Effects of chronic flunitrazepam treatment
schedule on therapy-induced sedation and motor impairment in mice
Sylwia Talarek, Jolanta Orzelska, Joanna Listos, Antonina Mazur, Sylwia Fidecka
Chair and Department of Pharmacology and Pharmacodynamics, Medical University of Lublin, ChodŸki 4A, PL 20-093, Lublin, Poland
Correspondence: Sylwia Talarek, e-mail: talareks@poczta.onet.pl
Abstract:
Background: The aim of the present study was to examine whether different treatment schedules could be associated with tolerance development to the ataxic and sedative effects of flunitrazepam in mice.
Methods: Effects of repeated flunitrazepam administration were studied in the rotarod and the chimney test for motor coordination and in a photocell apparatus for locomotor activity in mice. Flunitrazepam doses varied in particular types of injections or in different experiment duration periods.
Results: Repeated flunitrazepam administration (1 mg/kg, sc and 2 mg/kg, ip) for 8 consecutive days induced tolerance to the motor impairing effects of flunitrazepam in mice, both in the rotarod and the chimney test. In turn, no tolerance developed to sedative fluni- trazepam effects, regarding either dose level, injection type or treatment duration.
Conclusions: Those findings confirmed the previous observations that tolerance to benzodiazepines was not simultaneous for each pharmacological property of the drugs. Interestingly enough, an acute dose of flunitrazepam (1 mg/kg, sc) in our study enhanced lo- comotor activity of mice.
Key words:
flunitrazepam, tolerance, ataxia, sedation, treatment schedule
Introduction
Benzodiazepines (BZ) are useful therapeutic agents, prescribed for a variety of specific conditions, such as anxiety, sleep disorders or epilepsy. The drugs act on g-aminobutyric acid (GABA)Areceptors and enhance GABA-mediated neuronal inhibition [9, 35]. GABAA receptors exist as multisubunit (a16, b13, g13, d, e, j, p and r1-3), ligand-gated chloride channels [4, 29].
Unfortunately, despite the obvious advantages of BZ
as broad-spectrum drugs, they are also liable to de- velop tolerance and dependence in treated patients [21, 23].
Tolerance is defined as a gradual decrease of drug potential to produce same pharmacological effects in time [2]. The tolerance to diverse effects of BZ (ataxic, sedative, muscle relaxant, anxiolytic and anti- convulsant) after its prolonged administration is a well-documented issue in both clinical and animal studies [3, 13, 21, 23, 26]. However, the gradual re- sponse diminution to BZ, observed on time of its in-
take, is not simultaneous for each pharmacological property of the drug [3, 15, 21, 23]. For instance, the tolerance to the sedative and motor coordination affecting features of BZ develops more rapidly than that to their anticonvulsant or anxiolytic effects [3, 16, 24]. The mechanism, underlying the tolerance to BZ, is not fully understood but a growing body of evi- dence indicates its pharmacodynamic character, as it is determined by adaptive changes in the central nerv- ous system (CNS) [3, 17, 21]. Many studies have demonstrated GABAAreceptor down regulation, fol- lowing its exposure to BZ, what means that GABAA receptor’s ability to bind BZ and enhance GABA neu- rotransmission is gradually reduced in time with BZ administration [3, 17, 26, 36]. Moreover, it is thought that excitatory mechanisms (such as the glutamatergic system) are sensitized to compensate for BZ-induced chronic enhancement of GABAergic inhibition [1, 2, 6].
The purpose of the reported study was to examine the effects of different treatment schedules on toler- ance development to ataxic (motor impairing) and sedative effects of flunitrazepam (FNZ) in mice. FNZ was chosen as a full BZ agonist with highly hypnotic and sedative effects [9]. In many countries, FNZ has been reclassified into a dangerous drug. An increasing number of reports indicate FNZ abuse for its intoxi- cant and relaxant effects, often in combination with alcohol and/or illicit drugs (heroin, marijuana) [14, 28]. Moreover, FNZ is known as a date-rape drug be- cause sexual predators use it to chemically incapaci- tate their victims [14]. FNZ, as another BZ, can lead to physical dependence, addiction and to a state, known as the BZ withdrawal syndrome [8, 11, 18, 28].
In addition, a chronic intake of FNZ has been associ- ated with violent crimes, committed by people with history of violent behaviors [10].
