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: miroslav@pharmacy.bg.ac.rs

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

Benzodiazepines were introduced into clinical prac-

tice half a century ago and since then have been

widely used as anxiolytics, sedatives, hypnotics, mus-

cle relaxants and anticonvulsants. The rapid onset of

action, reasonable side-effects profile and good toler-

ability make the benzodiazepines the drugs of first

choice for short-term use. However, their long-term

use is hampered by the development of tolerance, li-

ability for physical and psychological dependence and

abuse potential [3, 30, 34]. Despite the efforts under-

taken to clarify these phenomena, the molecular and

neurobiological mechanisms underlying tolerance,

physical and psychological dependence to the effects

of benzodiazepines are still controversial [22, 34].

Development of tolerance, defined as a reduced re-

sponsiveness to a drug after repeated administration,

is an adaptive response of the body to the drug [17].

Tolerance to the various behavioral effects of benzo-

diazepines develops at the different rate and to differ-

ent extent [14], which might involve distinct mecha-

nisms and/or different subtypes of GABAA receptors

1116 Pharmacological Reports, 2012, 64, 1116�1125

Pharmacological Reports2012, 64, 1116�1125ISSN 1734-1140

Copyright © 2012by Institute of PharmacologyPolish Academy of Sciences

[6, 7, 29]. Namely, benzodiazepines act as positive al-

losteric modulators at four populations of GABAA re-

ceptors, containing the a1, a2, a3 or a5 subunit, and

the functional alterations of GABAA receptors have

been reported after protracted treatment [10, 35]. In-

volvement of different subtypes of GABAA receptors

in these adaptive neuronal changes is a matter of

intense investigation [22]. As an example, it was

pointed to a critical role of GABAA receptors contain-

ing the a5- in conjunction with the a1-subunit in the

development of tolerance to the sedative effect of

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

analgesic effect of a positive modulator of GABAAreceptors with neutral efficacy at a1-containing and

a partial positive efficacy at a2-, a3-, and a5- containing

GABAA receptors has not been demonstrated after

repeated exposure to the ligand [16].

The effects of benzodiazepines after protracted

administration vary considerably as a function of

treatment schedule. The duration of treatment, route

of administration, dose, and the behavioral paradigm

or model studied might affect the long-term treatment

effects and anxiolytic effects of benzodiazepines.

A number of studies suggest that tolerance to the anx-

iolytic effect of diazepam may occur when longer

duration of treatment (about 3 weeks) and continuous

exposure to a high-dose applied [4, 6, 9]. In contrast

to these studies, there are reports which have demon-

strated that long-term treatment with diazepam does

not induce tolerance to its anxiolytic effects [28], or

even may potentiate them [15]. On the other hand, tol-

erance to the sedative effect of benzodiazepines

reportedly develops in a few (3–7) days [6, 9]. There-

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-

sible outcomes – tolerance to the observed effect, no

changes, or even sensitization to this effect.

The aim of the present study was to assess the be-

havioral effects of acute challenge with 2 mg/kg of

diazepam in rats after protracted daily treatment with

one of the three selected doses of this high-efficacy,

non-selective positive allosteric modulator of GABAAreceptors. The doses of diazepam used during pro-

tracted treatment spanned the range from the beha-

viorally subeffective (0.5 mg/kg), over the acutely ef-

fective (2 mg/kg) to the acutely incapacitating dose

(10 mg/kg) [24, 25]. The behavioral paradigms used

were spontaneous locomotor activity test, the elevated

plus maze and the grip strength test, which were con-

sidered primarily predictive of the sedative, anxio-

lytic, and myorelaxant effects of benzodiazepines, re-

spectively. Since animal behavior may be affected by

handling and previous experimental experience [26],

the other aim of the present study was to determine

the influence of daily handling and ip route of admini-

stration on the different aspects of rat behavior and as-

sess applicability of the chosen behavioral paradigms.

Materials and Methods

Experiments were carried out on 66 male Wistar rats,

weighing 220–250 g on the test day. The animals

were group-housed (six per cage) in the standard

laboratory conditions with food and water available

ad libitum. The temperature of the animal room was

22 ± 1°C, the relative humidity 40–70%, the illumina-

tion 120 lx, and 12/12 h light/dark period (light on at

6:00 a.m.). All handling, daily administration of treat-

ment and testing took place during the light period,

between 9:00 a.m. and 1:00 p.m. All procedures in

the study conformed to EEC Directive 86/609 and

were approved by the Ethical Committee on Animal

Experimentation of the Faculty of Pharmacy in

Belgrade.

