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Behavioural Brain Research 284 (2015) 179–186

Contents lists available at ScienceDirect

Behavioural Brain Research

journa l homepage: www.e lsev ier .com/ locate /bbr

esearch report

ivergent effects of l-arginine-NO pathway modulators on diazepamnd flunitrazepam responses in NOR task performance

olanta Orzelska ∗, Sylwia Talarek, Joanna Listos, Sylwia Fideckahair and Department of Pharmacology and Pharmacodynamics, Medical University of Lublin, Chodzki 4A, 20-093 Lublin, Poland

i g h l i g h t s

Diazepam and flunitrazepam impaired recognition memory of rats.l-Arginine prevented diazepam-induced memory impairment.7-Nitroindazole induced the amnesic effects of diazepam.7-Nitroindazole inhibited the amnesic effects of flunitrazepam.

r t i c l e i n f o

rticle history:eceived 23 October 2014eceived in revised form 2 February 2015ccepted 6 February 2015vailable online 16 February 2015

eywords:iazepamlunitrazepamitric oxide

a b s t r a c t

The goal of the study was an evaluation of the degree, in which nitric oxide (NO) is involved in thebenzodiazepines (BZs)-induced recognition memory impairment in rats. The novel object recognition(NOR) test was used to examine recognition memory. The current research focused on the object memoryimpairing effects of diazepam (DZ; 0.5 and 1 mg/kg, sc) and flunitrazepam (FNZ; 0.1 and 0.2 mg/kg; sc) in1-hour delay periods in rats. It was found that acute ip injection of l-arginine (l-arg; 250 and 500 mg/kg;ip), 5 min before DZ administration (0.5 mg/kg, sc) prevented DZ-induced memory deficits. On the otherhand, it was also proven that l-arg (125, 250 and 500 mg/kg; ip) did not change the behaviour of ratsin the NOR test, following a combined administration with FNZ at a threshold dose (0.05 mg/kg; sc). Itwas also found that 7-nitroindazole (7-NI; 10, 20 and 40 mg/kg; ip) induced amnesic effects in DZ in

ovel object recognitionat

rats, submitted to the NOR test, following a combined administration of 7-NI with a threshold dose of DZ(0.25 mg/kg; sc). However, following a combined administration of 7-NI (10, 20 and 40 mg/kg; ip) withFNZ (0.1 mg/kg; sc), it was observed that 7-NI inhibited the amnesic effects of FNZ on rats in the NOR test.

Those findings led us to hypothesize that NO synthesis suppression may induce amnesic effects of DZ,while preventing FNZ memory impairment in rats, submitted to NOR tasks.

© 2015 Elsevier B.V. All rights reserved.

. Introduction

Although benzodiazepines (BZs) are commonly used inherapeutic practice for their anxiety-relieving, tranquilising, som-iferous, anticonvulsant and myorelaxant properties, they disruptemory performance [1]. The amnesic effects of BZs are regarded

o be rather detrimental in their harmful impact. For instance, aumber of elderly patients on BZ therapy are wrongly diagnosed asementia patients, whereas, in fact, they suffer from the amnesic

ffects of the therapy [1]. Generally, there is no doubt that memorys a key cognitive skill for work and life. Therefore, it is impor-ant to determine what aspects of memory are affected by BZs and

∗ Corresponding author. Tel.: +48 81 4487256.E-mail address: jolanta.orzelska@umlub.pl (J. Orzelska).

ttp://dx.doi.org/10.1016/j.bbr.2015.02.014166-4328/© 2015 Elsevier B.V. All rights reserved.

what neural mechanisms/processes are involved in their adverse,amnesic effects.

BZs have been reported to induce anterograde amnesia, bothin humans and in rodents (mice and rats) with selective mem-ory deficits caused by impaired acquisition of new information[1–4]. What is more, it is a well-established fact that acute BZs,such as diazepam (DZ) or triazolam impair the episodic mem-ory (a type of declarative memory) encoding in humans [5].Recognition is thought to be a critical component of declara-tive memory – a judgement of the prior occurrence [6]. While asubstantial amount of research has shown that BZs impair mem-ory paradigms in various rodents, e.g., radial maze [4], passive

avoidance [7], T-maze [8], water maze [3,7] and the modi-fied elevated plus-maze (mEPM) task [2], there are only a fewreports on the effects of BZs on recognition memory in rodents[9–11].

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The amnesic effect of BZs is mediated by the activation of specificeceptor sites on the gamma-aminobutyric acid (GABAA) receptoromplex, being thereby enhanced in the GABAergic transmissionn the central nervous system (CNS) [12,13]. Moreover, at theellular level, there is some evidence for the plastic mechanism,.g., long-term potentiation (LTP) involved in BZ-induced memorympairment [10,14]. However, the exact mechanism of BZ amnesicffects has not yet been fully unveiled.

Nitric oxide (NO) has been found to be a retrograde transmit-er which, via LTP, affects the learning and memory processes [15].urrent reports indicate that both NO donors and NOS inhibitorsINOS) are involved in the object recognition memory (for review,ee [16]). For instance, it has been demonstrated that NOS blocky different INOS, e.g., NG-nitro-l-arginine methyl ester (l-NAME;on-selective NOS inhibitors), 7-nitroindazole (7-NI; selective neu-onal NOS inhibitor) induce acquisition deficits in recognitionemory tasks in rats and these learning deficits are counteracted

y molsidomine, a NO donor [17,18]. Moreover, NO modulateshe release and the retrograde uptake of many neurotransmitters,ncluding GABA (for review, see [15]). For instance, at the cellularevel, Szabadits et al. [19] have provided evidence for NO playing aertain role in the control of hippocampal GABAergic transmission.he researchers have demonstrated that nNOS is present in hip-ocampal GABAergic synapses in adult rodents. What is more, ourehavioural studies also suggest some role of NO in anticonvulsant20], hypnotic [21] or antinociception [22] effects of BZs, as well asn the development of tolerance to the coordination disturbing [23]nd sedative [24] effects of BZs.

Our previous studies demonstrated that, in a spatial memoryask–mEPM–acquisition was impaired by DZ and flunitrazepamFNZ). But it should be noted that INOS enhanced DZ-induced,hile preventing FNZ-induced recognition memory impairment

2]. However, the interactions between NO and BZs in recognitionerformance has not yet been elucidated.