In our experiments, we studied effects of repeated FNZ administration on animal behaviors in the rota- rod and chimney tests, which are generally accepted as motor coordination assessment tests in animals [39]. Additionally, a photocell apparatus was used to monitor tolerance development to FNZ-induced seda- tion in mice [39]. The tolerance to BZ has been re- ported for both low and high doses of the drug if dos- ing frequency and administration duration are suffi- cient [3, 15, 16, 37]. Therefore, different FNZ dosing protocols were combined with different injection types or experiment duration periods.
Materials and Methods
Animals
Male albino Swiss mice were used with an initial body weight of 20–25 g at the study onset. The animals were housed in groups of 8–12 animals and maintained in a 12 h light-dark cycle and controlled temperature (21°C). They received standard food (Murigan pellets, Bacutil, Motycz, Poland) and tap water ad libitum. All behavioral experi- ments were carried out, according to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and to the European Community Directive for the Care and Use of Laboratory of 24 November 1986 (86/609/EEC). The study protocols were approved by the Local Ethics Committee (68/2009).
Drugs
Flunitrazepam of Sigma Chemicals (St. Louis, USA), was dissolved in 0.5% Tween 80 (1–2 drops), gently warmed and then diluted with 0.9% NaCl. A control group of mice received a vehicle, consisting of 0.5%
Tween 80 (1–2 drops) and 0.9% NaCl.
Experimental procedures
Experiment 1
The mice were treated subcutaneously (sc) with FNZ in dose of 1 mg/kg for 8 consecutive days and intrape- ritoneally (ip) by FNZ (1 or 2 mg/kg) to induce toler- ance to the motor impairing effect of FNZ. Motor co- ordination of the animals was measured on the 1st, 5th and 8th day of the experiment, using the rotarod and the chimney test, 30 min after the administration of either FNZ or 0.9% NaCl [18, 33, 34].
Experiment 2
The mice were treated according to one of the follow- ing patterns: FNZ – once a day (1 mg/kg, sc, 30 min before the test; 1 mg/kg, sc, 60 min before the test;
2 mg/kg, sc, 30 min before the test) and FNZ – twice a day (2 mg/kg, sc, 30 min before the test). The treat- ments lasted 11 consecutive days.
Locomotor activity of the animals was measured on the 1st, 2nd, 5th, 8thor 11thday of the experiment, us- ing individual circular alleys, 30 or 60 min after FNZ administration [18, 34, 37].
Chronic treatment with flunitrazepam, ataxia and sedation
Sylwia Talarek et al.
Rotarod test
The rotarod test apparatus consists of a circular rod (2 cm in diameter), rotating at constant speed (18 rpm). Animals, when placed on the rotating rod, naturally try to keep their balance and remain there rather than fall onto a platform some 30 cm below.
Before drug administration, the mice were trained daily for a 3-day period. During each training session, the mice were placed on the rotating rod for 3 min, being allowed an unlimited number of trials. All the animals from training sessions were used in subse- quent experiments. Drug testing was conducted in, at least, 24 h after final training. During the test, the mice had to remain on the rotating rod for as long as possible (a 60 s test was used for the test as maxi- mum).
Chimney test
The chimney test is a simple test for tranquilizing and muscle relaxant activity and can be used as an addi- tional test with other motor coordination determining tests. The animals had to climb backwards up a plastic tube (3 cm in lumen, 25 cm long). The mice were trained once daily for 3 days before the first day of ex- periment. Motor impairment was assessed as inability
[39].
Locomotor activity
Locomotor activity of individual mice was recorded, using a photocell apparatus (a round Plexi cage, 32 cm in diameter, Multiserv, Lublin, Poland). The animals were placed in individual cages for 60 or 30 min after FNZ injection. The cages were equipped with one row of infrared light-sensitive photocells (2 emitters and 2 sensors) located 1 cm above the floor. Locomotor activity was assessed as the number of photocell interruptions during a period of 30 min.
Motor activity measurement is a standard behavioral assay in testing the sedative effects of a drug [39].
Statistical analysis
All the results in the reported experiments were ana- lyzed by one-way ANOVA (for acute FNZ effect) and by two-way ANOVA (for tolerance development as- sessment). Post-hoc comparisons were carried out by Tukey’s test. The level of p < 0.05 was considered statistically significant. The data are presented as the means ± SEM. Each group consisted of 8–12 mice.
