg-’ i.p.) or ethanol (1, 2, 3 and 4 g kg-’ p.o.) were tested in both the elevated plus-maze and the early acquisition of shuttlebox avoidance. Both substances induced overall anxiolytic effects in the plus-maze, with the 0.75 mg kg-’ being the most effective alprazolam dose and l-3 g kg-’ being the most effective ethanol doses. Both drugs were also anxiolytic in the shuttlebox, since 1.75 mg kg-’ alprazolam and 2-3 g kg-’ ethanol improved acquisition of the task. Correlational and factor analysis showed that behaviour in the open arms of the plus-maze and efficiency in shuttlebox avoidance acquisition are positively associated, thus providing further support to the contention that the early acquisition of shuttlebox avoidance is an animal model of anxiety. KEY WORDS: alprazolam.
plus-maze,
shuttlebox
acquisition,
correlations,
anxiety
models,
ethanol,
INTRODUCTION A considerable amount of behavioural and pharmacological evidence has provided support for the contention that the early acquisition of two-way active (shuttlebox) avoidance is a valid animal model of anxiety. In fact, it has been shown that: (1) the initial steps of that task appear to involve a bidirectional (i.e. passive vs active) avoidance conflict in which an early conditioned emotional response (i.e. ‘conditioned fear’, see [l-3]) is responsible for a tendency to freeze (i.e. passive avoidance of the opposite compartment), which runs against actively learning to avoid the shock (i.e. to adaptatively cope by crossing to the opposite compartment [l-3]; (2) Several antianxiety drugs (diazepam, chlordiazepoxide, flurazepam, medazepam, nitrazepam, alprazolam, adinazolam, amobarbital, pentobarbital, etc) 0 1994 The Italian Pharmacological
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improve the early acquisition (i.e. during the initial stages or trials) of shuttlebox avoidance in rats and mice [4-131; (3) Manipulations which reduce emotional reactivity or fearfulness in rats, such as postnatal handling [14, 151 or repeated (14-day) handling of adolescent rats [6, 161 improve shuttlebox avoidance acquisition [6, 17-211; (4) The less fearful female rats are better in shuttlebox avoidance acquisition than the relatively more fearful male rats [22]; (5) Maudsley Reactive rats (genetically selected for high fearfulnes or emotionality in the open field test) are poorer performers in shuttlebox acquisition than Maudsley Nonreactive rats (genetically selected for low emotional reactivity in the open field; for a review see [23]), and, congruently, Roman high-avoidance rats (selected for good shuttlebox acquisition) are less emotionally reactive than Roman low-avoidance rats (selected for poor shuttlebox acquisition; see [23]; (6) Psychological manipulations leading to increased fearfulness, as well as the administration of anxiogenic drugs (FG 7 142, Ro 15-45 13, partial inverse agonists of benzodiazepine receptors), impair the acquisition of shuttlebox avoidance [S, 20, 21, 241; (7) The rate of acquisition of shuttlebox avoidance is inversely correlated with the rate of ultrasonic vocalization in rats [25]. The anxiolytic effects of drugs in the early acquisition of shuttlebox avoidance appear after very few (between the first 10 to 40) trials in a single session, and are typically reflected by an increase in the number of avoidances or a reduction of escape/avoidance latency, or both [4-7, 261. On the other hand, the elevated plus-maze, consisting of measures of spontaneous behaviour on two open and two enclosed arms forming a ‘+’ sign, is another well validated and much more widely used test of anxiety in rats and mice (see for instance [27-301). The anxiolytic effects of drugs are usually indicated by an increase in the number of entries made, and/or time spent, by the animals in the open arms [27-301. Benzodiazepines and ethanol, though acting at different sites of the GABAbenzodiazepine receptor complex, share a number of behavioural effects, one of which is an anti-anxiety action in animal models [see 28, 29, 31-371. Nevertheless, no study has thus far reported a similar anxiolytic-like effect of ethanol in the early acquisition of shuttlebox avoidance (that is, in a single short40 trials or less-session). Thus, the first objective of the present study was to extend the validation of the early acquisition of shuttlebox avoidance (i.e. one single short session) as an animal model of anxiety by testing the effects of several ethanol doses and comparing them with those of a benzodiazepine (in this case, alprazolam). The second objective was to evaluate the possible (and expected) correlations between shuttlebox avoidance acquisition and behaviour in the elevated plus-maze test 127,291, in order to provide evidence on the validity of the former by using an independent criterion. MATERIAL
AND METHODS
Atlimds
Forty-four male Sprague-Dawley rats, weighing 230-310 g (obtained from Iffa Credo, France) were used in the study. They were housed 2-3 per cage under
Phar.mac~olo~ic~ul Rescwrc~h. l’ol. 29, No. I, 1994
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regulated light/dark conditions (light, 08:OO to 20:00) with food and water freely available, and controlled temperature (22f2”C) and relative humidity (.50+10%). The experiment was carried out from 9:00 hrs to 14:00 hrs.
