Pharmacological Reports Copyright © 2012 2012, 64, 11161125 by Institute of Pharmacology ISSN 1734-1140 Polish Academy of Sciences
Tolerance liability of diazepam is dependent on the dose used for protracted treatment
Jovana Divljakoviæ, Marija Miliæ, Tamara Timiæ, Miroslav M. Saviæ
Department of Pharmacology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11221 Belgrade, Serbia
Correspondence: Miroslav M. Saviæ, e-mail: [email protected]
Abstract: Background: Behavioral effects of benzodiazepines following repeated exposure vary according to the intrinsic efficacy of the ben- zodiazepine studied, treatment schedule and the behavioral parameters evaluated. Methods: We applied the behavioral paradigms of spontaneous locomotor activity, elevated plus maze and grip strength to investi- gate the sedative, anxiolytic and myorelaxant effect of acute challenge with 2 mg/kg diazepam administered after 14 days of protracted treatment with 0.5, 2 or 10 mg/kg of diazepam. In addition, we studied the effects of everyday handling and intraperito- neal (ip) administration on animal behavior. Results: Tolerance to the sedative effect of 2 mg/kg diazepam ensued after 14 days of protracted treatment with 2 and 10 mg/kg of diazepam. In contrast, treatment with the lowest dose (0.5 mg/kg) of diazepam resulted in potentiation of the sedative effect of acute challenge with 2 mg/kg diazepam thus confounding the detection of the anxiolytic effect of diazepam. A sensitization-like response to the anxiolytic action of 2 mg/kg diazepam was seen after protracted treatment with the intermediate dose (2 mg/kg); however, anx- iolytic effect was absent after protracted administration of the highest dose. Partial tolerance to the muscle relaxant effect of 2 mg/kg diazepam ensued after protracted treatment with diazepam regardless of the dose. Daily handling or ip administration did not alter the behavioral response to acute challenge with 2 mg/kg diazepam in all the three behavioral paradigms studied. Conclusion: The presented results showed that behavioral effects of acute challenge with diazepam were differently affected by the dose administered during protracted treatment.
Key words: spontaneous locomotor activity, elevated plus maze, grip strength, tolerance
Introduction abuse potential [3, 30, 34]. Despite the efforts under- taken to clarify these phenomena, the molecular and neurobiological mechanisms underlying tolerance, Benzodiazepines were introduced into clinical prac- physical and psychological dependence to the effects tice half a century ago and since then have been of benzodiazepines are still controversial [22, 34]. widely used as anxiolytics, sedatives, hypnotics, mus- Development of tolerance, defined as a reduced re- cle relaxants and anticonvulsants. The rapid onset of sponsiveness to a drug after repeated administration, action, reasonable side-effects profile and good toler- is an adaptive response of the body to the drug [17]. ability make the benzodiazepines the drugs of first Tolerance to the various behavioral effects of benzo- choice for short-term use. However, their long-term diazepines develops at the different rate and to differ- use is hampered by the development of tolerance, li- ent extent [14], which might involve distinct mecha- ability for physical and psychological dependence and nisms and/or different subtypes of GABAA receptors
1116 Pharmacological Reports, 2012, 64, 11161125 Protracted treatment and behavioral effects of diazepam Jovana Divljakoviæ et al.