Taking into account the above-mentioned, somewhat contro-ersial results, the goal of the study was to evaluate the degree, inhich NO is involved in the BZ-induced recognition impairment

n rats. A novel object recognition (NOR) test was employed toxamine recognition memory. Memory performance in the NOR isased on the natural tendency of animals to explore novel objects.n important advantage of this task is that no aversive/stressfultimuli are needed [9]. In order to design a possible interactionetween NO activity and BZ responses in NOR tasks, performed byats, l-arginine (l-arg)–a precursor of NO and 7-NI – selective nNOSnhibitor – were applied [25]. DZ and FNZ were chosen as represen-ative members of BZ family. In addition, pilot study in NOR task wasonducted, to choose doses appropriate for further experiments.or this purpose, the effects of BZ and NO-related compounds (l-rg and 7-NI), given alone, on rats performance in NOR task werenvestigated. Moreover, in an attempt to exclude the possible seda-ive effect of BZ, l-arg or 7-NI, given alone or in combination withZ, motility of rats after administration of all the substances, wasvaluated.

. Materials and methods

.1. Animals

The examinations were carried out on 2-month-old male albinoistar rats (The Farm of Labolatory Animals, Z. Lipiec, Brwinow,

oland), weighing 200–250 g each. They were housed in groups of

ve and maintained on a 12 h light-dark cycle (lights on at 6:00 h)t controlled temperature (21 ◦C). The experiments were per-ormed between 9:00 h and 17:00 h. They received standard foodAgropol, Motycz, Poland) and tap water ad libitum. All behavioural

Research 284 (2015) 179–186

experiments were carried out, according to the National Instituteof Health Guidelines for the Care and Use of Laboratory Animalsand to the European Community Directive for the Care and Use ofLaboratory of 24 November 1986 (86/609/EEC), and approved bythe Local Ethics Committee (37/2010).

2.2. Drugs

l-arg and FNZ were purchased from Sigma Chemicals (St. Louis,USA). 7-NI (RBJ, Natick, USA) and FNZ were dissolved in 0.5%Tween-80 (1–2 drops), gently warmed and diluted with salinesolution (0.9% NaCl). DZ (Relanium, Polfa, Poland) was diluted in0.9% saline. l-arg was dissolved in saline solution. All drug sus-pensions/solutions were prepared immediately prior to use. l-argand 7-NI were given intraperitoneally (ip), whereas DZ and FNZsubcutaneously (sc). All the drugs were injected in a volume of0.2 ml/100 g body weight. Control animals were administred a cor-responding vehicle.

2.3. Novel object recognition test

The apparatus included a square open box, made of plexiglass(63 cm long × 44.5 cm high × 44 cm wide) and illuminated by alamp (light intensity–10 lx), suspended 50 cm above the box. Theobjects to be discriminated, made either of wood or plastic, were intwo different shapes: block and ball and too heavy to be displacedby the animals.

The object recognition test was performed as described else-where [9,26]. The day before the test, each rat was placed in theempty box for 2 min to get used to the environment. On the exper-imental day, the animals were submitted to two trials, spaced bya 1-h interval. The first trial (acquisition trial, T1) lasted 5 min andthe second one (test trial, T2) was 3 min long. During T1, the appa-ratus contained two identical objects (wooden blocks), placed intwo opposite corners, 10 cm from the sidewall. A rat was alwaysplaced in the middle of the box. After T1, the rat was put backinto its home cage. Subsequently, after 1 h, T2 was performed. Dur-ing T2, a new object (N) replaced one of the samples presented inT1, therefore, the rats were re-exposed to two objects: familiar (F)and new (N). In order to avoid the presence of olfactory trails, theapparatus and the objects were cleaned after each rat. The explo-ration looked as follows: directing the nose toward the object ata distance of no more than 2 cm and/or touching the object withnose. Turning around or sitting on the object was not considered asexploratory behaviour. The time periods, spent by rats in exploringeach object during T1 and T2 tests, were recorded manually witha stopwatch. The discrimination between F and N during T2 wasmeasured by comparing the time period, spent for exploration of Fwith that, spent for exploration of N. Memory was evaluated withthe discrimination index (DI), calculated for each animal by thefollowing formula: (N − F)/(N + F), corresponding to the differencebetween exploration time periods for N and F, adjusted for the totalexploration time period of both objects in T2. A higher discrimina-tion index is considered to reflect stronger memory retention forfamiliar objects.

2.4. Locomotor activity test

Locomotor activity of individual rats was recorded, using aphotocell device (plexiglass boxes - square cages, 60 cm on eachside; Porfex, Bialystok, Poland) at a sound-attenuated experi-mental room, under moderate illumination (10 lux). Ambulatory

activity (distance travelled) was measured by two rows of infraredlight-sensitivity photocells, installed along the long axis, 45 and100 mm above the floor. The animals were placed individually intocages, 30 min after DZ or FNZ injection and 35 min after l-arg or

J. Orzelska et al. / Behavioural Brain Research 284 (2015) 179–186 181

Table 1Effect of DZ or FNZ treatment on locomotor activity in rats.

Treatment Mean of thedistancetravelled ± SEM[m] within15 min

A Saline 17.77 ± 2.913

B DZ 0.25 mg/kg 19.72 ± 2.669DZ 0.5 mg/kg 20.43 ± 2.497DZ 1 mg/kg 13.24 ± 3.559

C FNZ 0.05 mg/kg 16.67 ± 3.012FNZ 0.1 mg/kg 13.50 ± 2.922FNZ 0.2 mg/kg 12.25 ± 2.07

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Treatment Mean of thedistancetravelled ± SEM[m] within15 min

A Saline 16.87 ± 2.48B DZ 0.5 mg/kg 20.32 ± 2.16C FNZ 0.05 mg/kg 19.71 ± 2.17

D l-arg 125 mg/kg 24.53 ± 1.47l-arg 250 mg/kg 21.99 ± 3.30l-arg 500 mg/kg 20.98 ± 3.86

E DZ 0.5 + l-arg 125 mg/kg 15.72 ± 2.25DZ 0.5 + l-arg 250 mg/kg 22.28 ± 2.58DZ 0.5 + l-arg 500 mg/kg 15.72 ± 3.43

F FNZ 0.05 + l-arg 125 mg/kg 22.14 ± 2.88FNZ 0.05 + l-arg 250 mg/kg 21.68 ± 4.26FNZ 0.05 + l-arg 500 mg/kg 19.59 ± 3.51

One-way ANOVA revealed statistically significant effects ofacute sc doses of DZ (0.5 and 1 mg/kg) on DI values [F(3,22) = 4,043;p = 0.0075]. The applied post hoc Bonferroni’s test revealed a

Table 3Effect of treatments on locomotor activity in rats treated with 7-NI and DZ or FNZ.