Behaviouaul tests Elellated plus-maze. Twenty minutes after drug administration, animals were tested for 5 min in the elevated plus-maze consisting in two open arms, 50x10 cm, and two enclosed arms, 50x10~40 cm, with an open square of 10x10 cm in the centre of the ‘+’ sign. The maze was elevated to a height of 50 cm [27]. Total arm entries (TE) and the number and duration of the entries into the open and enclosed arms were scored. From these parameters the percentage of open arm entries (% EOA=open arm entries/TExlOO) and of time spent in the open arms (%TOA=time open arms/[time open arms+time enclosed arms]xlOO) were calculated and used for analysis [27]. Two identical shuttlebox chambers (Letica Shuttlebox uvoidunce ucquisition. Inst.) were employed. The testing session included 10 min of habituation, immediately followed by a series of 20 trials of avoidance acquisition. Each trial consisted of 10 s of conditioned stimulus (CS, light and tone simultaneously), followed by a 0.6 mA footshock of 30 s as aversive unconditioned stimulus (US). The intertrial interval was 30 s. The number of avoidances (crossings in the presence of CS) during the first 10 trials (AVOIDlO) and during the whole sessions (AVOID20), as well as the respective mean escape latencies (LATlO and LAT20; mean time elapsing between the CS presentation and the response) were scored. The animals were introduced into the shuttlebox 30 min after drug administration and 5 min after testing in the elevated plus-maze.
Drug administration und experimental pinups In experiment 1, alprazolam (Upjohn Farmoquimica S.A.), at doses of 0.75 (n= 5) and 1.75 (n=5) mg kg-‘, was suspended in 1% carboxymethyl cellulose (cmc) and administered i.p. in a volume of 2 ml kg-‘. Control animals (n=5) received cmc at the same volume. In experiment 2, ethanol (Panreac), at doses of 1 (n=5), 2 (n=5), 3 (n=7) and 4 (n=5) g kg-‘, was given p.o. in a 20% v/v solution in tap water. Control animals (n=7) received tap water at the same volume. Both experiments were carried out within a 2-week period.
Stutisticctl unulysis One-way analysis of variance was applied to the data. Duncan’s Multiple Range tests were used for comparisons between groups. Correlational (Pearson’s coefficient) and factor analysis, including both plusmaze and shuttlebox testing measures, were applied to all the animals from the present two experiments (n=44) and to control animals (n=23). In the case of control animals, the number of rats was increased to 23 because an additional control group (n=l 1; treated and tested as in experiment 2 and during the same
40
Pharmacolo,~ical
period) from a separate experiment ‘n’ only for that analysis.
was included
Research,
with the purpose
Vol. 29, No. I, 1994
of increasing
RESULTS Alprazolam (experiment I, Table I) overall enhanced %EOA (F(2,12)=3.51, P= 0.06) and %TOA (F(2,12)=4.11, P~0.05) in the elevated plus-maze, the difference between 0.75 mg kg-’ alprazolam and vehicle groups reaching statistical significance for both parameters (P-cO.05, Duncan’s test), whereas a nonsignificant tendency was also observed at the 1.75 mg kg-’ dose (see Table I). The 1.75 mg kg-’ dose improved shuttlebox avoidance acquisition, as shown by a significant increase in AVOID10 (F(2,12)=3.64, P=O.O6; P~0.05 vs vehicle group, Duncan’s test) and showed a similar tendency in AVOID20 (F(2,12)=2.63, P= 0.11, but t=2.97, P~0.02 v,s vehicle group), as well as by significant reductions of LATlO (F(2,12)=3.23, P=O.O8; P~0.05 vs vehicle group, Duncan’s test) and LAT20 (F(2,12)=4.8, P~0.04; P~0.05 vs vehicle group, Duncan’s test). In experiment 2 (Table I), all ethanol doses increased %EOA (F(4,24)=7.5, PC 0.001) and %TOA (F(4,24)=17.4, P
ikk!anS~SEM
Exp. 1 Alprazolam (mg kg-‘) Vehicle 0.75
TE
%EOA
%TOA
AVOID10
AVOID20
LATI 0
LAT20
16.6 (1.8) 11.6 (2.9)
26.2 (5.3) 48.8* (4.7) 46.4 (9.0)
17.5 (4.9) .57.7* (10.9) 38.3 (12.3)
1.2 (0.4) 1.5 (0.5)
3.0 (1.0) 4.7 (1.9) 6.8 (0.8)
19.8 (4.8) 14.2 (2.6) 9.5* (0.5)
18.7 (2.7) 13.5 (3.2)
9.4 (1.1) 21.0* (2.3) 26.0* (3.8) 26.7* (4.0) 13.2 (2.5)
21.9 (2.3) 56.3* (3.9) 60.3* (1.1) 60.5* (3.4) 47.9* (19.3)
7.1 (1.3) 56.9* (6.0) 58.5* (1.5) 66.0* (3.2) 52.0* (16.7)
1.7 (0.6) 3.0 (0.8) 5.6* (1.7) 5.1* (1.4) 1.6 (0.4)
13.0 (0.8) 12.3 (1.0) Il.7 (0.9) 15.1 (1.7) 32.3* (3.5)
12.1 (1.0) Il.5 (0.7) 11.3 (0.7) 13.0 (1.4) 31.2* (4.1)
1.75
Exp 2 Ethanol (g kg-’ ) Vehicle I
2 3 4
Table I of plus-maze and shuttlebox acquisition
*P
test).