[6, 7, 29]. Namely, benzodiazepines act as positive al- (10 mg/kg) [24, 25]. The behavioral paradigms used losteric modulators at four populations of GABAA re- were spontaneous locomotor activity test, the elevated ceptors, containing the a1, a2, a3 or a5 subunit, and plus maze and the grip strength test, which were con- the functional alterations of GABAA receptors have sidered primarily predictive of the sedative, anxio- been reported after protracted treatment [10, 35]. In- lytic, and myorelaxant effects of benzodiazepines, re- volvement of different subtypes of GABAA receptors spectively. Since animal behavior may be affected by in these adaptive neuronal changes is a matter of handling and previous experimental experience [26], intense investigation [22]. As an example, it was the other aim of the present study was to determine pointed to a critical role of GABAA receptors contain- the influence of daily handling and ip route of admini- ing the a5- in conjunction with the a1-subunit in the stration on the different aspects of rat behavior and as- development of tolerance to the sedative effect of sess applicability of the chosen behavioral paradigms. diazepam [31]. Furthermore, there is an evidence that long-term activation of a1-containing GABAA recep- tors is essential for the development of tolerance to anticonvulsant effect [1, 12]. Finally, tolerance to the Materials and Methods analgesic effect of a positive modulator of GABAA receptors with neutral efficacy at a1-containing and a partial positive efficacy at a2-, a3-, and a5- containing Experiments were carried out on 66 male Wistar rats, GABAA receptors has not been demonstrated after weighing 220–250 g on the test day. The animals repeated exposure to the ligand [16]. were group-housed (six per cage) in the standard The effects of benzodiazepines after protracted laboratory conditions with food and water available administration vary considerably as a function of ad libitum. The temperature of the animal room was treatment schedule. The duration of treatment, route 22 ± 1°C, the relative humidity 40–70%, the illumina- of administration, dose, and the behavioral paradigm tion 120 lx, and 12/12 h light/dark period (light on at or model studied might affect the long-term treatment 6:00 a.m.). All handling, daily administration of treat- effects and anxiolytic effects of benzodiazepines. ment and testing took place during the light period, A number of studies suggest that tolerance to the anx- between 9:00 a.m. and 1:00 p.m. All procedures in iolytic effect of diazepam may occur when longer the study conformed to EEC Directive 86/609 and duration of treatment (about 3 weeks) and continuous were approved by the Ethical Committee on Animal exposure to a high-dose applied [4, 6, 9]. In contrast Experimentation of the Faculty of Pharmacy in to these studies, there are reports which have demon- Belgrade. strated that long-term treatment with diazepam does Diazepam (Galenika, Serbia) was suspended in not induce tolerance to its anxiolytic effects [28], or a solvent containing 85% distilled water, 14% propyl- even may potentiate them [15]. On the other hand, tol- ene glycol, and 1% Tween 80. On the beginning of erance to the sedative effect of benzodiazepines experiment, animals were randomly distributed reportedly develops in a few (3–7) days [6, 9]. There- among five treatment groups, as shown in Table 1. fore, it can be hypothesized that the different patterns of adaptive mechanisms may be triggered depending on the treatment schedule, giving rise to various pos- Tab. 1. Protracted treatment groups sible outcomes – tolerance to the observed effect, no changes, or even sensitization to this effect. Treatment code Protracted treatment Acute challenge The aim of the present study was to assess the be- (days 1–14) (day 15) havioral effects of acute challenge with 2 mg/kg of SOL-SOL solvent solvent diazepam in rats after protracted daily treatment with one of the three selected doses of this high-efficacy, SOL-DZP 2 solvent 2 mg/kg diazepam non-selective positive allosteric modulator of GABAA DZP 0.5-DZP 2 0.5 mg/kg diazepam 2 mg/kg diazepam receptors. The doses of diazepam used during pro- tracted treatment spanned the range from the beha- DZP 2-DZP 2 2 mg/kg diazepam 2 mg/kg diazepam viorally subeffective (0.5 mg/kg), over the acutely ef- DZP 10-DZP 2 10 mg/kg diazepam 2 mg/kg diazepam fective (2 mg/kg) to the acutely incapacitating dose