Diazepam (Galenika, Serbia) was suspended in

a solvent containing 85% distilled water, 14% propyl-

ene glycol, and 1% Tween 80. On the beginning of

experiment, animals were randomly distributed

among five treatment groups, as shown in Table 1.

Pharmacological Reports, 2012, 64, 1116�1125 1117

Protracted treatment and behavioral effects of diazepamJovana Divljakoviæ et al.

Tab. 1. Protracted treatment groups

Treatment code Protracted treatment(days 1–14)

Acute challenge(day 15)

SOL-SOL solvent solvent

SOL-DZP 2 solvent 2 mg/kg diazepam

DZP 0.5-DZP 2 0.5 mg/kg diazepam 2 mg/kg diazepam

DZP 2-DZP 2 2 mg/kg diazepam 2 mg/kg diazepam

DZP 10-DZP 2 10 mg/kg diazepam 2 mg/kg diazepam

Rats from the three of these groups were protractedly

treated with diazepam at the doses of 0.5 mg/kg,

2 mg/kg or 10 mg/kg in a volume of 1 ml/kg ip once

daily, during 14 consecutive days. The other two

groups received solvent, ip once daily, during this

period. On the test day, the animals from the diaze-

pam-treated groups and from one of the solvent

groups were acutely challenged with 2 mg/kg diaze-

pam 5 min before the beginning of successive testing

in three behavioral tasks. The rats from the other

solvent-treated group received an additional dose of

solvent on the testing day. Such a short interval

between dosing and testing was chosen in accordance

with the fast onset of diazepam action (Cmax » 10 min)

and its relatively short plasma and cerebrospinal fluid

half-lives in rats [33]. The sequence of tasks (sponta-

neous locomotor activity – SLA – test followed by the

elevated plus maze – EPM, and then by assessment of

the grip strength) was chosen to proceed from less to

more invasive procedures. In order to assess the influ-

ence of the solvent administration and daily handling

on the animal’s behavior, two additional groups were

studied, where the rats were exposed to single ip ad-

ministration of 2 mg/kg diazepam or solvent, without

any previous manipulations.

SLA test

Rats were placed in individual Plexiglas chambers

(40 × 25 × 35 cm) for the measurement of motor activ-

ity. Animal behavior under dim red light was recorded

for a total of 20 min without any habituation period.

The central 20% of the chamber was virtually set as

a central zone. The minimum percentage of the ani-

mal that must have been in the zone for an entry to oc-

cur was set at 70%, and 50% of the animal must have

remained in the zone for an exit not to occur. Using

the ANY-maze tracking system (Stoelting Co., Wood

Dale, IL, USA) the total distance travelled and the

distances travelled in the central and in the peripheral

zone of the chamber were measured.

EPM test

The apparatus was constructed of sheet metal, with

a black rubber floor. It consisted of two opposed open

arms (50 × 10 cm) with ledges (0.3 cm high) and two

opposed enclosed arms (50 × 10 × 40 cm), connected

by the junction area (10 × 10 cm). The whole appara-

tus was elevated 50 cm above the floor. Illumination

in the experimental room was provided with one red

neon tube fixed on the ceiling above the maze. Light

intensity was 10 lx on the surface of the closed arms.

At the beginning of the experiment, single rats were

placed in the centre of the maze, facing one of the en-

closed arms and allowed to freely explore the appara-

tus during 5 min. Animal’s behavior was recorded and

analyzed using ANY-maze software. The choice of

behavioral parameters in the elevated plus maze test

was made in accordance with factor analysis per-

formed by several groups of authors. The parameters

negatively correlated with anxiety are percentage of

open arm entries (i.e., 100 × open arm entries/total

entries) percentage of time spent on the open arms

(i.e., 100 × time spent in open arms/300) and time

spent in distal part of open arms. Additionally, the

time in the central square is probably related to deci-

sion making process and/or risk assessment behavior

[2, 21]. Furthermore, we analyzed the motor activity-

related parameters, namely total distance travelled

and the number of closed arm entries. The distal part

of open arms was a virtually set area of the most dis-

tant 30% of open arms. An entry into an open arm,

closed arm, or distal part of open arm was scored

when 90% of the animal crossed the virtual line sepa-

rating zones, whereas an exit occurred when more

than 90% of the animal left the respective zone.