1.6 (0.7) 2.0 (0.7) 1.7
(0.6) 0.6 (0.2)
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AVOID20 (F(4,24)=2.9, P
Table II between sbuttlebox acquisition and performance in the elevated plus-maze test
Correlations 1 1. TE 2. %EOA 3. %TOA 4. AVOID10 5. AVOID20 6. LATlO 7. LAT20
-0.29 0.41t 0.65t 0.38t 0.6lj 0.20 0.44* 0.19 0.16 -0.05 0.20 -0.09
2
3
4
5
6
7
0.11 0.86-t -0.16 0.23 0.01 0.23 -0.22 -0.33* -0.26 -0.36*
0.43+ 0.31* 0.41+ 0.25 -0.15 -0.18 -0.12 -0.16
0.75t 0.70t -0.20 -0.44t -0.10 -0.34*
-0.15 -0.35* -0.28 -0.41*
-0.897 -0.94t
-
Upper values are control animals; n=23 because an additional control group, treated and tested as control rats from experiment 2 and during the same two-week period, has been included in order to increase the number of animals for performing correlations. Lower values are all the animals (n=44; vehicle- plus drug-treated groups) used in the present two experiments. +P
Table III Factor (principal-components) analysis between plus-maze measures from control animals and from the whole sample Control
Variables (loadings) TE %EOA %TOA AVOID10 AVOID20 LATlO LAT20 Percentage of explained variance
animals ._.~ 1
-
and shuttlebox (‘All animals’) All animals
I
0.15 -0.08 0.74 0.87 0.82 -0.30 -0.21
0.42 0.72 0.66 0.69 0.67 -0.74 -0.74
30%
45%
‘Control animals’ (n=23) and ‘all animals’ (n=44) as in Table II. Symbols and II. Loadings 2.30 are considered as significant.
as in Tables
I
DISCUSSION As in previous studies, which involved only shuttlebox avoidance testing ([4-71, and two more unpublished replications), alprazolam (a well known anxiolytic triazolobenzodiazepine) improved shuttlebox avoidance acquisition (by increasing avoidances and reducing latencies) and also showed the expected [30] anxiolytic effects in the plus-maze test. Since alprazolam (Experiment 1) was also included to test the reliability of the present (repeated testing) design, the results indicate that the present procedure (i.e. plus-maze testing plus shuttlebox avoidance testing) retains the sensitivity of both tests (as when they are used independently) for detecting the anxiolytic effects of drugs [4-7, 29, 301, and thus it is unlikely that the previous plus-maze administration could have produced artifactual influences on shuttlebox testing. On the other hand, the obvious advantage of the present procedure is that of testing the predicted possible correlations between the performance in both anxiety models (see below). The effects of ethanol during the process of acquisition of shuttlebox avoidance in random bred rats were studied by Chesher [38]. However, in that study, rats were tested in six consecutive IO-trial shuttlebox avoidance sessions (24-h interval between sessions [38]) after ethanol pretreatment (one injection of 1.5 or 2 g kg-’ i.p., in Wistar or Sprague-Dawley rats respectively, before each test). The results indicated that ethanol improved avoidance behavior only after the third training session [38]. In a different study Prune11 et al. [39] reported that acute ethanol (2 g kg-’ p.o.) shortened avoidance latencies in overtrained rats (their performance being 100% of avoidances when the treatment was administered). But, due to the procedures used, none of those studies provided evidence on the issue of whether ethanol could improve shuttlebox avoidance acquisition during its very early stages (as benzodiazepines do [4-7]), since it is during that phase when the conflict present in the task involves the maximum anxiety levels, and the