Pharmacological Reports, 2012, 64, 11161125 1117 Rats from the three of these groups were protractedly in the experimental room was provided with one red treated with diazepam at the doses of 0.5 mg/kg, neon tube fixed on the ceiling above the maze. Light 2 mg/kg or 10 mg/kg in a volume of 1 ml/kg ip once intensity was 10 lx on the surface of the closed arms. daily, during 14 consecutive days. The other two At the beginning of the experiment, single rats were groups received solvent, ip once daily, during this placed in the centre of the maze, facing one of the en- period. On the test day, the animals from the diaze- closed arms and allowed to freely explore the appara- pam-treated groups and from one of the solvent tus during 5 min. Animal’s behavior was recorded and groups were acutely challenged with 2 mg/kg diaze- analyzed using ANY-maze software. The choice of pam 5 min before the beginning of successive testing behavioral parameters in the elevated plus maze test in three behavioral tasks. The rats from the other was made in accordance with factor analysis per- solvent-treated group received an additional dose of formed by several groups of authors. The parameters solvent on the testing day. Such a short interval negatively correlated with anxiety are percentage of between dosing and testing was chosen in accordance open arm entries (i.e., 100 × open arm entries/total with the fast onset of diazepam action (Cmax » 10 min) entries) percentage of time spent on the open arms and its relatively short plasma and cerebrospinal fluid (i.e., 100 × time spent in open arms/300) and time half-lives in rats [33]. The sequence of tasks (sponta- spent in distal part of open arms. Additionally, the neous locomotor activity – SLA – test followed by the time in the central square is probably related to deci- elevated plus maze – EPM, and then by assessment of sion making process and/or risk assessment behavior the grip strength) was chosen to proceed from less to [2, 21]. Furthermore, we analyzed the motor activity- more invasive procedures. In order to assess the influ- related parameters, namely total distance travelled ence of the solvent administration and daily handling and the number of closed arm entries. The distal part on the animal’s behavior, two additional groups were of open arms was a virtually set area of the most dis- studied, where the rats were exposed to single ip ad- tant 30% of open arms. An entry into an open arm, ministration of 2 mg/kg diazepam or solvent, without closed arm, or distal part of open arm was scored any previous manipulations. when 90% of the animal crossed the virtual line sepa- rating zones, whereas an exit occurred when more SLA test than 90% of the animal left the respective zone. An animal was considered to be in the central square, Rats were placed in individual Plexiglas chambers when it was not in any other zone. (40 × 25 × 35 cm) for the measurement of motor activ- ity. Animal behavior under dim red light was recorded Grip strength for a total of 20 min without any habituation period. The central 20% of the chamber was virtually set as Muscle strength was assessed by the grip strength me- a central zone. The minimum percentage of the ani- ter (Ugo Basile, Milan, Italy, model 47105). The mal that must have been in the zone for an entry to oc- measurement was conducted by allowing the animals cur was set at 70%, and 50% of the animal must have to grasp the device with forelimbs and then they were remained in the zone for an exit not to occur. Using gently pulled by the tail until releasing the grid. The the ANY-maze tracking system (Stoelting Co., Wood apparatus measured the peak force of experimenter’s Dale, IL, USA) the total distance travelled and the pull (in g) necessary to overcome the strength of the distances travelled in the central and in the peripheral animal’s forelimbs grip. The grip strength was calcu- zone of the chamber were measured. lated as the median value of three consecutive trials corrected for body weight and expressed in grams per EPM test kg of animal’s weight.
The apparatus was constructed of sheet metal, with Statistical analysis a black rubber floor. It consisted of two opposed open arms (50 × 10 cm) with ledges (0.3 cm high) and two All numerical data presented in the figures were given opposed enclosed arms (50 × 10 × 40 cm), connected as the mean ± SEM. Data obtained from the experi- by the junction area (10 × 10 cm). The whole appara- ment with repeated administration of treatment were tus was elevated 50 cm above the floor. Illumination assessed by a one-way ANOVA. If the ANOVA was
1118 Pharmacological Reports, 2012, 64, 11161125 Protracted treatment and behavioral effects of diazepam Jovana Divljakoviæ et al.