Treatment Mean of thedistancetravelled ± SEM[m] within 15 min

A Saline 15.01 ± 3.08B DZ 0.25 mg/kg 19.62 ± 2.31C FNZ 0.1 mg/kg 21.70 ± 2.35

D 7-NI 10 mg/kg 16.41 ± 1.667-NI 20 mg/kg 18.55 ± 1.897-NI 40 mg/kg 14.52 ± 2.65

E DZ 0.25 + 7-NI 10 mg/kg 21.49 ± 5.20DZ 0.25 + 7-NI 20 mg/kg 22.56 ± 0.79DZ 0.25 + 7-NI 40 mg/kg 15.39 ± 2.38

F FNZ 0.1 + 7-NI 10 mg/kg 19.85 ± 1.28FNZ 0.1 + 7-NI 20 mg/kg 14.40 ± 2.09

Z and FNZ were injected sc 30 min before the test. The data are expressed asean ± SEM of total distance travelled in meters within 15 min.

-NI injection. Total horizontal activity (the distance travelled ineters) was recorded for a 15-minute time period [26,27].

.5. Treatment

Different doses of DZ (0.25, 0.5 and 1 mg/kg, sc) [9] and FNZ (0.05,.1 and 0.2 mg/kg, sc) [28] were administered on 30 min before T1.-arg (125, 250 and 500 mg/kg, ip) [29,30] and 7-NI (10, 20 and0 mg/kg, ip) [18,30] were administered 35 min before T1, alone.

n order to evaluate the influence of l-arg or 7-NI on DZ or FNZ-reated rats, l-arg or 7-NI were administered 5 min prior to DZ orNZ injections. T2 was carried out always 1 h after T1.

.6. Statistical analysis

The data, relative to DI values and the distance, travelled for DZnd FNZ, given alone, were analysed by the one-way analysis ofariances (ANOVA). DI values for the co-administration of “l-argnd DZ or FNZ”, and also of “7-NI and DZ or FNZ”, were made bywo-way ANOVA, with the drug treatment (saline and DZ or FNZ)s factor 1 and the drug pre-treatment (saline, l-arg or 7-NI) as fac-or 2. In those cases that the interaction between treatment andre-treament was significant or quite signicant, Bonferroni’s postoc test was applied. The level of p < 0.05 was considered statisti-ally significant. The data are presented as means ± standard errorsf means (S.E.M.) of DI values or distance segments, travelled ineters. Each group of animals consisted of 8 rats. All figures were

repared using GraphPad Prism version 5.00 for Windows, Graph-ad Software (San Diego, California, USA), www.graphpad.com.

. Results

No difference was observed in any group during Test 1 (T1),hen exploration time periods were compared for location of two

dentical objects in two opposite corners (the data not shown).

.1. Effects of treatments on the locomotor activity of rats

No significant difference was observed between the groups,egarding the effects of either a single DZ (0.25, 0.5, and 1 mg/kg) orNZ (0.05, 0.1 and 0.2 mg/kg) injection on the total distance, trav-lled in meters within 15 minutes by rats, using the photocell deviceone-way ANOVA; F(3,25) = 1.168; p = 0.3416 for DZ, see Table 1A and; F(3,28) = 0.8881; p = 0.4593 for FNZ, see Table 1A and C].

Two-way ANOVA revealed that a treatment with DZ (0.5 mg/kg)r saline [F(1,49) = 1.62] and pre-treatment with l-arg (125, 250 and00 mg/kg) or saline [F(3,49) = 0.74] did not affect the total distanceravelled by rats (Table 2A, B, D and E).

l-arg was injected ip 5 min prior to administration of BZ, whereas BZ were injectedsc 30 min prior to the test. The data are expressed as mean ± SEM of total distancetravelled in meters within 15 min.

Two-way ANOVA revealed that a treatment with FNZ(0.05 mg/kg) or saline [F(1,48) = 0.02] and pre-treatment with l-arg(125, 250 and 500 mg/kg) or saline [F(3,48) = 0.89] did not affect thetotal distance travelled by rats (Table 2A, C, D and F).

Two-way ANOVA revealed that a treatment with DZ(0.25 mg/kg) or saline [F(1,46) = 3.34] and pre-treatment with7-NI (10, 20 and 40 mg/kg) or saline [F(3,46) = 1.47] did not affectthe total distance travelled by rats (Table 3A, B, D and E).

Two-way ANOVA revealed that a treatment with FNZ(0.1 mg/kg) or saline [F(1,52) = 0.4] did not affect locomotor activ-ity of rats whereas pre-treatment with 7-NI (10, 20 and 40 mg/kg)or saline [F(3,52) = 5,48] affected the total distance travelled by rats(Table 3A, C, D and F). The post hoc Bonferroni’s test showed thatco-administration of FNZ with 7-NI (40 mg/kg) decreased the totaldistance travelled by rats compared to FNZ-treated group (p < 0.05).

3.2. Effects of diazepam and flunitrazepam administration on ratperformance in the object recognition task

FNZ 0.1 + 7-NI 40 mg/kg 9.49 ± 1.36*

7-NI was injected ip 5 min prior to administration of BZ, whereas BZ were injectedsc 30 min prior to the test. The data are expressed as mean ± SEM of total distancetravelled in meters within 15 min. *p < 0.05 vs. FNZ 0.1 mg/kg (Bonferroni’s test).

182 J. Orzelska et al. / Behavioural Brain Research 284 (2015) 179–186

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ig. 1. Effects of DZ (0.25, 0.5 and 1 mg/kg, sc) administration on rats performance ibjects in T2 (B)). BZs or saline were injected 30 min before the first trial. Data are e

ignificant decrease of DI by DZ (0.5 and 1 mg/kg), as comparedith the saline-treated control group [p < 0.05], confirming that DZid disrupt memory processes (see Fig. 1A), while the lowest dosef DZ (0.25 mg/kg) did not affect DI.