improving effects of environmental or pharmacological anxiolytic treatments are more evident [4,7, 10, II, 20, 211. Thus, the present study is, to our knowledge, the first dose-response study reporting positive effects of ethanol in the early acquisition (i.e. in a single short session) of shuttlebox avoidance, this being in agreement with the contention that moderate doses of ethanol display anxiolytic-like effects. It is, however, clear that the highest ethanol dose (4 g kg-‘) induces differential effects in both tests (i.e. it is anxiolytic in the plus-maze but has detrimental effects in shuttlebox acquisition), which is probably due to the different behavioural requirements involved in each situation, particularly to the fact that the perceptual and associative processes involved in the shuttlebox task (but not in the plus-maze) are probably disrupted by such a high ethanol dose. The moderate (but significant) correlations observed between plus-maze and shuttlebox acquisition measures (Table I), as well as their association in the principal-components analysis (Table Il), provide additional support for the hypothesis that the early acquisition of shuttlebox avoidance is an anxietymediated behaviour [l-7, 22-261. The most relevant associations between both tests indicate that, in control animals (n=23) AVOID10 and AVOID20 are positively correlated with %TOA, and, confirming this, the three measures have high loadings (0.74 to 0.87) of the same sign in the factor analysis. The consistency of those relationships is further confirmed by the fact that, in the whole sample of animals from both experiments (n=44), AVOID10 and %TOA are positively correlated and have positive loadings in the factor analysis (0.69 and 0.66, respectively), whereas, congruently, LATlO and LAT20 are inversely correlated with %EOA and also load inversely in the factor analysis (-0.74, -0.74 and 0.72. respectively). Also interesting is the fact that in control animals AVOID10 and AVOID20 correlated positively with TE, and the three measures loaded positively in the factor analysis (0.87, 0.82 and 0.75, respectively). However, those relationships became non-significant (see Table I) when including the drug-treated animals (n= 44), thus suggesting that there is little (if any) relationship between the effects of the drugs on general activity (i.e. TE) and their action on avoidance acquisition. This agrees with results from several previous studies which have indicated that shuttlebox avoidance acquisition cannot be completely accounted for by general activity levels 14, 5, 7, 20, 221. The within-test correlations show essentially the patterns that should be expected according to previous studies (see [4, 26, 27-29]), that is: (I) there are mostly positive correlations between the three plus-maze measures and positive loadings in the factor analysis (see [27-291); and (2) there are overall negative correlations between latency and avoidance parameters and inverse loadings of these measures in the factor analysis (see [4, 251). The only relevant exception to these patterns is the absence of correlation between %EOA and %TOA in control animals, which is probably due to the fact that %EOA scores in control animals were clearly higher than %TOA scores (Table I), whereas the values of both measures were overall much closer to each other in drug-treated rats (i.e. treatments led to similar within-group values in both %EOA and %TOA; see Table I). On the other hand, as indicated by pharmacological studies, both
44
Pharmacological Research, Vol. 29, No. 1, 1994
measures (i.e. %EOA and %TOA) are not always associated since experimental conditions can affect them differently [29]. Therefore, both the correlational and factor analyses (Tables I, II) show overall a positive association between behaviour in the open arms of the plus-maze and efficiency in shuttlebox acquisition. Thus, to summarize, two main new findings are reported in the present study which provide further evidence supporting the early acquisition of shuttlebox avoidance as an animal model of anxiety: (1) ethanol, like benzodiazepines, improves the early shuttlebox avoidance acquisition in rats and (2) positive relationships are shown between behaviour in the open arms of the plus-maze and the ability to solve the initial stages of the shuttlebox avoidance acquisition task.
ACKNOWLEDGEMENTS This work was supported by the DGICYT (PM88-007.5, PM92/0071). M.P. received a grant from the ‘Consejeria de Education de1 Gobierno de las Islas Canarias’, and R.M.E. was supported by the FISSs (92/0712).