1 5 8 1 5 8 1 5 8 1 5 8
0 10 20 30 40 50 60 70
###
###
### ###
## ###
***
*** p < 0.001 vs. 0.9% NaCl (1 day)
### p < 0.001 vs. a dose of FNZ (1 day)
0.9% NaCl FNZ
1 mg/kg sc FNZ 1 mg/kg ip
FNZ 2 mg/kg ip Day of experiment
Timeonrotarod[s]
***
***
A
1 5 8 1 5 8 1 5 8 1 5 8
0 10 20 30 40 50 60 70
### ###
0.9% NaCl FNZ 1 mg/kg sc
FNZ 1 mg/kg ip
FNZ 2 mg/kg ip
*** p < 0.001 vs. 0.9% NaCl (1 day)
### p < 0.001 vs. a dose of FNZ (1 day)
Day of experiment
Timeinchimney[s] ***
*** ***
st B
st st
st
Fig. 1. Effects of acute and chronic administration of FNZ on motor performance in the rotarod test (A) and in the chimney test (B). FNZ (1 mg/kg, sc; 1 mg/kg, ip and 2 mg/kg, ip) was injected for 8 consecutive days. The test was performed 30 min after the injection of FNZ, on the 1st, 5thand 8thday of the experiment. The results are expressed as the means ± SEM (n = 10–12 mice/group). ### p < 0.001 vs. FNZ (1stday),
*** p < 0.001 vs. 0.9% NaCl (1stday) (Tukey’s test)
Results
Effects of acute and chronic administration of FNZ on motor performance in the rotarod test (Fig. 1A)
FNZ administration in a single dose (1 mg/kg, sc;
1 mg/kg, ip and 2 mg/kg, ip) on day 1 impaired the motor coordination of mice (p < 0.001) in the rotarod test, while, the repeated (5- or 8-day) treatment of mice with FNZ (1 mg/kg, sc; 1 mg/kg, ip and 2 mg/kg, ip) reduced their motor incoordination. The two-way ANOVA showed a significant effect for both days and the drug and a significant day × drug interaction (see Tab. 1). A post-hoc comparison confirmed tolerance development to the motor impairing effect of FNZ by significant differences between the FNZ-acutely treated groups (day 1) and FNZ-chronically treated mice (5 or 8 days) (p < 0.001).
Effects of acute and chronic administration of FNZ on motor performance in mice, measured by the chimney test (Fig. 1B)
FNZ administration in a single dose (1 mg/kg, sc;
1 mg/kg, ip and 2 mg/kg, ip) on day 1 impaired the mo- tor coordination of mice (p < 0.001) in the chimney test.
The two-way ANOVA showed a significant effect of the drug and day and of the drug × day interaction for FNZ (1 mg/kg, sc and 2 mg/kg, ip). The injection of FNZ (1 mg/kg, ip) resulted in a significant interac-
tion, observed in the two-way ANOVA (see Tab. 2).
A post-hoc comparison showed significant differ- ences between the FNZ-acutely treated (1 mg/kg, sc and 2 mg/kg, ip) (day 1) and the FNZ-chronically treated (1 mg/kg, sc and 2 mg/kg, ip) mice (8 days) (p < 0.001) in the chimney test. That result clearly demonstrated tolerance development to the motor im- pairing effect of FNZ (1 mg/kg, sc and 2 mg/kg, ip) after 8 days of treatment. In turn, the post-hoc com- parison did not show any significant differences be- tween the FNZ-treated (1 mg/kg, sc and ip; 2 mg/kg, ip) groups on day 1 and the FNZ-treated (1 mg/kg, sc and ip; 2 mg/kg, ip) groups on day 5 or between FNZ treatment (1 mg/kg, ip) on day 1 and FNZ treatment (1 mg/kg, ip) on day 8 in the chimney test.
Effects of acute or chronic administration of FNZ on mice performance in the photocell test (Fig. 2A, Fig. 2B)
The acute (the 1st day of experiment) administration of FNZ (1 mg/kg, sc), 60 min before the test, in- creased locomotor activity of the mice in a photocell apparatus (p < 0.05), whereas the acute administra- tion of FNZ (1 mg/kg, sc and 2 mg/kg, sc), 30 min be- fore the test, had no influence on the locomotor activ- ity of mice in the photocell apparatus.