Similarly, one-way ANOVA revealed statistically significantffects of the acute sc doses of FNZ (0.1 and 0.2 mg/kg) on DIalues [F(3,38) = 6,481; p = 0.0012]. The post hoc Bonferroni’s testevealed that FNZ (0.1 and 0.2 mg/kg) significantly decreased DI,s compared with the saline-treated control group (p < 0.05), thusonfirming FNZ to be a memory process disrupting factor (Fig. 2A).NZ, when given at the dose of 0.05 mg/kg, did not impair DI in theOR test.

As shown in Fig. 1B and Fig. 2B in this set of experiments, datanalysis indicated that all doses of DZ and FNZ did not change totalxploration time in T2.

.3. l-Arginine effects on DZ (0.5 mg/kg, sc)-induced memorympairment of rats in the NOR test

Two-way ANOVA revealed statistically significant effects ofZ or saline treatment [F(1.37) = 4.68; p = 0.037] and interactionetween l-arg pre-treatment and DZ treatment [F(3.37) = 3.13;= 0.037].

The acute ip injection of l-arg (250 and 500 mg/kg), 5 min beforeZ administration (0.5 mg/kg, sc) prevented DZ-induced memoryeficits, as the “l-arg (250 or 500 mg/kg) and DZ (0.5 mg/kg)”-

reated rats discriminated much better the novel objects vs. theamiliar objects during T2, when juxtaposed with their coun-erparts on saline and DZ (0.5 mg/kg) (p < 0.05, the post hoconferroni’s test; see Fig. 3A).

ig. 2. Effects of FNZ (0.05, 0.1 and 0.2 mg/kg, sc) administration on rats performance inoth objects in T2 (B)). BZs or saline were injected 30 min before the first trial. Data are ex

object recognition task (discrimination index (A) and total exploration time of bothsed as mean ± SEM values. *p < 0.05 vs. saline control group (Bonferroni’s test).

As shown in Fig. 3B in this set of experiments total explorationtime was unchanged.

3.4. l-Arginine effects on FNZ (0.05 mg/kg, sc)-treated rats in theNOR test

Two-way ANOVA revealed no statistically significant effect,either of FNZ or saline treatment and no interaction between l-argpre-treatment and FNZ treatment or between FNZ or saline pre-treatment (Fig. 4A). As shown in Fig. 4B in this set of experimentstotal exploration time was unchanged.

3.5. 7-NI effects on DZ (0.25 mg/kg, sc)-treated rats in the NORtest

Two-way ANOVA revealed statistically significant effects of DZor saline treatment [F(1.49) = 38.05; p < 0.0001] and DZ or saline pre-treatment [F(3.49) = 4.53; p = 0.007]. The interaction between 7-NIpre-treatment and DZ treatment was considered not quite signifi-cant [F(3.49) = 2.6; p = 0.0623].

The acute ip injection of 7-NI (10, 20 and 40 mg/kg), 5 min beforeDZ administration (0.25 mg/kg) induced memory deficits, as “7-NI(10, 20 or 40 mg/kg) and DZ (0.25 mg/kg)”-treated rats did not dis-criminate between novel and familiar objects during T2 test withrespect to their counterparts on saline and DZ (0.25 mg/kg) (p < 0.05

for 7-NI 10 and 20 mg/kg, p < 0.01 for 7-NI 40 mg/kg, the post hocBonferroni’s test; see Fig. 5A).

As shown in Fig. 5B in this set of experiments total explorationtime was unchanged.

the object recognition task (discrimination index (A) and total exploration time ofpressed as mean ±SEM values. *p < 0.05 vs. saline control group (Bonferroni’s test).

J. Orzelska et al. / Behavioural Brain Research 284 (2015) 179–186 183

Fig. 3. The influence of l-arg pretreatment (125, 250 and 500 mg/kg, ip) on DZ(0.5 mg/kg, sc)-induced deficits in the object recognition task (A). Total explorationtime displayed by different groups of rats in the object recognition task in T2 (B).l-arg was injected 5 min prior to administration of DZ, whereas DZ was injected3ut

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Fig. 4. The influence of l-arg pretreatment (125, 250 and 500 mg/kg, ip) on ratsperformance in the object recognition task after FNZ (0.05 mg/kg, sc) treatment (A).Total exploration time displayed by different groups of rats in the object recognition

0 min prior to testing in the first trial. The data are expressed as mean ± SEM val-es. **p < 0.01 vs. saline control group; #p < 0.05 vs. DZ-treated group (Bonferroni’sest).

.6. 7-NI effects on FNZ (0.1 mg/kg, sc)-induced memorympairment of rats in the NOR test

Two-way ANOVA revealed statistically significant effects ofNZ or saline treatment [F(1.47) = 6.47; p = 0.0143]. The interactionetween the 7-NI pre-treatment and FNZ treatment [F(3.47) = 2.79;= 0.0508] was not quite significant.

The acute ip injection of 7-NI (10, 20 and 40 mg/kg) 5 minefore FNZ administration (0.1 mg/kg, sc) prevented FNZ-inducedemory deficits, as the “7-NI (10, 20 or 40 mg/kg) and FNZ

0.1 mg/kg)”-treated rats discriminated better the novel objectss. the familiar objects during T2 test, when compared with theirounterparts on saline and FNZ (0.1 mg/kg) (p < 0.05, the post hoconferroni’s test; see Fig. 6A).

As shown in Fig. 6B in this set of experiments total explorationime was unchanged.

. Discussion

The reported research indicated object memory impairingffects of either DZ (0.5 and 1 mg/kg) or FNZ (0.1 and 0.2 mg/kg)n a 1-h delay in rats. In the reported experiments, the animals

ere injected with BZ before the first trial, therefore, BZ could

ave disrupted the acquisition phase of memory. Those results areonsistent with the prior findings of BZ amnesic effects in rodentsuring NOR tasks [9–11]. Additionally, BZ-impaired memory washown in different rodent models (models for spatial [2–4,7] and

task in T2 (B). l-arg was injected 5 min prior to administration of FNZ, whereasFNZ was injected 30 min prior to testing in the first trial. The data are expressed asmean ± SEM values.

for recognition memory [9–11], therefore, the present data confirmthe amnesic effects of BZs to be not specific for one learning type.

What is more, the observed object memory impairing activi-ties of BZs were not associated with any changes in the locomotoractivity, since BZs did not alter the total distance, travelled by ratsin performed test. The data are in line with the reports, in whichthe amnesic effects of BZs are not related to their sedative action[9,11].