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ANXIOLYTIC PROFILES OF ALPRAZOLAM AND ETHANOL IN THE ELEVATED PLUS-MAZE TEST AND THE EARLY ACQUISITION OF SHUTTLEBOX AVOIDANCE M. PRUNELL*,
R. M. ESCORIHUELAt, A. FERNANDEZ-TERUELt, J. F. NfiREZt and A. TOBERAt
“Laboratorio de Fisiologia Animal, Facultad de Biologia, Universidad de la Laguna, Tenerife, Spain and I‘Unitat de Psicologia Mbdica, Dept. de Farmacologia i Psiquiatria, Facultat de Medicina, Universidad Autbnoma de Barcelona, 08193-Bellaterra, Barcelona, Spain Received in final form
27 July 1993
SUMMARY Rats pretreated either with the anxiolytic triazolobenzodiazepine alprazolam (0.75 and 1.75 mg kg-’ i.p.) or ethanol (1, 2, 3 and 4 g kg-’ p.o.) were tested in both the elevated plus-maze and the early acquisition of shuttlebox avoidance. Both substances induced overall anxiolytic effects in the plus-maze, with the 0.75 mg kg-’ being the most effective alprazolam dose and l-3 g kg-’ being the most effective ethanol doses. Both drugs were also anxiolytic in the shuttlebox, since 1.75 mg kg-’ alprazolam and 2-3 g kg-’ ethanol improved acquisition of the task. Correlational and factor analysis showed that behaviour in the open arms of the plus-maze and efficiency in shuttlebox avoidance acquisition are positively associated, thus providing further support to the contention that the early acquisition of shuttlebox avoidance is an animal model of anxiety. KEY WORDS: alprazolam.
plus-maze,
shuttlebox
acquisition,
correlations,
anxiety
models,
ethanol,
INTRODUCTION A considerable amount of behavioural and pharmacological evidence has provided support for the contention that the early acquisition of two-way active (shuttlebox) avoidance is a valid animal model of anxiety. In fact, it has been shown that: (1) the initial steps of that task appear to involve a bidirectional (i.e. passive vs active) avoidance conflict in which an early conditioned emotional response (i.e. ‘conditioned fear’, see [l-3]) is responsible for a tendency to freeze (i.e. passive avoidance of the opposite compartment), which runs against actively learning to avoid the shock (i.e. to adaptatively cope by crossing to the opposite compartment [l-3]; (2) Several antianxiety drugs (diazepam, chlordiazepoxide, flurazepam, medazepam, nitrazepam, alprazolam, adinazolam, amobarbital, pentobarbital, etc) 0 1994 The Italian Pharmacological
Society
3x
Pha~niac,olo,~icrrl
Rrseamh.
Vol. 29, No. I. 1994
improve the early acquisition (i.e. during the initial stages or trials) of shuttlebox avoidance in rats and mice [4-131; (3) Manipulations which reduce emotional reactivity or fearfulness in rats, such as postnatal handling [14, 151 or repeated (14-day) handling of adolescent rats [6, 161 improve shuttlebox avoidance acquisition [6, 17-211; (4) The less fearful female rats are better in shuttlebox avoidance acquisition than the relatively more fearful male rats [22]; (5) Maudsley Reactive rats (genetically selected for high fearfulnes or emotionality in the open field test) are poorer performers in shuttlebox acquisition than Maudsley Nonreactive rats (genetically selected for low emotional reactivity in the open field; for a review see [23]), and, congruently, Roman high-avoidance rats (selected for good shuttlebox acquisition) are less emotionally reactive than Roman low-avoidance rats (selected for poor shuttlebox acquisition; see [23]; (6) Psychological manipulations leading to increased fearfulness, as well as the administration of anxiogenic drugs (FG 7 142, Ro 15-45 13, partial inverse agonists of benzodiazepine receptors), impair the acquisition of shuttlebox avoidance [S, 20, 21, 241; (7) The rate of acquisition of shuttlebox avoidance is inversely correlated with the rate of ultrasonic vocalization in rats [25]. The anxiolytic effects of drugs in the early acquisition of shuttlebox avoidance appear after very few (between the first 10 to 40) trials in a single session, and are typically reflected by an increase in the number of avoidances or a reduction of escape/avoidance latency, or both [4-7, 261. On the other hand, the elevated plus-maze, consisting of measures of spontaneous behaviour on two open and two enclosed arms forming a ‘+’ sign, is another well validated and much more widely used test of anxiety in rats and mice (see for instance [27-301). The anxiolytic effects of drugs are usually indicated by an increase in the number of entries made, and/or time spent, by the animals in the open arms [27-301. Benzodiazepines and ethanol, though acting at different sites of the GABAbenzodiazepine receptor complex, share a number of behavioural effects, one of which is an anti-anxiety action in animal models [see 28, 29, 31-371. Nevertheless, no study has thus far reported a similar anxiolytic-like effect of ethanol in the early acquisition of shuttlebox avoidance (that is, in a single short40 trials or less-session). Thus, the first objective of the present study was to extend the validation of the early acquisition of shuttlebox avoidance (i.e. one single short session) as an animal model of anxiety by testing the effects of several ethanol doses and comparing them with those of a benzodiazepine (in this case, alprazolam). The second objective was to evaluate the possible (and expected) correlations between shuttlebox avoidance acquisition and behaviour in the elevated plus-maze test 127,291, in order to provide evidence on the validity of the former by using an independent criterion. MATERIAL
AND METHODS
Atlimds
Forty-four male Sprague-Dawley rats, weighing 230-310 g (obtained from Iffa Credo, France) were used in the study. They were housed 2-3 per cage under
Phar.mac~olo~ic~ul Rescwrc~h. l’ol. 29, No. I, 1994
39
regulated light/dark conditions (light, 08:OO to 20:00) with food and water freely available, and controlled temperature (22f2”C) and relative humidity (.50+10%). The experiment was carried out from 9:00 hrs to 14:00 hrs.