Fig. 1. The influence of different treatments on the SLA test. Graphs a) and b) represent the acute effects of treatment with solvent (SOL) or 2 mg/kg diazepam (DZP 2) in naive (gray bars) and protracted solvent-treated (dark gray bars) rats. Data are represented as total distance travelled (a) and distance travelled in the central (hatched bars) and peripheral zone (open bars) of the activity chamber (b). The lines above bars represent the significant main effect of factor treatment (SOL vs. DZP 2). Graphs c) and d) represent the acute effects of SOL or DZP 2 administered to rats that received 14 days protracted treatment with SOL (SOL-SOL and SOL-DZP 2), 0.5 mg/kg (DZP 0.5-DZP 2), 2 mg/kg (DZP 2-DZP 2), or 10 mg/kg (DZP 10-DZP 2) of DZP. Data are represented as total distance travelled (c) and distance travelled in the central (hatched bars) and peripheral zone (open bars) of the activity chamber (d). * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the respective control group (SOL or SOL-SOL). The number of animals per treatment was 8 to 10
significant (p < 0.05), post-hoc Tukey’s test was per- Results formed. The two-way ANOVA was used to assess the influence on behavioral parameters of the handling accompanying daily solvent exposure vs. non- SLA test handling, in rats given either solvent or diazepam prior testing. The animals that fell from the EPM ap- As shown in Figure 1a and 1b, the single administra- paratus were excluded from further data analysis. Sta- tion of 2 mg/kg diazepam induced a significant activity- tistical analyses were performed with ANY-maze depressing effect in the SLA test. According to two- software, where applicable, while SigmaPlot 11.0 way ANOVA, the single administration of diazepam (Systat Software Inc., San Jose, CA, USA) was used (treatment as a factor) decreased the total distance elsewhere. travelled [F (1, 32) = 12.861, p = 0.001; Fig. 1a]. Fig-
Pharmacological Reports, 2012, 64, 11161125 1119 Fig. 2. The influence of different treatments on the EPM test. Graphs a), b), c) and d) represent the acute effects of treatment with solvent (SOL) or 2 mg/kg diazepam (DZP 2) in naive (gray bars) and protracted solvent-treated (dark gray bars) rats. Data are represented as percent time in the open arms (a), percent of open arm entries (b), time in distal part of open arms (c), and time in the central square (d) of the EPM. The lines above bars represent the significant main effect of factor treatment (SOL vs. DZP 2). Graphs e), f), g) and h) represent the acute effects of SOL or DZP 2 administered to rats that received 14 days protracted treatment with SOL (SOL-SOL and SOL-DZP 2), 0.5 mg/kg (DZP 0.5-DZP 2), 2 mg/kg (DZP 2-DZP 2), or 10 mg/kg (DZP 10-DZP 2) of DZP. Data are represented as percent of open time in the open arms (e), percent of open arm entries (f), time in distal part of open arms (g), and time in the central square (h); * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the respective control group (SOL or SOL-SOL); † p < 0.05 compared to DZP 10-DZP 2 group. The number of animals per group was 8–10
1120 Pharmacological Reports, 2012, 64, 11161125 Protracted treatment and behavioral effects of diazepam Jovana Divljakoviæ et al.
ure 1b shows that diazepam-induced decrease in dis- mg/kg diazepam between groups protractedly treated tance travelled was more prominent in the peripheral with diazepam (DZP 0.5-DZP 2, DZP 2-DZP 2 and zone [F (1, 32) = 13.418, p < 0.001] than in the central DZP 10-DZP 2) and solvent (SOL-DZP 2) in any of zone [F (1, 32) = 7.261, p = 0.011] of activity cham- the SLA measures. ber. There was not a significant influence of repeated administration of solvent accompanied with daily EPM test handling on any of the three analyzed parameters, and interaction between factors (treatment × handling) did As shown in Figure 2a, 2b and 2c, the single admini- not reach statistical significance. stration of 2 mg/kg diazepam induced a significant As presented in Figure 1c and 1d, the influence of anxiolytic-like effect, indicated by increases in the protracted administration of different doses of diaze- percentage of time spent in the open arm [F (1, 31) = pam on the effect of acute challenge with this benzo- 5.426, p = 0.027], percentage of open arm entries diazepine on total distance travelled in the SLA test [F (1, 31) = 5.231, p = 0.029] and the time spent in the varied between groups. The one-way ANOVA re- distal part of open arms [F (1, 31) = 9.724, p = 0.004], vealed a significant main effect of treatment on the to- compared to control. Diazepam did not affect the time tal distance travelled during 20 min of monitoring spent in the central square of