An animal was considered to be in the central square,

when it was not in any other zone.

Grip strength

Muscle strength was assessed by the grip strength me-

ter (Ugo Basile, Milan, Italy, model 47105). The

measurement was conducted by allowing the animals

to grasp the device with forelimbs and then they were

gently pulled by the tail until releasing the grid. The

apparatus measured the peak force of experimenter’s

pull (in g) necessary to overcome the strength of the

animal’s forelimbs grip. The grip strength was calcu-

lated as the median value of three consecutive trials

corrected for body weight and expressed in grams per

kg of animal’s weight.

Statistical analysis

All numerical data presented in the figures were given

as the mean ± SEM. Data obtained from the experi-

ment with repeated administration of treatment were

assessed by a one-way ANOVA. If the ANOVA was

1118 Pharmacological Reports, 2012, 64, 1116�1125

significant (p < 0.05), post-hoc Tukey’s test was per-

formed. The two-way ANOVA was used to assess the

influence on behavioral parameters of the handling

accompanying daily solvent exposure vs. non-

handling, in rats given either solvent or diazepam

prior testing. The animals that fell from the EPM ap-

paratus were excluded from further data analysis. Sta-

tistical analyses were performed with ANY-maze

software, where applicable, while SigmaPlot 11.0

(Systat Software Inc., San Jose, CA, USA) was used

elsewhere.

Results

SLA test

As shown in Figure 1a and 1b, the single administra-

tion of 2 mg/kg diazepam induced a significant activity-

depressing effect in the SLA test. According to two-

way ANOVA, the single administration of diazepam

(treatment as a factor) decreased the total distance

travelled [F (1, 32) = 12.861, p = 0.001; Fig. 1a]. Fig-

Pharmacological Reports, 2012, 64, 1116�1125 1119

Protracted treatment and behavioral effects of diazepamJovana 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) or2 mg/kg diazepam (DZP 2) in naive (gray bars) and protracted solvent-treated (dark gray bars) rats. Data are represented as total distancetravelled (a) and distance travelled in the central (hatched bars) and peripheral zone (open bars) of the activity chamber (b). The lines abovebars represent the significant main effect of factor treatment (SOL vs. DZP 2). Graphs c) and d) represent the acute effects of SOL or DZP 2administered 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 respectivecontrol group (SOL or SOL-SOL). The number of animals per treatment was 8 to 10

1120 Pharmacological Reports, 2012, 64, 1116�1125

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 inthe 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 linesabove bars represent the significant main effect of factor treatment (SOL vs. DZP 2). Graphs e), f), g) and h) represent the acute effects of SOLor 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 ofopen 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 therespective 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

ure 1b shows that diazepam-induced decrease in dis-

tance travelled was more prominent in the peripheral

zone [F (1, 32) = 13.418, p < 0.001] than in the central

zone [F (1, 32) = 7.261, p = 0.011] of activity cham-

ber. There was not a significant influence of repeated

administration of solvent accompanied with daily

handling on any of the three analyzed parameters, and

interaction between factors (treatment × handling) did

not reach statistical significance.

As presented in Figure 1c and 1d, the influence of

protracted administration of different doses of diaze-

pam on the effect of acute challenge with this benzo-

diazepine on total distance travelled in the SLA test

varied between groups. The one-way ANOVA re-

vealed a significant main effect of treatment on the to-

tal distance travelled during 20 min of monitoring

[F (4, 45) = 6.675, p < 0.001]. According to Tukey’s

test, administration of the test dose of diazepam

(2 mg/kg) decreased the total distance travelled only

in groups exposed to solvent for 14 days (SOL-DZP

2) or 0.5 mg/kg diazepam (DZP 0.5-DZP 2) (p = 0.019

and p < 0.001 related to SOL-SOL group, respectively;

Fig. 1c). Figure 1d shows a significant influence of

treatment on the distance travelled in the central zone

[F (4, 45) = 4.006, p = 0.007] and distance travelled in

the peripheral zone [F (4, 45) = 7.231, p < 0.001].

Post-hoc test revealed that 2 mg/kg diazepam signifi-

cantly decreased the distance travelled in the central

zone of the chamber only in the group protractedly

treated with the lowest dose of diazepam (0.5 mg/kg).