Behaviouaul tests Elellated plus-maze. Twenty minutes after drug administration, animals were tested for 5 min in the elevated plus-maze consisting in two open arms, 50x10 cm, and two enclosed arms, 50x10~40 cm, with an open square of 10x10 cm in the centre of the ‘+’ sign. The maze was elevated to a height of 50 cm [27]. Total arm entries (TE) and the number and duration of the entries into the open and enclosed arms were scored. From these parameters the percentage of open arm entries (% EOA=open arm entries/TExlOO) and of time spent in the open arms (%TOA=time open arms/[time open arms+time enclosed arms]xlOO) were calculated and used for analysis [27]. Two identical shuttlebox chambers (Letica Shuttlebox uvoidunce ucquisition. Inst.) were employed. The testing session included 10 min of habituation, immediately followed by a series of 20 trials of avoidance acquisition. Each trial consisted of 10 s of conditioned stimulus (CS, light and tone simultaneously), followed by a 0.6 mA footshock of 30 s as aversive unconditioned stimulus (US). The intertrial interval was 30 s. The number of avoidances (crossings in the presence of CS) during the first 10 trials (AVOIDlO) and during the whole sessions (AVOID20), as well as the respective mean escape latencies (LATlO and LAT20; mean time elapsing between the CS presentation and the response) were scored. The animals were introduced into the shuttlebox 30 min after drug administration and 5 min after testing in the elevated plus-maze.
Drug administration und experimental pinups In experiment 1, alprazolam (Upjohn Farmoquimica S.A.), at doses of 0.75 (n= 5) and 1.75 (n=5) mg kg-‘, was suspended in 1% carboxymethyl cellulose (cmc) and administered i.p. in a volume of 2 ml kg-‘. Control animals (n=5) received cmc at the same volume. In experiment 2, ethanol (Panreac), at doses of 1 (n=5), 2 (n=5), 3 (n=7) and 4 (n=5) g kg-‘, was given p.o. in a 20% v/v solution in tap water. Control animals (n=7) received tap water at the same volume. Both experiments were carried out within a 2-week period.
Stutisticctl unulysis One-way analysis of variance was applied to the data. Duncan’s Multiple Range tests were used for comparisons between groups. Correlational (Pearson’s coefficient) and factor analysis, including both plusmaze and shuttlebox testing measures, were applied to all the animals from the present two experiments (n=44) and to control animals (n=23). In the case of control animals, the number of rats was increased to 23 because an additional control group (n=l 1; treated and tested as in experiment 2 and during the same
40
Pharmacolo,~ical
period) from a separate experiment ‘n’ only for that analysis.