FNZ administration on day 2 (1 mg/kg sc; 2 mg/kg sc, 30 min before the test; 1 mg/kg sc 60 min before the test and 2 mg/kg sc, 30 min before the test but after twice daily injection) caused sedation in the
Chronic treatment with flunitrazepam, ataxia and sedation
Sylwia Talarek et al.
nd
Fig. 2. Effects of acute and chronic administration of FNZ on mouse performance in the photocell apparatus (a single injection of FNZ – A;
a double injection of FNZ – B). FNZ (1 or 2 mg/kg, sc) was administered for 11 consecutives days. The test was performed 30 or 60 min after FNZ injection, on the 1st, 2nd, 5th, 8thand 11thday of the experiment. The results are expressed as the means ± SEM (n = 10–12 mice/group).
* p < 0.05, ** p < 0.01, *** p < 0.001 vs. 0.9% NaCl (2ndday); # p < 0.05 vs. 0.9% NaCl (1stday) (Tukey’s test)
mice, measured in the photocell apparatus (p < 0.001, p < 0.01, p < 0.05 and p < 0.001, respectively).
The two-way ANOVA showed a significant effect of the drug × day interaction (FNZ 2 mg/kg, sc, once a day, 30 min before the test), drug (FNZ 2 mg/kg, sc, once a day, 30 min before the test; FNZ 1 mg/kg, sc, once a day, 30 and 60 min before the test; FNZ 2 mg/kg, twice a day, 30 min before the test) and day (FNZ 1 mg/kg, sc, once a day 60 min before the test;
FNZ 2 mg/kg, twice a day, 30 min before the test) (see Tab. 3).
However, a post-hoc comparison did not show any statistically significant differences between locomotor activity of the FNZ-treated mice on day 2 and 5, 8 or 11 measured in the photocell apparatus.
Discussion
A long-term use of BZ results in the development of tolerance to these drugs, both in animals and humans, while one of the most striking features is such that this tolerance develops at different rates with regards to different behavioral effects [3, 16, 23]. In the reported experiment, the ataxic effect of FNZ after its repeated administration was examined in the rotarod and chim- ney tests. Those tests have proven useful in the as- sessment of motor impairment induced by a number of central depressive drugs [27, 30, 32, 39]. Following our results, the acute FNZ (1 mg/kg; sc or ip and 2 mg/kg; ip) administration produced significant mo-
Tab. 3. Tabular results (two-way ANOVA) of chronic FNZ administration on mice performance, measured in the photocell apparatus
Variation source
FNZ doses and the route of injections
FNZ 1 mg/kg, sc (30 min) FNZ 1 mg/kg, sc (60 min) FNZ 2 mg/kg, sc (30 min) FNZ 2 mg/kg, sc (30 min);
twice a day Interaction F3,68= 1.189; p = 0.3204 F3,72= 0.123; p = 0.9457 F3,88= 2.95; p = 0.0371 F3,82= 1.552; p = 0.2074 Drug F1,68= 290.7; p < 0.0001 F1,72= 45.10; p < 0.0001 F1,88= 143.3; p < 0.0001 F1,82= 109.0; p < 0.0001 Day F3,68= 0.926; p = 0.4328 F3,72= 2.878; p = 0.0419 F3,88= 0.519; p = 0.6702 F3,82= 5.502; p = 0.0017 Tab. 2. Tabular results (two-way ANOVA) of chronic FNZ administration on motor performance in mice, measured by the chimney test
Variation source
FNZ doses and the route of injections
FNZ 1 mg/kg, sc FNZ 1 mg/kg, ip FNZ 2 mg/kg, ip
Interaction F2,42= 4.417; p = 0.0182 F2,42= 0.6212; p = 0.5422 F2,42= 5.469; p = 0.0077 Drug F1,42= 46.76; p < 0.0001 F1,42= 104.5; p < 0.0001 F1,42= 39.46; p < 0.0001 Day F2,42= 4.019; p = 0.0253 F2,42= 0.3076; p = 0.7368 F2,42= 4.894; p = 0.0123 Variation source
FNZ 1 mg/kg, sc FNZ 1 mg/kg, ip FNZ 2 mg/kg, ip
Interaction F2,47= 15.55; p < 0.0001 F2,47= 13.31; p < 0.0001 F2,50= 28.08; p < 0.0001 Drug F1,47= 88.99; p < 0.0001 F1,47= 53.05; p < 0.0001 F1,50= 79.37; p < 0.0001 Day F2,47= 14.44; p < 0.0001 F2,47= 12.72; p < 0.0001 F2,50= 26.67; p < 0.0001
tor impairments in mice in both tests, whereas the chronic administration of FNZ in the same doses brought tolerance to the motor impairing effect of FNZ, observed on day 5 and day 8 of the experiment.