The diazepam-induced decrease in the distance trav-

elled in the peripheral zone of chamber was observed

in all treatment groups, the respective p values for

SOL-DZP 2, DZP 0.5-DZP 2, DZP 2-DZP 2 and DZP

10-DZP 2 groups being p = 0.014, p < 0.001, p =

0.048 and p = 0.041, respectively, related to SOL-

SOL group. The post-hoc test revealed that there were

no differences in the effect of acute challenge with 2

mg/kg diazepam between groups protractedly treated

with diazepam (DZP 0.5-DZP 2, DZP 2-DZP 2 and

DZP 10-DZP 2) and solvent (SOL-DZP 2) in any of

the SLA measures.

EPM test

As shown in Figure 2a, 2b and 2c, the single admini-

stration of 2 mg/kg diazepam induced a significant

anxiolytic-like effect, indicated by increases in the

percentage of time spent in the open arm [F (1, 31) =

5.426, p = 0.027], percentage of open arm entries

[F (1, 31) = 5.231, p = 0.029] and the time spent in the

distal part of open arms [F (1, 31) = 9.724, p = 0.004],

compared to control. Diazepam did not affect the time

spent in the central square of the EPM [F (1, 31) =

1.037, p = 0.316; Fig. 2d]. The influence of 2 mg/kg

diazepam on the total distance travelled and closed

arm entries, which are both related to activity (Tab. 2),

was not significant (statistics not shown). According

to two-way ANOVA, handling accompanied with the

daily administration of solvent, as a factor, was not

statistically significant for any of the analyzed pa-

rameters in the EPM test; the interactions between

factors were also not statistically significant.

Figure 2e, 2f and 2g show the influence of repeated

administration of different doses of diazepam on the

effect of acute challenge with this benzodiazepine on

the anxiety-related parameters. The one-way ANOVA

revealed a significant effect of treatment on the per-

centage of time spent on open arms [F (4, 41) =

2.6271, p = 0.048], the percentage of open arm entries

[F (4, 41) = 2.6158, p = 0.049] and the time spent in

the distal part of open arms [F (4, 41) = 3.9654, p =

0.008]. Post-hoc tests revealed that administration of

2 mg/kg diazepam on the test day after 14 days of treat-

ment with the same diazepam dose (DZP 2-DZP 2

group) significantly increased the percentage of time

Pharmacological Reports, 2012, 64, 1116�1125 1121

Protracted treatment and behavioral effects of diazepamJovana Divljakoviæ et al.

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-treatedrats (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 distancetravelled (m)

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

Closed armentries

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

* p < 0.05, *** p < 0.001 compared to the control group (SOL- SOL); + p < 0.05 compared to SOL-DZP2 group

spent in the open arms (p = 0.045) and time spent in

the distal part of open arms (p = 0.003), and also

tended to increase the percentage of open arm entries

(p = 0.084), compared to the control group (SOL-

SOL). There were no differences in the effect of acute

challenge with diazepam (2 mg/kg) between groups

protractedly treated with diazepam (DZP 0.5-DZP 2,

DZP 2-DZP 2 and DZP 10-DZP 2) and solvent

(SOL-DZP 2) for any of the anxiety-related parame-

ters.

Figure 2h presents a significant effect of repeated ad-

ministration of diazepam on the time spent in the central

square [F (4, 41) = 4.2722, p = 0.006]. According to the

post-hoc test, administration of 2 mg/kg diazepam to the

group repeatedly exposed to the same dose of the benzo-

diazepine significantly decreased the time spent in the

central square, compared to the control group (p = 0.016)

as well as to the DZP 10-DZP 2 group (p = 0.032).

The one-way ANOVA showed that the influence of

treatment on the total distance travelled in the EPM

test (Tab. 2) did not reach statistical significance

[F (4, 41) = 2.2507, p = 0.080]. Nonetheless, the total

distance travelled in the group DZP 0.5-DZP 2 was

decreased in comparison with the control group. Fur-

thermore, the one-way ANOVA showed a significant

influence of treatment on the closed arm entries [F

(4, 41) = 6.2916, p < 0.001]. Post-hoc test revealed

that acute challenge with 2 mg/kg diazepam after pro-

tracted treatment with the lowest (0.5 mg/kg) and the

intermediate dose (2 mg/kg) significantly decreased

the closed arm entries compared to the control group

(p < 0.001 and p = 0.026, respectively). Moreover, the

number of closed arm entries was significantly de-

creased in the group protractedly treated with the low-

est dose of diazepam (DZP 0.5-DZP 2) in comparison

with SOL-DZP 2 group (p = 0.014) (Tab. 2).