NOR task has been considered to be a pure memory test in whichanimals are able to discriminate between familiar and novel objectsafter some retention interval [26]. Since the NOR paradigm, in com-parison to other animal learning and memory models, does notrequire lengthy training nor does it induce high arousal and stresslevels, it provides closer conditions to those under which humanrecognition memory is measured [26]. In our experiments, 1-hourdelays were employed, according to literature data, indicating goodobject memory performance of rats with 1-hour intervals betweensubsequent trials [9].

The reported investigations examined the effects of l-arginineand 7-NI, a NO precursor and a nNOS inhibitor, respectively, on theperformance of rats, subjected to DZ and FNZ injections in NORtasks. Acute ip injections of l-arg (250 and 500 mg/kg), admin-istered in 5 min before DZ (0.5 mg/kg, sc), prevented DZ-inducedmemory deficits. On the other hand, it was also proven that l-arg(125, 250 and 500 mg/kg) did not change the behaviour of rats inthe NOR test, following it’s combined administration with FNZ at athreshold dose (0.05 mg/kg). It was also found that 7-NI (10, 20 and

40 mg/kg) induced amnesic effects of DZ in rats, participating in theNOR test, following it’s combined administration with a thresholddose of DZ (0.25 mg/kg). However, following the combined admin-istration of 7-NI (10, 20 and 40 mg/kg) with FNZ (0.1 mg/kg), it was

184 J. Orzelska et al. / Behavioural Brain Research 284 (2015) 179–186

Fig. 5. The influence of 7-NI pretreatment (10, 20 and 40 mg/kg, ip) on rats per-formance in the object recognition task after DZ (0.25 mg/kg, sc) treatment (A).Total exploration time displayed by different groups of rats in the object recog-nDm

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Fig. 6. The influence of 7-NI pretreatment (10, 20 and 40 mg/kg, ip) FNZ (0.1 mg/kg,sc)-induced deficits in the object recognition task (A). Total exploration time dis-played by different groups of rats in the object recognition task in T2 (B). 7-NI was

ition task in T2 (B). 7-NI was injected 5 min prior to administration of DZ, whereas

Z was injected 30 min prior to testing in the first trial. The data are expressed asean ± SEM values. #p < 0.05, ##p < 0.01 vs. DZ-treated group (Bonferroni’s test).

bserved that 7-NI inhibited the amnesic effects of FNZ on rats inhe NOR test.

The presented results, while indicating a different orientation ofhe interactions between NO and DZ or FNZ are consistent with therevious data [2]. Our earlier studies demonstrated acquisition toe impaired by DZ and FNZ in a mEPM task and that INOS (l-NAMEnd 7-NI) enhanced DZ-induced, but prevented FNZ-induced mem-ry impairment [2]. An mEPM task evaluates spatial and emotionalearning performance because it is based on the natural aversionf rodents to open and elevated spaces [31]. Scientists suggested aichotomy in the temporal lobe and in the prefrontal structures,ediating object and spatial memory. It is, therefore, plausible

hat recognition memory and spatial memory processes activateifferent parts of rat brain [32].

Our findings of the interactions between l-arg and DZ aren agreement with a lot of literature data, indicating that NOonors (molsidomine, S-nitroso-N-acetylpenicillamine (SNAP), theovel NO donor (3-(4-hydroxy-3-methoxyphenyl)-2-propenoiccid 5-(nitrooxy) butyl ester) NCX-2057) counteract the memorycquisition deficits induced by other compounds, shown in rats inhe NOR task [33–36]. For instance, it was shown that systemicdministration (ip) of molsidomine reversed scopolamine [33] -nd baclofen [34] - induced acquisition deficits in NOR. Moreover,ildiz Akar et al. [25] showed that l-arg (200 mg/kg, ip) counter-cted the negative effects of 7-NI (5 mg/kg, ip) on memory in ratsn a three-panel runway task. In our experimental conditions, l-arg125, 250 and 500 mg/kg), when given alone, had no impact on theOR behaviour.

Similarly, our findings of the interactions between 7-NI and DZre comparable with a number of previous reports, indicating that-NI did inhibit the acquisition phase of learning in different rodentodels (passive avoidance, mEPM, 14-unit T-maze, three-panel

injected 5 min prior to administration of FNZ, whereas FNZ was injected 30 min priorto testing in the first trial. The data are expressed as mean ± SEM values. **p < 0.01vs. saline control group; #p < 0.05 vs. FNZ-treated group (Bonferroni’s test).

runway test, Y-maze and object recognition test) [25,30,37–39].What is more, Hölscher [40] showed that a systematic administra-tion of 7-NI exerts an almost complete block of LTP.

Moreover, the present proamnesic effect of 7-NI on DZ-treatedrats and the suppressed effect of l-arg on the amnesic action ofDZ are consistent with the results of earlier experiments, car-ried out at our Laboratory. Previous studies have indicated thatsome effects of BZs, such as anticonvulsant, hypnotic or antinoci-ceptive, seem to be modulated by the NO system. Talarek et al.[20–22] showed the administration of both non-selective INOS: l-NAME, NG-nitro-l-arginine (l-NOARG) and selective NOS inhibitor:7-NI, to significantly increase the duration of DZ-, chlordiazepoxide(CDZ)- and clonazepam (CZ)-induced sleep, in the anticonvulsanteffect of DZ and in the antinociceptive activity of DZ, CDZ and CZ.What is more, most of those effects were reversed by l-arg.

Our findings, showing that 7-NI successfully (at all used doses)reversed the FNZ (0.1 mg/kg)-induced memory deficit in the ratssubjected to NOR task, are difficult to explain. That effect wascompletely opposite to the interactions between 7-NI and DZ, aspresented in this paper. But, this is consistent with the recent evi-dence reporting that peripheral acute administration of l-NAME,at a low dose range (1–3 mg/kg, ip) reversed recognition mem-ory deficits produced by the NMDA receptor antagonists MK-801and ketamine in the NOR task in rats [41]. Also, both l-NAME (1and 3 mg/kg, ip) and 7-NI (1 and 3 mg/kg, ip) antagonized perfor-mance deficits-induced by dopamine agonist apomorphine in NORtest in rats [42]. Importantly, pre-test administration of l-NAME,

at the low dose (0.25 �g/mouse, intra-CA1), reversed the mem-ory impairment induced by muscimol - GABAA receptor agonistin mice, in the passive avoidance test [43]. In addition, it has shownthat l-NAME decreased the memory impairment of mice induced

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y morphine, in the water maze task in mice [44] and in rats, inhe inhibitory avoidance task [45]. Also, it was determined that l-AME (10 mg/kg, ip) reversed the beneficial effects of pioglitazonen acquisition memory impaired by morphine in mice, in both Y-aze and passive avoidance tasks [46] and by scopolamine [47] inpassive avoidance test.