Additionally, two different injection routes were used for further comparisons. After ip administration, the injected substance is absorbed through the perito- neum, getting quicker into circulation than after sc ad- ministration [20]. However, an ip administration may cause an untypical, weak response to injected com- pound when the needle has by mistake been inserted into the intestine, while not passing between the vis- cera. In general, it may lead to the lack of repeatabil- ity of the response to administered compound, i.e., to failure in the reproducibility of results [20, 22].
Therefore, the lack of injection failure risk is an ad- vantage of sc injection over ip injection. In addition, Inoue et al. [22] showed that sc injection was safe as it did not cause any local damage at injection site in mice after 7-day treatment. In the reported study, a lack of significant correlation was observed between the development of tolerance to the motor impairing effect of FNZ in mice and the ways of drug admini- stration (sc or ip) in the rotarod test.
In the chimney test, a tolerance to FNZ motor im- pairment was observed only after the repeated ad- ministration of the drug (1 mg/kg, sc and 2 mg/kg, ip) for 8 days. There were no significant results after the chronic FNZ (1 mg/kg, ip) treatment in the chimney test. It is difficult to interpret the discrepancies, ob- served between the two motor deficit models (chim- ney and rotarod). The rotarod test is used to assess not only motor coordination but also the rodent’s sense of balance [19, 32, 39]. Moreover, motor coordination deficits, identified by the rotarod test, are subtle and provisional in many experiments, including the re- ported one [7, 32], while the chimney test can be used only as complementary to other tests which determine muscle relaxant activity [39]. In this context, we may suggest that chronic treatment with FNZ in dose of 1 mg/kg, sc or ip, for 5 days, is the most appropriate treatment schedule to obtain tolerance to the ataxic ef- fect of the drug, measured only in the rotarod test.
Our results support the literature data, in which the tolerance to the motor impairment inducing effects of BZ developed during a relatively short time, i.e., 5–14 days of BZ treatment at a wide range of doses [13, 23, 26, 31, 33]. For instance, tolerance to the ataxic effect of lorazepam (a BZ agonist which has an intermediate half-time similar to that of FNZ) developed between day 4 and day 7, and the tolerance was not dose-de-
pendent within the range of used doses (1, 2, 4 and 10 mg/kg) [26]. Moreover, a tolerance to the motor in- coordination effect of diazepam (a BZ agonist with very long half-life: 40-120 h) developed after a 10- day treatment [33].
The other purpose of the reported study was to de- termine a treatment schedule necessary to develop tol- erance to the sedative effects of FNZ. It is widely known that FNZ produces relatively strong sedation and amnesia in humans, in comparison with other BZ [9]. Interestingly enough, the animals in the reported experiment showed an increased locomotor activity after the first injection of FNZ (1 mg/kg, sc, 60 min before the test) but on the 2nd day of the experiment the same injection of FNZ caused a significant de- crease in the locomotor activity of the mice. An ex- planation of this result is difficult because BZ are used clinically, mainly to treat anxiety, muscular spasms and sleep disorders. However, irritability and aggressive outbursts have been reported in patients treated with BZ, a phenomenon referred to as “para- doxical effects” [9, 12]. FNZ, when used illegally, has been linked to a high incident of barbaric crimes, in- volving rape and violence against other individuals [10]. It is known that the paradoxical effects of FNZ are not related to its blood concentration levels. Con- sequently, certain individuals (a 6% subgroup out of 415 cases) could be more susceptible to aggressive or paradoxical reactions induced by FNZ [8]. Moreover, these paradoxical effects have been observed in most experimental models with low doses of BZ, for exam- ple, an increased spontaneous locomotor activity in rats after alprazolam injection (0.0037 mg/kg) [5].