Grip strength test

The data obtained from the grip strength test are illus-

trated in Figure 3. The two-way ANOVA showed that

single administration of 2 mg/kg diazepam signifi-

cantly reduced the grip strength of rats [F (1, 31) =

24.169, p < 0.001]. Handling accompanied with daily

administration of solvent, as a factor, as well as inter-

action between factors were not significant.

According to one-way ANOVA, the influence

of treatment on the grip strength was significant

[F (4, 41) = 3.008, p = 0.029; Fig. 3b]. Post-hoc Tuk-

ey’s test revealed that acute challenge with 2 mg/kg

diazepam significantly decreased the grip strength (p

= 0.032) only in animals repeatedly exposed to sol-

vent (SOL-DZP 2), compared to the control (SOL-

SOL group). The decrease in grip strength in DZP

0.5-DZP 2 group was close to significant (p = 0.053).

The post-hoc test did not reveal differences in the ef-

1122 Pharmacological Reports, 2012, 64, 1116�1125

Fig. 3. The influence of different treatments on the grip strength. Graph a) represents the acute effects of treatment with solvent (SOL) or2 mg/kg diazepam (DZP 2) in naive (gray bars) and protracted solvent-treated (dark gray bars) rats. The line above bars represents thesignificant main effect of factor treatment (SOL vs. DZP 2). Graph b) represents the acute effects of SOL or DZP 2 administered to rats thatreceived 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 numberof animals per treatment was 8 to 10

fect of 2 mg/kg diazepam on the grip strength in

groups protractedly treated with diazepam (DZP 0.5-

DZP 2, DZP 2-DZP 2 and DZP 10-DZP 2) in com-

parison with solvent (SOL-DZP 2).

Discussion

Procedures for behavioral investigation of molecular

mechanisms underlying the development of tolerance

to the different behavioral effects elicited by benzodi-

azepines and related drugs that positively modulate

the action of GABA at different GABAA receptors are

not straightforward. Behavioral test batteries reduce

the number of subjects and may be more sensitive to

treatment effects in comparison to the separately per-

formed tests [27]. The battery of behavioral tasks in

the present study comprised of the SLA test, the EPM

test and the grip strength and was similar to some pre-

viously reported batteries, also characterized by the

consecutive running of multiple tests [20].

Repeated handling of animals for several days can

change the baseline-anxiety level of solvent-treated

animals in both directions [21]. Moreover, prior ma-

nipulation may modify pharmacological response to

certain agents even in the absence of differences in

the baseline-anxiety level [13, 21]. It is known that

the previous ip treatment and daily handling of ani-

mals can alter behavioral response in the test such as

EPM [26, 32]. The present results indicate that ip ad-

ministration of solvent and gentle daily manipulation

of animals did not affect various aspects of behavior

(motor activity, anxiety level and grip strength) in ani-

mals acutely challenged with solvent. Also, be-

havioral response to 2 mg/kg diazepam was preserved

in all three behavioral tasks employed. Notably, the

prior exposure to the SLA test did not affect the sub-

sequent activity in the EPM test, when it was com-

pared with the EPM activity of experimentally naive

animals [24]. In general, it has been suggested that di-

azepam’s sedative effect is prone to rapid develop-

ment of tolerance (within 3 to 7 days), which is unre-

lated to the administered dose, route of administration

and the brain concentration of diazepam [6, 9]. In the

present SLA test, the lack of significant decrease in

total distance travelled when acute challenge with

2 mg/kg of diazepam was administered in animals

protractedly treated with the equal or higher (10 mg/kg)

dose also pointed to tolerance to its sedative effect.

Nonetheless, reduction in distance travelled in the pe-

ripheral zone of the chamber indicated a mild but de-

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

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

chamber, induced by acute challenge with diazepam.

These findings suggest that the sedative effect of di-

azepam may be potentiated if it is administered after

protracted treatment with a lower dose of the same

benzodiazepine.