the EPM [F (1, 31) = [F (4, 45) = 6.675, p < 0.001]. According to Tukey’s 1.037, p = 0.316; Fig. 2d]. The influence of 2 mg/kg test, administration of the test dose of diazepam diazepam on the total distance travelled and closed (2 mg/kg) decreased the total distance travelled only arm entries, which are both related to activity (Tab. 2), in groups exposed to solvent for 14 days (SOL-DZP was not significant (statistics not shown). According 2) or 0.5 mg/kg diazepam (DZP 0.5-DZP 2) (p = 0.019 to two-way ANOVA, handling accompanied with the and p < 0.001 related to SOL-SOL group, respectively; daily administration of solvent, as a factor, was not Fig. 1c). Figure 1d shows a significant influence of statistically significant for any of the analyzed pa- treatment on the distance travelled in the central zone rameters in the EPM test; the interactions between [F (4, 45) = 4.006, p = 0.007] and distance travelled in factors were also not statistically significant. the peripheral zone [F (4, 45) = 7.231, p < 0.001]. Figure 2e, 2f and 2g show the influence of repeated Post-hoc test revealed that 2 mg/kg diazepam signifi- administration of different doses of diazepam on the cantly decreased the distance travelled in the central effect of acute challenge with this benzodiazepine on zone of the chamber only in the group protractedly the anxiety-related parameters. The one-way ANOVA treated with the lowest dose of diazepam (0.5 mg/kg). revealed a significant effect of treatment on the per- The diazepam-induced decrease in the distance trav- centage of time spent on open arms [F (4, 41) = elled in the peripheral zone of chamber was observed 2.6271, p = 0.048], the percentage of open arm entries in all treatment groups, the respective p values for [F (4, 41) = 2.6158, p = 0.049] and the time spent in SOL-DZP 2, DZP 0.5-DZP 2, DZP 2-DZP 2 and DZP the distal part of open arms [F (4, 41) = 3.9654, p = 10-DZP 2 groups being p = 0.014, p < 0.001, p = 0.008]. Post-hoc tests revealed that administration of 0.048 and p = 0.041, respectively, related to SOL- 2 mg/kg diazepam on the test day after 14 days of treat- SOL group. The post-hoc test revealed that there were ment with the same diazepam dose (DZP 2-DZP 2 no differences in the effect of acute challenge with 2 group) significantly increased the percentage of time
Tab. 2. The acute effects (mean ± SEM) of treatment with solvent or 2 mg/kg diazepam in naive (SOL or DZP 2) and protracted solvent-treated rats (SOL-SOL, SOL-DZP 2, DZP 0.5-DZP 2, DZP 2-DZP 2, DZP 10-DZP 2) on the activity-related parameters in the EPM test
SOL DZP 2 SOL- SOL SOL- DZP 2 DZP 0.5- DZP 2 DZP2- DZP2 DZP10- DZP2 Total distance 8.67 ± 0.96 10.02 ± 1.31 10.32 ± 0.75 10.34 ± 0.85 6.86 ± 1.25 10.22 ± 1.00 9.59 ± 0.90 travelled (m) Closed arm 6.00 ± 0.68 6.25 ± 0.70 7.40 ± 0.62 6.22 ± 0.83 3.00 ± 0.60 ***, + 4.56 ± 0.73* 5.50 ± 0.45 entries
* p < 0.05, *** p < 0.001 compared to the control group (SOL- SOL); + p < 0.05 compared to SOL-DZP2 group
Pharmacological Reports, 2012, 64, 11161125 1121 spent in the open arms (p = 0.045) and time spent in tracted treatment with the lowest (0.5 mg/kg) and the the distal part of open arms (p = 0.003), and also intermediate dose (2 mg/kg) significantly decreased tended to increase the percentage of open arm entries the closed arm entries compared to the control group (p = 0.084), compared to the control group (SOL- (p < 0.001 and p = 0.026, respectively). Moreover, the SOL). There were no differences in the effect of acute number of closed arm entries was significantly de- challenge with diazepam (2 mg/kg) between groups creased in the group protractedly treated with the low- protractedly treated with diazepam (DZP 0.5-DZP 2, est dose of diazepam (DZP 0.5-DZP 2) in comparison DZP 2-DZP 2 and DZP 10-DZP 2) and solvent with SOL-DZP 2 group (p = 0.014) (Tab. 2). (SOL-DZP 2) for any of the anxiety-related parame- ters. Grip strength test Figure 2h presents a significant effect of repeated ad- ministration of diazepam on the time spent in the central The data obtained from the grip strength test are illus- square [F (4, 41) = 4.2722, p = 0.006]. According to the trated in Figure 3. The two-way ANOVA showed that post-hoc test, administration of 2 mg/kg diazepam to the single administration of 2 mg/kg diazepam signifi- group repeatedly exposed to the same dose of the benzo- cantly reduced the grip strength of rats [F (1, 31) = diazepine significantly decreased the time spent in the 24.169, p < 0.001]. Handling accompanied with daily central square, compared to the control group (p = 0.016) administration of solvent, as a factor, as well as inter- as well as to the DZP 10-DZP 2 group (p = 