What is more, De Almeida et al. [11] showed that FNZ at low doses (0.01, 0.03, 0.1 and 0.18 mg/kg, ip) tended to increase aggressive behaviors in rats. The mechanism of paradoxical effects has not yet been ex- plained but, probably, these reactions are coupled with the activity of BZ site on the GABAAreceptor complex. Weinbroum et al. [40] showed that the midazolam-induced paradox phenomenon was re- versible by flumazenil (an antagonist of the GABAA receptor). It is worth noting that no paradoxical effect was observed in the reported study after acute FNZ injection on day 1 of the experiment in both the chim- ney and the rotarod test.
A substantial decrease in locomotor activity of the studied mice, measured by a photocell apparatus, was observed on the second day of the experiment.
That diminution of locomotor activity was found in
Chronic treatment with flunitrazepam, ataxia and sedation
Sylwia Talarek et al.
the sedative effect of FNZ. Because the treatment with single daily injection of 1 mg/kg did not signifi- cantly alter the potency of FNZ, a higher dose of FNZ (2 mg/kg) and two injections daily were used in the treatment regimen of the experiment. That treatment was chosen upon the results of van Rijnsoever et al.
[37], which had shown that a repeated (for 9 days) treatment of mice with diazepam (10 + 5 mg/kg/day;
on day 9 – only 10 mg/kg; sc) resulted in a develop- ment of tolerance to its sedative effect, observed as an increased locomotor activity of mice, measured in a photocell apparatus. Similar conditions – twice daily injections, were used by Gerak [18] to induce tolerance to FNZ (1 mg/kg, ip) action in rats. But, in the reported study, no tolerance developed to the seda- tive effect of FNZ after twice daily injections of FNZ (2 mg/kg, sc), either. The literature data provide oppo- site results for tolerance to BZ-induced sedative activ- ity. For example, it was demonstrated in rodents that tolerance to the sedative action of a BZ with shorter activity duration, i.e., lorazepam, developed rapidly, within 3-5 days of chronic administration [16, 24], whereas tolerance to the sedative action of diazepam (a long-acting BZ) developed after 7–9 days of treat- ment [15, 37]. However, De Almeida et al. [11]
showed that tolerance to the initially sedative effect of FNZ (0.3 mg/kg, ip) developed in rats after 42 days of administration, once a day. Therefore, one of possible explanations for our results can be the too short treat- ment to develop tolerance to the sedative effect of FNZ, which has a high affinity with central BZ recep- tors and affects them profoundly [9].
Because the tolerance to BZ develops gradually in time, lasting from days to weeks, it would be sugges- tive of some structural changes of the GABAArecep- tor, which may lead to decreased BZ capacity to phar- macologically enhance the GABA response (i.e., tol- erance causes uncoupling and desensitization) [38].
But there are some opinions that a specific desensi- tization-resistant population of GABAAreceptors are present at postsynaptic sites, what was found in in vi- trostudies [25]. Mellor and Randall [25] showed that FNZ was completely ineffective in unmasking transmitter-induced desensitization, what could par- tially explain the lack of tolerance to the sedative ef- fect of FNZ.
erty of the drug [3, 23]. Many behavioral studies have shown rapidly developing tolerance to sedative and motor coordination deficits, whereas tolerance not always develops to the anxiolytic or memory impair- ing effects of BZ-type drugs after long periods of their use [13, 15, 33, 34, 37]. Bateson [3] suggests that dif- ferences in the time scale of tolerance development, while being dependent upon receptor subtype and/or the brain region, are responsible for various tolerance degrees to particular BZ effects.
It may then be concluded that a chronic treatment with BZ produces tolerance to different behavioral ef- fects, induced by these drugs, and that the tolerance develops along different time courses. We demon- strated in the reported study that tolerance to motor disturbances, exerted by FNZ, occurred rapidly, whereas no tolerance developed to the sedative effect of FNZ. Further investigations are needed to better explain the observed processes and mechanisms, re- sponsible for the development of tolerance to BZ ef- fects.
References:
1. Aitta-Aho T, Vekovischeva OY, Neuvonen PJ, Korpi ER:
Reduced benzodiazepine tolerance, but increased flumazenil-precipitated withdrawal in AMPA-receptor GluR-A subunit-deficient mice. Pharmacol Biochem Behav, 2009, 92, 283–290.
2. Allison C, Pratt JA: Neuroadaptive processes in GABA- ergic and glutamatergic systems in benzodiazepine de- pendence. Pharmacol Ther, 2003, 98, 171–195.
3. Bateson AN: Basic pharmacologic mechanisms involved in benzodiazepine tolerance and withdrawal. Curr Pharm Des, 2002, 8, 5–21.