However, there is a controversy over the precise role of INOSn memory processes. It should be underlined that INOS wereeported to disrupt acquisition of new tasks but these findingsre discrepant in this context. Some studies have found that INOSl-NAME, 7-NI, l-NOARG) given systemically prior to training, doot change learning performance in different rodents models ofemory [48–50]. In our experimental conditions, 7-NI (10, 20 and

0 mg/kg), when given alone, had no impact on the NOR behaviour.he recent evidence reporting that peripheral acute administrationf INOS, at a low dose range (1, 3 and 10 mg/kg) did not affectnimal’s performance in the NOR test [41]. Interestingly, a pre-est administration of l-NAME, at the low dose (0.25 �g/mouse,ntra-CA1), alone cannot affect memory, whereas at the higheroses (0.5 and 1 �g/mouse, intra-CA1) impaired learning perfor-ance of mice [43]. In this context, research into the role of INOS

n the induction of LTP is also discrepant. Block of NOS has beeneported to impair the induction of LTP in some studies. Othertudies observed a partial block of LTP, while yet other stud-es did not find and effect of INOS under any conditions tested51]. Despite considerable evidence for NO involvement in at leastome forms of memory processing its required function is notnown.

It should be noted that much remains unknown, regarding thexact role of the NO signalling system in the CNS [15]. Some reportsssume the release of GABA to be biphasically dependent on NOoncentrations. Getting et al. [52] showed that l-NAME at low con-entration increased, whereas at higher concentration decreasedasal GABA release. On the contrary, high concentrations of theO donor (SNAP) enhanced GABA outflow [53]. In addition, the

nteraction between BZs and NO seems to be a more complex pro-ess, as NO also modulates the release of other neurotransmittersnvolved in memory processes, such as acetylcholine and glutamatefor review, see [51]).

The above-mentioned discrepancy between NO and DZ or FNZight have been associated with their selective actions at spe-

ific GABAA receptor subunits. Each GABAA receptor subtype hasot a distinct pattern of expression within the mammalian brain,uggesting a predefined physiological role. The �5 subunit of theABAA receptor is found mainly in the hippocampus and it is sug-ested to be involved in memory process. Genetically modifiedice with a partial or full deficit of �5GABAA receptors showed an

mproved performance in various associative learning and mem-ry tasks [54,55]. Other results have indicated that DZ-inducednterograde amnesia is mediated by �1GABAA receptors [56].

FNZ is very potent agonist of GABAA receptor with a high abilityo cause anterograde amnesia, thus called a date-rape drug [57,58].n addition, our previous results concerning FNZ were also unex-ected because of increased locomotor activity of mice after therst injection of FNZ (1 mg/kg), whereas the first injection of DZ10 mg/kg) caused sedative effect in mice, in the same experiment2]. Moreover, Hauser et al. [59] suggested that FNZ could act asither an agonist or inverse agonist, depending on the GABAA recep-or configuration.

It is important to note the lack of l-arg effect on rat performancen the NOR task after FNZ injection (0.05 mg/kg). A subthresholdose of FNZ was used because, based on previous results [2], l-arg

ffects were expected.

What is more, the pharmacokinetic interaction between thesed modulator of NO action and BZs may be one contributingactor in the incongruities of experimental findings [60]. However

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Research 284 (2015) 179–186 185

future studies are needed, to check BZ concentration in rat bloodafter co-administration of l-arg or 7-NI with DZ and FNZ.

Even more, our results rule out the possibility that the inter-actions between NO -related compounds with BZs in NOR taskmight be caused by motivational factors. Total exploration timesdisplayed by all groups during the choice trail (T2) were unchanged.

Additionally, it is important to mention that l-arg and 7-NI givenalone and in combination with BZs did not affect rats motility.Therefore, interactions between l-arg or 7-NI with BZs in NOR task,do not seem to be modified by non-specific factors. But, it is oneexception–the inhibitory effect of 7-NI at a dose of 40 mg/kg onthe amnesic action of FNZ (0.1 mg/kg). The combination of 7-NI(40 mg/kg) with FNZ (0.1 mg/kg) decreased locomotor activity ofrats. In this case, it could not be excluded that other factors mighthave affected animals’ performance in NOR test.

One may consider that the effect of 7-NI on DZ or FNZ perfor-mance in the NOR task is due to its influence on blood pressure butit has been shown that 7-NI does not affect blood pressure at dosesup to 80 mg/kg [61].

To sum up, these findings led us hypothesize that the inhibitionof NO synthesis may induce the amnesic effects of DZ, while pre-venting FNZ memory impairment of rats, submitted to NOR taskactivities.

Acknowledgement

The reported study was supported by Grant No. NN 405091740from the Polish Ministry of Science and Higher Education.

References

[1] Buffett-Jerrott SE, Stewart SH. Cognitive and sedative effects of benzodiazepineuse. Curr Pharm Des 2002;8(1):45–58.

[2] Orzelska J, Talarek S, Listos J, Fidecka S. Effects of NOS inhibitors on thebenzodiazepines-induced memory impairment of mice in the modified ele-vated plus-maze task. Behav Brain Res 2013;244:100–6.

[3] Timic T, Joksimovic S, Milic M, Divljakovic J, Batinic B, Savic MM. Midazolamimpairs acquisition and retrieval, but not consolidation of reference memoryin the Morris water maze. Behav Brain Res 2013;241:198–205.

[4] White AM, Simson PE, Best PJ. Comparison between the effects of ethanol anddiazepam on spatial working memory in the rat. Psychopharmacology (Berl)1997;133(3):256–61.

[5] Savic MM, Obradovic DI, Ugresic ND, Bokonjic DR. Memory effects of benzodi-azepines: memory stages and types versus binding-site subtypes. Neural Plast2005;12(4):289–98.

[6] Winters BD, Saksida LM, Bussey TJ. Object recognition memory: neurobiologicalmechanisms of encoding, consolidation and retrieval. Neurosci Biobehav Rev2008;32(5):1055–70.

[7] Noguchi H, Kitazumi K, Mori M, Shiba T. Effect of zaleplon on learning andmemory in rats. Naunyn Schmiedebergs. Arch Pharmacol 2002;366(2):183–8.