was included
Research,
with the purpose
Vol. 29, No. I, 1994
of increasing
RESULTS Alprazolam (experiment I, Table I) overall enhanced %EOA (F(2,12)=3.51, P= 0.06) and %TOA (F(2,12)=4.11, P~0.05) in the elevated plus-maze, the difference between 0.75 mg kg-’ alprazolam and vehicle groups reaching statistical significance for both parameters (P-cO.05, Duncan’s test), whereas a nonsignificant tendency was also observed at the 1.75 mg kg-’ dose (see Table I). The 1.75 mg kg-’ dose improved shuttlebox avoidance acquisition, as shown by a significant increase in AVOID10 (F(2,12)=3.64, P=O.O6; P~0.05 vs vehicle group, Duncan’s test) and showed a similar tendency in AVOID20 (F(2,12)=2.63, P= 0.11, but t=2.97, P~0.02 v,s vehicle group), as well as by significant reductions of LATlO (F(2,12)=3.23, P=O.O8; P~0.05 vs vehicle group, Duncan’s test) and LAT20 (F(2,12)=4.8, P~0.04; P~0.05 vs vehicle group, Duncan’s test). In experiment 2 (Table I), all ethanol doses increased %EOA (F(4,24)=7.5, PC 0.001) and %TOA (F(4,24)=17.4, P
ikk!anS~SEM
Exp. 1 Alprazolam (mg kg-‘) Vehicle 0.75
TE
%EOA
%TOA
AVOID10
AVOID20
LATI 0
LAT20
16.6 (1.8) 11.6 (2.9)
26.2 (5.3) 48.8* (4.7) 46.4 (9.0)
17.5 (4.9) .57.7* (10.9) 38.3 (12.3)
1.2 (0.4) 1.5 (0.5)
3.0 (1.0) 4.7 (1.9) 6.8 (0.8)
19.8 (4.8) 14.2 (2.6) 9.5* (0.5)
18.7 (2.7) 13.5 (3.2)
9.4 (1.1) 21.0* (2.3) 26.0* (3.8) 26.7* (4.0) 13.2 (2.5)
21.9 (2.3) 56.3* (3.9) 60.3* (1.1) 60.5* (3.4) 47.9* (19.3)
7.1 (1.3) 56.9* (6.0) 58.5* (1.5) 66.0* (3.2) 52.0* (16.7)
1.7 (0.6) 3.0 (0.8) 5.6* (1.7) 5.1* (1.4) 1.6 (0.4)
13.0 (0.8) 12.3 (1.0) Il.7 (0.9) 15.1 (1.7) 32.3* (3.5)
12.1 (1.0) Il.5 (0.7) 11.3 (0.7) 13.0 (1.4) 31.2* (4.1)
1.75
Exp 2 Ethanol (g kg-’ ) Vehicle I
2 3 4
Table I of plus-maze and shuttlebox acquisition
*P
test).
1.6 (0.7) 2.0 (0.7) 1.7
(0.6) 0.6 (0.2)
pharmacological
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41
AVOID20 (F(4,24)=2.9, P
Table II between sbuttlebox acquisition and performance in the elevated plus-maze test
Correlations 1 1. TE 2. %EOA 3. %TOA 4. AVOID10 5. AVOID20 6. LATlO 7. LAT20
-0.29 0.41t 0.65t 0.38t 0.6lj 0.20 0.44* 0.19 0.16 -0.05 0.20 -0.09
2
3
4
5
6
7
0.11 0.86-t -0.16 0.23 0.01 0.23 -0.22 -0.33* -0.26 -0.36*
0.43+ 0.31* 0.41+ 0.25 -0.15 -0.18 -0.12 -0.16
0.75t 0.70t -0.20 -0.44t -0.10 -0.34*
-0.15 -0.35* -0.28 -0.41*
-0.897 -0.94t
-
Upper values are control animals; n=23 because an additional control group, treated and tested as control rats from experiment 2 and during the same two-week period, has been included in order to increase the number of animals for performing correlations. Lower values are all the animals (n=44; vehicle- plus drug-treated groups) used in the present two experiments. +P
Table III Factor (principal-components) analysis between plus-maze measures from control animals and from the whole sample Control
Variables (loadings) TE %EOA %TOA AVOID10 AVOID20 LATlO LAT20 Percentage of explained variance
animals ._.~ 1
-
and shuttlebox (‘All animals’) All animals
I
0.15 -0.08 0.74 0.87 0.82 -0.30 -0.21
0.42 0.72 0.66 0.69 0.67 -0.74 -0.74
30%
45%
‘Control animals’ (n=23) and ‘all animals’ (n=44) as in Table II. Symbols and II. Loadings 2.30 are considered as significant.