4. Bateson AN: The benzodiazepine site of the GABA A receptor: an old target with new potential? Sleep Med, 2004, 5, Suppl. 1, S9–S15.
5. Bentué-Ferrer, Reymann JM, Tribut O, Allain H, Vasar E, Bourin M: Role of dopaminergic and serotonergic sys- tems on behavioral stimulatory effects of low-dose alpra- zolam and lorazepam. Eur Neuropsychopharmacol, 2001, 11, 41–50.
6. Bonavita CD, Bisagno V, Bonelli CG, Acosta GB, Rubio MC, Wikinski SI: Tolerance to the sedative effect of lo- razepam correlates with a diminution in cortical release and affinity for glutamate. Neuropharmacology, 2002, 42, 619–625.
7. Bouët V, Freret T, Toutain J, Divoux D, Boulouard M, Schumann-Bard P: Sensorimotor and cognitive deficits
after transient middle cerebral artery occlusion in the mouse. Exp Neurol, 2007, 203, 555–567.
8. Bramness JG, Skurtveit S, Morland J: Flunitrazepam:
Psychomotor impairment, agitation and paradoxical re- actions. Forensic Sci Int, 2006, 159, 83–91.
9. Charney DS, Mihic SJ, Harris RA: Hypnotics and seda- tives. In: Goodman and Gilman’s the Pharmacological Basis of Therapeutics. 11thedn., Eds. Brunton LL, Chabner BA, Knollmann BC, Medical Publishing Divi- sion, USA, 2006, 402–414.
10. Dåderman AM, Fredriksson B, Krustiansson M, Nilsson LH, Lidberg L: Violent behavior, impulsive decision- making, and anterograde amnesia while intoxicated with flunitrazepam and alcohol or other drugs: a case study in forensic psychiatric patients. J Am Acad Psychiatry Law, 2002, 30, 238–251.
11. De Almeida RMM, Benini Q, Betat JS, Hipólide DC, Miczek KA, Svensson AI: Heightened aggression after chronic flunitrazepam in male rats: potential links to cor- tical and caudate-putamem-binding sites. Psychopharma- cology, 2008, 197, 309–318.
12. DiMascio A: The effects of benzodiazepines on aggres- sion: reduced or increased. Psychopharmacologia, 1973, 30, 95–102.
13. Divljakoviæ J, Miliæ M, Timiæ T, Saviæ MM: Tolerance liability of diazepam is dependent on the dose used for pro- tracted treatment. Pharmacol Rep, 2012, 64, 1116–1125.
14. Druid H, Holmgren P, Ahlner J: Flunitrazepam: an evaluation of use, abuse and toxicity. Forensic Sci Int, 2001, 122, 136–141.
15. Fernandes C, Arnot MI, Irvine EE, Bateson AN, Martin IL, File SE: The effect of treatment regimen on the de- velopment of tolerance to the sedative and anxiolytic ef- fects of diazepam. Psychopharmacology, 1999, 145, 251–259.
16. File SE: Tolerance to the behavioral actions of benzodi- azepines. Neurosci Biobehav Rev, 1985, 9, 113–121.
17. Gallager DW, Lakoski JM, Gonsalves SF, Rauch SL:
Chronic benzodiazepine treatment decreases postsynap- tic GABA sensitivity. Nature, 1984, 308, 74–77.
18. Gerak LR: Selective changes in sensitivity to benzodi- azepines, and not other positive GABAAmodulators, in rats receiving flunitrazepam chronically. Psychopharma- cology, 2009, 204, 667–677.
19. Hamm RJ, Pike BR, O’Dell DM, Lyeth BG, Jenkins LW, Jenkins LW: The rotarod test: an evaluation of its effec- tiveness in assessing motor deficits following traumatic brain injury. J Neurotrauma, 1994, 11, 187–196.
20. Hayward AM, Lemke LB, Bridgeford EC, There EJ, Jackson CN, Cumliffe-Beamer TL, Marini RP: Biometh- odology and surgical techniques. In: The Mouse in Bio- medical Research. 2ndedn., Eds. Fox JG, Barthold SW, Davisson MT, Newcomer CE, Quimby FW, Smith AL, Academic Press, USA, 2007, 444–454.