[8] Quintero S, Buckland C, Gray JA, McNaughton N, Mellanby J. The effects ofcompounds related to gamma-aminobutyrate and benzodiazepine receptorson behavioural responses to anxiogenic stimuli in the rat: choice behaviour inthe T-maze. Psychopharmacology (Berl) 1985;86(3):328–33.

[9] Bertaina-Anglade V, Enjuanes E, Morillon D, Drieu la RC. The object recognitiontask in rats and mice: a simple and rapid model in safety pharmacology to detectamnesic properties of a new chemical entity. J Pharmacol Toxicol Methods2006;54(2):99–105.

10] Wan H, Warburton EC, Zhu XO, Koder TJ, Park Y, Aggleton JP, et al. Benzodi-azepine impairment of perirhinal cortical plasticity and recognition memory.Eur J Neurosci 2004;20(8):2214–24.

11] Longone P, Impagnatiello F, Guidotti A, Costa E. Reversible modification ofGABAA receptor subunit mRNA expression during tolerance to diazepam-induced cognition dysfunction. Neuropharmacology 1996;35(9-10):1465–73.

12] Bateson AN. The benzodiazepine site of the GABAA receptor: an old target withnew potential? Sleep Med 2004;5 Suppl 1:S9–15.

13] Chapouthier G, Venault P. GABA-A receptor complex and memory processes.Curr Top Med Chem 2002;2(8):841–51.

14] Xu JY, Sastry BR. Benzodiazepine involvement in LTP of the GABA-ergic IPSC in

rat hippocampal CA1 neurons. Brain Res 2005;1062(1-2):134–43.

15] Vincent SR. Nitric oxide neurons and neurotransmission. Prog Neurobiol2010;90(2):246–55.

16] Pitsikas N. The role of nitric oxide in the object recognition memory. BehavBrain Res 2014.

1 Brain

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

86 J. Orzelska et al. / Behavioural

17] Pitsikas N, Rigamonti AE, Bonomo SM, Cella SG, Muller EE. Molsidomine antag-onizes l-NAME-induced acquisition deficits in a recognition memory task inthe rat. Pharmacol Res 2003;47(4):311–5.

18] Prickaerts J, Steinbusch HW, Smits JF, de VJ. Possible role of nitric oxide-cyclicGMP pathway in object recognition memory: effects of 7-nitroindazole andzaprinast. Eur J Pharmacol 1997;337(2-3):125–36.

19] Szabadits E, Cserep C, Szonyi A, Fukazawa Y, Shigemoto R, Watanabe M, et al.NMDA receptors in hippocampal GABAergic synapses and their role in nitricoxide signaling. J Neurosci 2011;31(16):5893–904.

20] Talarek S, Fidecka S. Role of nitric oxide in anticonvulsant effects of benzodi-azepines in mice. Pol J Pharmacol 2003;55(2):181–91.

21] Talarek S, Fidecka S. Involvement of nitricoxidergic system in the hypnoticeffects of benzodiazepines in mice. Pol J Pharmacol 2004;56(6):719–26.

22] Talarek S, Fidecka S. Role of nitric oxide in benzodiazepines-induced antinoci-ception in mice. Pol J Pharmacol 2002;54(1):27–34.

23] Talarek S, Listos J, Fidecka S. Role of nitric oxide in the development oftolerance to diazepam-induced motor impairment in mice. Pharmacol Rep2008;60(4):475–82.

24] Talarek S, Orzelska J, Listos J, Fidecka S. Effects of sildenafil treatment on thedevelopment of tolerance to diazepam-induced motor impairment and seda-tion in mice. Pharmacol Rep 2010;62(4):627–34.

25] Yildiz AF, Celikyurt IK, Ulak G, Mutlu O. Effects of L-arginine on 7-nitroindazole-induced reference and working memory performance of rats. Pharmacology2009;84(4):211–8.

26] Ennaceur A, Delacour J. A new one-trial test for neurobiological studies ofmemory in rats. 1: Behavioral data. Behav Brain Res 1988;31(1):47–59.

27] Koltunowska D, Gibula-Bruzda E, Kotlinska JH. The influence of ionotropicand metabotropic glutamate receptor ligands on anxiety-like effect ofamphetamine withdrawal in rats. Prog Neuropsychopharmacol Biol Psychiatry2013;45:242–9.

28] Amato RJ, Moerschbaecher JM, Winsauer PJ. Effects of pregnanolone and fluni-trazepam on the retention of response sequences in rats. Pharmacol BiochemBehav 2011;99(3):391–8.

29] Volke V, Soosaar A, Koks S, Vasar E, Mannisto PT. L-Arginine abolishes theanxiolytic-like effect of diazepam in the elevated plus-maze test in rats. EurJ Pharmacol 1998;351(3):287–90.

30] Yildiz AF, Ulak G, Tanyeri P, Erden F, Utkan T, Gacar N. 7-Nitroindazole, aneuronal nitric oxide synthase inhibitor, impairs passive-avoidance and ele-vated plus-maze memory performance in rats. Pharmacol Biochem Behav2007;87(4):434–43.

31] Itoh J, Nabeshima T, Kameyama T. Utility of an elevated plus-maze for theevaluation of memory in mice: effects of nootropics, scopolamine and elec-troconvulsive shock. Psychopharmacology (Berl) 1990;101(1):27–33.

32] Steckler T, Drinkenburg WH, Sahgal A, Aggleton JP. Recognition memory inrats—I. Concepts and classification. Prog Neurobiol 1998;54(3):289–311.

33] Pitsikas N, Rigamonti AE, Cella SG, Locatelli V, Sala M, Muller EE. Effects ofmolsidomine on scopolamine-induced amnesia and hypermotility in the rat.Eur J Pharmacol 2001;426(3):193–200.

34] Pitsikas N, Rigamonti AE, Cella SG, Muller EE. The GABAB receptor and recog-nition memory: possible modulation of its behavioral effects by the nitrergicsystem. Neuroscience 2003;118(4):1121–7.

35] Boultadakis A, Liakos P, Pitsikas N. The nitric oxide-releasing derivative of fer-ulic acid NCX 2057 antagonized delay-dependent and scopolamine-inducedperformance deficits in a recognition memory task in the rat. Prog Neuropsy-chopharmacol Biol Psychiatry 2010;34(1):5–9.