0.032). action between factors were not significant. The one-way ANOVAshowed that the influence of According to one-way ANOVA, the influence treatment on the total distance travelled in the EPM of treatment on the grip strength was significant test (Tab. 2) did not reach statistical significance [F (4, 41) = 3.008, p = 0.029; Fig. 3b]. Post-hoc Tuk- [F (4, 41) = 2.2507, p = 0.080]. Nonetheless, the total ey’s test revealed that acute challenge with 2 mg/kg distance travelled in the group DZP 0.5-DZP 2 was diazepam significantly decreased the grip strength (p decreased in comparison with the control group. Fur- = 0.032) only in animals repeatedly exposed to sol- thermore, the one-way ANOVA showed a significant vent (SOL-DZP 2), compared to the control (SOL- influence of treatment on the closed arm entries [F SOL group). The decrease in grip strength in DZP (4, 41) = 6.2916, p < 0.001]. Post-hoc test revealed 0.5-DZP 2 group was close to significant (p = 0.053). that acute challenge with 2 mg/kg diazepam after pro- The post-hoc test did not reveal differences in the ef-
Fig. 3. The influence of different treatments on the grip strength. Graph a) represents the acute effects of treatment with solvent (SOL) or 2 mg/kg diazepam (DZP 2) in naive (gray bars) and protracted solvent-treated (dark gray bars) rats. The line above bars represents the significant main effect of factor treatment (SOL vs. DZP 2). Graph b) represents the acute effects of SOL or DZP 2 administered to rats that received 14 days protracted treatment with SOL (SOL-SOL and SOL-DZP 2), 0.5 mg/kg (DZP 0.5-DZP 2), 2 mg/kg (DZP 2-DZP 2), or 10 mg/kg (DZP 10-DZP 2) of DZP; * p < 0.05 compared to control group (SOL-SOL), *** p < 0.001 compared to the respective control group. The number of animals per treatment was 8 to 10
1122 Pharmacological Reports, 2012, 64, 11161125 Protracted treatment and behavioral effects of diazepam Jovana Divljakoviæ et al.
fect of 2 mg/kg diazepam on the grip strength in dose also pointed to tolerance to its sedative effect. groups protractedly treated with diazepam (DZP 0.5- Nonetheless, reduction in distance travelled in the pe- DZP 2, DZP 2-DZP 2 and DZP 10-DZP 2) in com- ripheral zone of the chamber indicated a mild but de- parison with solvent (SOL-DZP 2). tectable sedative-like effect, which was less promi- nent even than sedation induced by acute administra- tion of 1.25 mg/kg diazepam [23]. On the other hand, protracted daily administration of the lowest dose of Discussion diazepam (0.5 mg/kg) was connected with a further reduction in total distance travelled and distances travelled in the peripheral and central zone of the Procedures for behavioral investigation of molecular chamber, induced by acute challenge with diazepam. mechanisms underlying the development of tolerance These findings suggest that the sedative effect of di- to the different behavioral effects elicited by benzodi- azepam may be potentiated if it is administered after azepines and related drugs that positively modulate protracted treatment with a lower dose of the same the action of GABA at different GABAA receptors are benzodiazepine. not straightforward. Behavioral test batteries reduce Development of tolerance to the anxiolytic effect the number of subjects and may be more sensitive to of benzodiazepines is a matter of debate [14]. The treatment effects in comparison to the separately per- present results indicated preserved sensitivity to the formed tests [27]. The battery of behavioral tasks in anxiolytic effect of the test dose after protracted treat- the present study comprised of the SLA test, the EPM ment with the intermediate, but not the other two test and the grip strength and was similar to some pre- doses of diazepam. The assessment of anxiolytic-like viously reported batteries, also characterized by the effects in the EPM is confounded by an increase or consecutive running of multiple tests [20]. decrease in locomotor activity [5]. In our study, the Repeated handling of animals for several days can activity-depressing effect was consistently observed change the baseline-anxiety level of solvent-treated in the group protractedly treated with the lowest dose animals in both directions [21]. Moreover, prior ma- of diazepam, and this may have caused the loss of the nipulation may modify pharmacological response to anxiolytic-like effect in this group. In the group re- certain agents even in the absence of differences in peatedly treated with the intermediate dose of diaze- the baseline-anxiety level [13, 21]. It is known that pam, closed arm entries, but not the total distance the previous ip treatment and daily handling of ani- travelled, were