as in Tables
I
DISCUSSION As in previous studies, which involved only shuttlebox avoidance testing ([4-71, and two more unpublished replications), alprazolam (a well known anxiolytic triazolobenzodiazepine) improved shuttlebox avoidance acquisition (by increasing avoidances and reducing latencies) and also showed the expected [30] anxiolytic effects in the plus-maze test. Since alprazolam (Experiment 1) was also included to test the reliability of the present (repeated testing) design, the results indicate that the present procedure (i.e. plus-maze testing plus shuttlebox avoidance testing) retains the sensitivity of both tests (as when they are used independently) for detecting the anxiolytic effects of drugs [4-7, 29, 301, and thus it is unlikely that the previous plus-maze administration could have produced artifactual influences on shuttlebox testing. On the other hand, the obvious advantage of the present procedure is that of testing the predicted possible correlations between the performance in both anxiety models (see below). The effects of ethanol during the process of acquisition of shuttlebox avoidance in random bred rats were studied by Chesher [38]. However, in that study, rats were tested in six consecutive IO-trial shuttlebox avoidance sessions (24-h interval between sessions [38]) after ethanol pretreatment (one injection of 1.5 or 2 g kg-’ i.p., in Wistar or Sprague-Dawley rats respectively, before each test). The results indicated that ethanol improved avoidance behavior only after the third training session [38]. In a different study Prune11 et al. [39] reported that acute ethanol (2 g kg-’ p.o.) shortened avoidance latencies in overtrained rats (their performance being 100% of avoidances when the treatment was administered). But, due to the procedures used, none of those studies provided evidence on the issue of whether ethanol could improve shuttlebox avoidance acquisition during its very early stages (as benzodiazepines do [4-7]), since it is during that phase when the conflict present in the task involves the maximum anxiety levels, and the
improving effects of environmental or pharmacological anxiolytic treatments are more evident [4,7, 10, II, 20, 211. Thus, the present study is, to our knowledge, the first dose-response study reporting positive effects of ethanol in the early acquisition (i.e. in a single short session) of shuttlebox avoidance, this being in agreement with the contention that moderate doses of ethanol display anxiolytic-like effects. It is, however, clear that the highest ethanol dose (4 g kg-‘) induces differential effects in both tests (i.e. it is anxiolytic in the plus-maze but has detrimental effects in shuttlebox acquisition), which is probably due to the different behavioural requirements involved in each situation, particularly to the fact that the perceptual and associative processes involved in the shuttlebox task (but not in the plus-maze) are probably disrupted by such a high ethanol dose. The moderate (but significant) correlations observed between plus-maze and shuttlebox acquisition measures (Table I), as well as their association in the principal-components analysis (Table Il), provide additional support for the hypothesis that the early acquisition of shuttlebox avoidance is an anxietymediated behaviour [l-7, 22-261. The most relevant associations between both tests indicate that, in control animals (n=23) AVOID10 and AVOID20 are positively correlated with %TOA, and, confirming this, the three measures have high loadings (0.74 to 0.87) of the same sign in the factor analysis. The consistency of those relationships is further confirmed by the fact that, in the whole sample of animals from both experiments (n=44), AVOID10 and %TOA are positively correlated and have positive loadings in the factor analysis (0.69 and 0.66, respectively), whereas, congruently, LATlO and LAT20 are inversely correlated with %EOA and also load inversely in the factor analysis (-0.74, -0.74 and 0.72. respectively). Also interesting is the fact that in control animals AVOID10 and AVOID20 correlated positively with TE, and the three measures loaded positively in the factor analysis (0.87, 0.82 and 0.75, respectively). However, those relationships became non-significant (see Table I) when including the drug-treated animals (n= 44), thus suggesting that there is little (if any) relationship between the effects of the drugs on general activity (i.e. TE) and their action on avoidance acquisition. This agrees with results from several previous studies which have indicated that shuttlebox avoidance acquisition cannot be completely accounted for by general activity levels 14, 5, 7, 20, 221. The within-test correlations show essentially the patterns that should be expected according to previous studies (see [4, 26, 27-29]), that is: (I) there are mostly positive correlations between the three plus-maze measures and positive loadings in the factor analysis (see [27-291); and (2) there are overall negative correlations between latency and avoidance parameters and inverse loadings of these measures in the factor analysis (see [4, 251). The only relevant exception to these patterns is the absence of correlation between %EOA and %TOA in control animals, which is probably due to the fact that %EOA scores in control animals were clearly higher than %TOA scores (Table I), whereas the values of both measures were overall much closer to each other in drug-treated rats (i.e. treatments led to similar within-group values in both %EOA and %TOA; see Table I). On the other hand, as indicated by pharmacological studies, both
44
Pharmacological Research, Vol. 29, No. 1, 1994
measures (i.e. %EOA and %TOA) are not always associated since experimental conditions can affect them differently [29]. Therefore, both the correlational and factor analyses (Tables I, II) show overall a positive association between behaviour in the open arms of the plus-maze and efficiency in shuttlebox acquisition. Thus, to summarize, two main new findings are reported in the present study which provide further evidence supporting the early acquisition of shuttlebox avoidance as an animal model of anxiety: (1) ethanol, like benzodiazepines, improves the early shuttlebox avoidance acquisition in rats and (2) positive relationships are shown between behaviour in the open arms of the plus-maze and the ability to solve the initial stages of the shuttlebox avoidance acquisition task.
ACKNOWLEDGEMENTS This work was supported by the DGICYT (PM88-007.5, PM92/0071). M.P. received a grant from the ‘Consejeria de Education de1 Gobierno de las Islas Canarias’, and R.M.E. was supported by the FISSs (92/0712).
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