36] Furini CR, Rossato JI, Bitencourt LL, Medina JH, Izquierdo I, Cammarota M.Beta-adrenergic receptors link NO/sGC/PKG signaling to BDNF expression dur-ing the consolidation of object recognition long-term memory. Hippocampus2010;20(5):672–83.

37] Meyer RC, Spangler EL, Patel N, London ED, Ingram DK. Impaired learning inrats in a 14-unit T-maze by 7-nitroindazole, a neuronal nitric oxide synthaseinhibitor, is attenuated by the nitric oxide donor, molsidomine. Eur J Pharmacol1998;341(1):17–22.

38] Yamada K, Hiramatsu M, Noda Y, Mamiya T, Murai M, Kameyama T, et al.

Role of nitric oxide and cyclic GMP in the dizocilpine-induced impairment ofspontaneous alternation behavior in mice. Neuroscience 1996;74(2):365–74.

39] Mutlu O, Ulak G, Belzung C. Effects of nitric oxide synthase inhibitors 1-(2-trifluoromethylphenyl)–imidazole (TRIM) and 7-nitroindazole (7-NI) onlearning and memory in mice. Fundam Clin Pharmacol 2011;25(3):368–77.

[

Research 284 (2015) 179–186

40] Holscher C. Different strains of rats show different sensitivity to block oflong-term potentiation by nitric oxide synthase inhibitors. Eur J Pharmacol2002;457(2-3):99–106.

41] Boultadakis A, Pitsikas N. Effects of the nitric oxide synthase inhibitor L-NAME on recognition and spatial memory deficits produced by different NMDAreceptor antagonists in the rat. Neuropsychopharmacology 2010;35(12):2357–66.

42] Gourgiotis I, Kampouri NG, Koulouri V, Lempesis IG, Prasinou MD, GeorgiadouG, et al. Nitric oxide modulates apomorphine-induced recognition memorydeficits in rats. Pharmacol Biochem Behav 2012;102(4):507–14.

43] Jafari-Sabet M, Khodadadnejad MA, Ghoraba S, Ataee R. Nitric oxide in thedorsal hippocampal area is involved on muscimol state-dependent mem-ory in the step-down passive avoidance test. Pharmacol Biochem Behav2014;117:137–43.

44] Kahveci N, Gulec G, Ozluk K. Effects of intracerebroventricularly-injected mor-phine on anxiety, memory retrieval and locomotor activity in rats: involvementof vasopressinergic system and nitric oxide pathway. Pharmacol BiochemBehav 2006;85(4):859–67.

45] Zarrindast MR, Piri M, Nasehi M, Ebrahimi-ghiri M. Nitric oxide in the nucleusaccumbens is involved in retrieval of inhibitory avoidance memory by nicotine.Pharmacol Biochem Behav 2012;101(1):166–73.

46] Babaei R, Javadi-Paydar M, Sharifian M, Mahdavian S, Almasi-Nasrabadi M,Norouzi A, et al. Involvement of nitric oxide in pioglitazone memory improve-ment in morphine-induced memory impaired mice. Pharmacol Biochem Behav2012;103(2):313–21.

47] Allami N, Javadi-Paydar M, Rayatnia F, Sehhat K, Rahimian R, Norouzi A, et al.Suppression of nitric oxide synthesis by L-NAME reverses the beneficial effectsof pioglitazone on scopolamine-induced memory impairment in mice. Eur JPharmacol 2011;650(1):240–8.

48] Knepper BR, Kurylo DD. Effects of nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester on spatial and cued leaning. Neuroscience1998;83(3):837–41.

49] Tobin JR, Gorman LK, Baxter MG, Traystman RJ. Nitric oxide synthase inhibitiondoes not impair visual or spatial discrimination learning. Brain Res 1995;694(1-2):177–82.

50] Wiley JL, Willmore CB. Effects of nitric oxide synthase inhibitors on timing andshort-term memory in rats. Behav Pharmacol 2000;11(5):421–9.

51] Prast H, Philippu A. Nitric oxide as modulator of neuronal function. Prog Neu-robiol 2001;64(1):51–68.

52] Getting SJ, Segieth J, Ahmad S, Biggs CS, Whitton PS. Biphasic modulation ofGABA release by nitric oxide in the hippocampus of freely moving rats in vivo.Brain Res 1996;717(1–2):196–9.

53] Getting SJ, Segieth J, Ahmad S, Biggs CS, Whitton PS. Biphasic modulation ofGABA release by nitric oxide in the hippocampus of freely moving rats in vivo.Brain Res 1996;717(1-2):196–9.

54] Collinson N, Kuenzi FM, Jarolimek W, Maubach KA, Cothliff R, Sur C, et al.Enhanced learning and memory and altered GABAergic synaptic transmis-sion in mice lacking the alpha 5 subunit of the GABAA receptor. J Neurosci2002;22(13):5572–80.

55] Crestani F, Keist R, Fritschy JM, Benke D, Vogt K, Prut L, et al. Trace fear con-ditioning involves hippocampal alpha5 GABA(A) receptors. Proc Natl Acad SciUSA 2002;99(13):8980–5.

56] Rudolph U, Crestani F, Benke D, Brunig I, Benson JA, Fritschy JM, et al. Benzo-diazepine actions mediated by specific gamma-aminobutyric acid(A) receptorsubtypes. Nature 1999;401(6755):796–800.

57] Druid H, Holmgren P, Ahlner J. Flunitrazepam: an evaluation of use, abuse andtoxicity. Forensic Sci Int 2001;122(2-3):136–41.

58] Negrusz A, Moore CM, Stockham TL, Poiser KR, Kern JL, Palaparthy R, et al.Elimination of 7-aminoflunitrazepam and flunitrazepam in urine after a singledose of Rohypnol. J Forensic Sci 2000;45(5):1031–40.

59] Hauser CA, Wetzel CH, Berning B, Gerner FM, Rupprecht R. Flunitrazepam hasan inverse agonistic effect on recombinant alpha6beta2gamma2-GABAA recep-tors via a flunitrazepam-binding site. J Biol Chem 1997;272(18):11723–7.

60] Mandrioli R, Mercolini L, Raggi MA. Benzodiazepine metabolism: an analytical

perspective. Curr Drug Metab 2008;9(8):827–44.

61] Moore PK, Wallace P, Gaffen Z, Hart SL, Babbedge RC. Characteriza-tion of the novel nitric oxide synthase inhibitor 7-nitro indazole andrelated indazoles: antinociceptive and cardiovascular effects. Br J Pharmacol1993;110(1):219–24.