Development of tolerance to the anxiolytic effect

of benzodiazepines is a matter of debate [14]. The

present results indicated preserved sensitivity to the

anxiolytic effect of the test dose after protracted treat-

ment with the intermediate, but not the other two

doses of diazepam. The assessment of anxiolytic-like

effects in the EPM is confounded by an increase or

decrease in locomotor activity [5]. In our study, the

activity-depressing effect was consistently observed

in the group protractedly treated with the lowest dose

of diazepam, and this may have caused the loss of the

anxiolytic-like effect in this group. In the group re-

peatedly treated with the intermediate dose of diaze-

pam, closed arm entries, but not the total distance

travelled, were lower than the control values. Signifi-

cant decrease of closed arm entries in this group may

have been a consequence of the accompanying disin-

hibition of open arm activity. A decreased time spent

in the central square adds to the impression that the

repeated treatment with the intermediate dose aug-

mented the anxiolytic potential of diazepam. A poten-

tiation of anxiolytic effect observed in this group

could be correlated with a potentiated anxiolytic ef-

fect in the light/dark box after two weeks of repeated

administration of 2 mg/kg diazepam [15]. On the

other hand, protracted treatment with the highest dose

of diazepam (10 mg/kg) was not followed by signifi-

cant anxiolytic effect of the acute challenge with

2 mg/kg of diazepam, despite the fact that the motor

activity, assessed by both the SLA test and motor-

activity related parameters in the EPM test, was on

the control level. Therefore, it can be concluded that

only the long-term treatment with 10 mg/kg diazepam

has led to development of tolerance-like phenomenon

Pharmacological Reports, 2012, 64, 1116�1125 1123

Protracted treatment and behavioral effects of diazepamJovana Divljakoviæ et al.

to the anxiolytic effect of 2 mg/kg diazepam, devoid

of any confounding influence of motor changes.

In line with published reports, acute administration

of 2 mg/kg diazepam induced a clear myorelaxant ef-

fect, measured by the grip strength test [11]. Only

a few studies have investigated the myorelaxant effect

of diazepam after repeated administration in rats. Tol-

erance to muscle relaxation has ensued after repeated

administration of diazepam during 7 days in a dose

range from 2.5 mg/kg to 10 mg/kg [18], while no

signs of tolerance were observable after 10 days of re-

peated iv administration of 10 mg/kg diazepam [19].

The present results indicated that the prolonged ad-

ministration of diazepam has led to loss of the muscle

relaxant effect of acute challenge with 2 mg/kg diaze-

pam. However, the grip strength of protractedly

treated groups was not significantly different from

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-

tracted treatment. It appears that the emergence of

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-

tracted administration are also under strong influence

of treatment schedule. Evident discrepancies between

studies that investigated the phenomenon of tolerance

may be at least partly related to different experimental

conditions in the various studies. It has been sug-

gested that different treatment regimens during

chronic benzodiazepine treatment produced signifi-

cantly different adaptive changes at specific GABAAreceptors [6]. Furthermore, it has been demonstrated

that tolerance to the anxiolytic effect was favored

when continuous exposure to the drug is achieved [6].

It is worthy to note that in the present study once daily

administration of treatment was applied. Although it

is known that metabolism of diazepam is much faster

in rats than in humans, it has been found that pro-

tracted ip administration results in accumulation of di-

azepam after daily treatment for 7 days [8], which im-

plies more complete receptor occupancy than it would

be expected [33].

In conclusion, the consecutive running of three be-

havioral tests in the present study has enabled simul-

taneous assessment of the diazepam effects on the dif-

ferent aspects of rat’s behavior. Daily handling of ani-

mals or ip route of administration did not alter the

behavioral response to acute challenge with 2 mg/kg

diazepam in any of the three behavioral paradigms

employed. The results indicated that the dose used

during protracted treatment differentially affected the

behavioral actions of 2 mg/kg diazepam, depending

on the parameters or assays evaluated. The relation-

ship between the dose used during the long-term treat-

ment and sedative and anxiolytic effects of acute chal-

lenge with 2 mg/kg diazepam was bidirectional, with

elements not only of tolerance, but also sensitization

to the effect of diazepam. It can be hypothesized that

protracted administration of diazepam has induced

neurochemical changes dependent on the dose, proba-

bly affecting the expression of different GABAA re-

ceptor subunits. The involvement of specific GABAAreceptor subtypes in processes underlying the devel-

opment of tolerance and other long-term effects of

benzodiazepines needs to be elucidated further.

Acknowledgment:

This work was supported by The Ministry of Science, R. Serbia –

Grant No. 175076.

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Received: August 31, 2011; in the revised form: March 12, 2012;

accepted: May 23, 2012.

Pharmacological Reports, 2012, 64, 1116�1125 1125

Protracted treatment and behavioral effects of diazepamJovana Divljakoviæ et al.