lower than the control values. Signifi- mals can alter behavioral response in the test such as EPM [26, 32]. The present results indicate that ip ad- cant decrease of closed arm entries in this group may ministration of solvent and gentle daily manipulation have been a consequence of the accompanying disin- of animals did not affect various aspects of behavior hibition of open arm activity. A decreased time spent (motor activity, anxiety level and grip strength) in ani- in the central square adds to the impression that the mals acutely challenged with solvent. Also, be- repeated treatment with the intermediate dose aug- havioral response to 2 mg/kg diazepam was preserved mented the anxiolytic potential of diazepam. A poten- in all three behavioral tasks employed. Notably, the tiation of anxiolytic effect observed in this group prior exposure to the SLA test did not affect the sub- could be correlated with a potentiated anxiolytic ef- sequent activity in the EPM test, when it was com- fect in the light/dark box after two weeks of repeated pared with the EPM activity of experimentally naive administration of 2 mg/kg diazepam [15]. On the animals [24]. In general, it has been suggested that di- other hand, protracted treatment with the highest dose azepam’s sedative effect is prone to rapid develop- of diazepam (10 mg/kg) was not followed by signifi- ment of tolerance (within 3 to 7 days), which is unre- cant anxiolytic effect of the acute challenge with lated to the administered dose, route of administration 2 mg/kg of diazepam, despite the fact that the motor and the brain concentration of diazepam [6, 9]. In the activity, assessed by both the SLA test and motor- present SLA test, the lack of significant decrease in activity related parameters in the EPM test, was on total distance travelled when acute challenge with the control level. Therefore, it can be concluded that 2 mg/kg of diazepam was administered in animals only the long-term treatment with 10 mg/kg diazepam protractedly treated with the equal or higher (10 mg/kg) has led to development of tolerance-like phenomenon
Pharmacological Reports, 2012, 64, 11161125 1123 to the anxiolytic effect of 2 mg/kg diazepam, devoid diazepam in any of the three behavioral paradigms of any confounding influence of motor changes. employed. The results indicated that the dose used In line with published reports, acute administration during protracted treatment differentially affected the of 2 mg/kg diazepam induced a clear myorelaxant ef- behavioral actions of 2 mg/kg diazepam, depending fect, measured by the grip strength test [11]. Only on the parameters or assays evaluated. The relation- a few studies have investigated the myorelaxant effect ship between the dose used during the long-term treat- of diazepam after repeated administration in rats. Tol- ment and sedative and anxiolytic effects of acute chal- erance to muscle relaxation has ensued after repeated lenge with 2 mg/kg diazepam was bidirectional, with administration of diazepam during 7 days in a dose elements not only of tolerance, but also sensitization range from 2.5 mg/kg to 10 mg/kg [18], while no to the effect of diazepam. It can be hypothesized that signs of tolerance were observable after 10 days of re- protracted administration of diazepam has induced peated iv administration of 10 mg/kg diazepam [19]. neurochemical changes dependent on the dose, proba- The present results indicated that the prolonged ad- bly affecting the expression of different GABAA re- ministration of diazepam has led to loss of the muscle ceptor subunits. The involvement of specific GABAA relaxant effect of acute challenge with 2 mg/kg diaze- receptor subtypes in processes underlying the devel- pam. However, the grip strength of protractedly opment of tolerance and other long-term effects of treated groups was not significantly different from benzodiazepines needs to be elucidated further. that of the acutely treated group, suggesting that par- tial tolerance to the muscle relaxant effect has devel- oped, irrespective of the diazepam dose used for pro- Acknowledgment: tracted treatment. It appears that the emergence of This work was supported by The Ministry of Science, R. Serbia – Grant No. 175076. tolerance to myorelaxant effect of diazepam is de- pendent on the duration of treatment, route of admini- stration and animal test studied, while the sedative and anxiolytic effect of benzodiazepines after pro- References: tracted administration are also under strong influence of treatment schedule. Evident discrepancies between studies that investigated the phenomenon of tolerance 1. 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