21. Hutchinson MA, Smith F, Darlington CL: The behav- ioural and neural effects of the chronic administration of benzodiazepine anxiolytic and hypnotic drugs. Prog Neurobiol, 1996, 49, 73–97.
22. Inoue Y, Kiryu S, Izawa K, Watanabe M, Tojo A, Ohtomo K: Comparison of subcutaneous and aperitoneal
injection of D-luciferin for in vivo bioluminescence im- aging. Eur J Nucl Med Mol Imaging, 2009, 36, 771–779.
23. Licata SC, Rowlett JK: Abuse and dependence liability of benzodiazepine-type drugs: GABAAreceptor modula- tion and beyond. Pharmacol Biochem Behav, 2008, 90, 74–89.
24. Marin RH, Salvatierra NA, Ramirez OA: Rapid toler- ance to benzodiazepine modifies rat hippocampal synap- tic plasticity. Neurosci Lett, 1996, 215, 149–152.
25. Mellor JR, Randall AD: Synaptically released neuro- transmitter fails to desensitize postsynaptic GABAAre- ceptors in cerebellar cultures. J Neurophysiol, 2001, 85, 1847–1857.
26. Miller LG, Greenblatt DJ, Barnhill JG, Shader RI:
Chronic benzodiazepine administration. I. Tolerance is associated with benzodiazepine receptor downregulation and decreased g-aminobutyric acidAreceptor function.
J Pharmacol Exp Ther, 1988, 246, 170–176.
27. Pérez-Raya MD, Risco S, Navarro C, Jimenez J, Cabo J:
Changes in chlorpromazine induced CNS effects when associated with other neuroleptics and anxiolytics.
Pharm Acta Helv, 1989, 64, 146–150.
28. Ricaurte GA, McCann UD: Recognition and manage- ment of complications of new recreational drug use.
Lancet, 2005, 365, 2137–2145.
29. Rudolph U, Möhler H: GABA-based therapeutic ap- proaches: GABAAreceptor subtype functions. Curr Opin Pharmacol, 2006, 6, 18–23.
30. Simiand J, Keane PE, Biziere K, Soubrie P: Comparative study in mice of tetrazepam and other centrally active skeletal muscle relaxants. Arch Int Pharmacodyn Ther, 1989, 297, 272–285.
31. Smith MA, Gergans SR, Lyle MA: The motor-impairing ef- fects of GABAAand GABABagonists in g-hydroxybutyrate (GHB)-treated rats: cross-tolerance to baclofen but not flu- nitrazepam. Eur J Pharmacol, 2006, 552, 83–89.
32. Stephens DN, Schneider HH, Kehr W, Andrews JS, Retting KJ, Turski L, Schmiechen R: Abecarnil, a metab- olically stable, anxioselective b-carboline acting at ben- zodiazepine receptors. J Pharmacol Exp Ther, 1990, 253, 334–343.
33. Talarek S, Listos J, Fidecka S: Role of nitric oxide in the development of tolerance to diazepam-induced motor impairment in mice. Pharmacol Rep, 2008, 60, 475–482.
34. Talarek S, Orzelska J, Listos J, Fidecka S: Effects of sildenafil treatment on the development of tolerance to diazepam-induced motor impairment and sedation in mice. Pharmacol Rep, 2010, 62, 627–634.
35. Tallman JF, Gallager DW: The GABAergic system:
a locus of benzodiazepine action. Annu Rev Neurosci, 1985, 8, 21–44.
36. Tietz EI, Chiu TH, Rosenberg HC: Regional GABA/ben- zodiazepine receptor/chloride channel coupling after acute and chronic benzodiazepine treatment. Eur J Phar- macol, 1989, 167, 57–65.
37. Van Rijnsoever C, Täuber M, Choulli MK, Keist R, Rudolph U, Möhler H, Fritschy JM, Crestani F: Require- ment of a5-GABAAreceptors for the development of tol- erance to the sedative action of diazepam in mice. J Neu- rosci, 2004, 24, 6785–6790.
Chronic treatment with flunitrazepam, ataxia and sedation
Sylwia Talarek et al.
39. Vogel HG: Psychotropic and neurotropic activity.
In: Vogel HG. Drug discovery and evaluation: Pharma- cological Assays. Springer-Verlag, Berlin. Heidelberg.
New York, 2008, 579–580.
18, 789–797.
Received: December 13, 2011; in the revised form: September 13, 2012; accepted: October 8, 2012.
