A Comparison of Cholinesterase Inhibitors in the Treatment of Quinuclidinyl Benzilate-Induced Behavioural Deﬁcit in Rats Performing the Multiple T-Maze

journal of applied biomedicine 12 (2014) 211–217

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Original Research Article A comparison of cholinesterase inhibitors in the treatment of quinuclidinyl benzilate-induced behavioural deﬁcit in rats performing the multiple T-maze

Jan Misik *, Jiri Kassa

University of Defence, Faculty of Military Health Sciences, Department of Toxicology, Třebešská 1575, 500 01 Hradec Králové, Czech Republic article info abstract

Article history: Cholinesterase inhibitors are beneficial in the treatment of Alzheimer's disease via indirect Received 23 October 2013 increase of cholinergic neuro-transmission. The aim of the present study was to evaluate the Received in revised form potency of inhibitors tacrine, rivastigmine and donepezil to reverse cholinergic depletion 2 January 2014 induced by 3-quinuclidinyl benzilate (QNB, 2 mg kg 1) in Wistar rats performing the multiple Accepted 20 January 2014 T-maze test. The effect of QNB on retention was compared to the effect of standard amnesic Available online 4 February 2014 drug, scopolamine, at the dose of 0.3 mg kg 1. Well-trained rats were treated intra-perito- neally with QNB, followed by another injection containing saline or tacrine (10 mg kg 1)or 1 1 Keywords: rivastigmine (1.2 mg kg ) or donepezil (2.65 mg kg ) 15 min later. Rats were subjected to Tacrine the T-maze task 30 min and 24 h following QNB administration. The passage time and fi Rivastigmine number of errors were observed. QNB signi cantly impaired the performance of rats in both Donepezil tested times in contrast to short-lasting effect of scopolamine (30 min only). The inhibitors fi Neurotransmission rivastigmine and donepezil signi cantly attenuated QNB-induced behavioural impairment Alzheimer's disease in the 30 min tests, whereas tacrine failed to have the same effect. Moreover, the perfor- fi Spatial orientation mance of tacrine-treated rats was worse due to cholinergic over-stimulation. Bene cial Acetylcholinesterase effects of all tested inhibitors including tacrine were evident in the 24 h test. # Acetylcholine 2014 Faculty of Health and Social Studies, University of South Bohemia in Ceske Muscarinic receptors Budejovice. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. Memory

strength and duration of neurotransmission, thus ChEIs can Introduction be used therapeutically for the symptomatic treatment of some neurodegenerative diseases such as Alzheimer's disease Inhibitors of cholinesterase (ChEIs) are a varied group of both (AD). Given that ACh is a key compound involved in learning natural and synthetic compounds which are able to inhibit and memory, ChEIs could potentially be considered as cholinesterase cleavage of the neurotransmitter acetylcholine cognitive enhancers. There are a number of substances with (ACh). Decreased degradation of ACh leads to increased possible beneﬁcial effects on AD patients and their cognitive

* Corresponding author. Tel.: +420 973 255 160; fax: +420 495 512 430. E-mail addresses: [email protected], [email protected] (J. Misik). 1214-021X/$ – see front matter # 2014 Faculty of Health and Social Studies, University of South Bohemia in Ceske Budejovice. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.jab.2014.01.006 212 journal of applied biomedicine 12 (2014) 211–217

functions including, for example, vitamin E, selegiline, non- steroidal anti-inflammatory drugs, statin drugs or omega-3 Materials and methods fatty acids, however the number of approved drugs is limited (Howland, 2011). Thus current therapeutic protocols based on Animals ChEIs, such as donepezil, galantamine and rivastigmine, are still valuable in the clinical treatment of AD (Wynn and Male Wistar rats (8–10-week-old, 150–200 g b.w.) were Cummings, 2004; Isensee et al., 2007; Pohanka, 2012) even if obtained from Velaz (Czech Republic). The animals were the effect of these inhibitors is palliative rather than curative. housed in groups of 6 in temperature- and light-controlled Centrally-acting ChEIs influence the cholinergic system in the breeding units at an approved animal facility. The animals brain, which is greatly affected by the pathological processes received standard rodent diet (Cerea corp.) and drinking water occurring in AD. Lack of neurotransmission is correlated with ad libitum. The food supply was limited to approximately 75% progressive cognitive decline and so the elevation of ACh ofthefreefeedingrateandtheacclimatizationperiodwasa transmission indirectly, via drugs such as ChEIs, can reduce minimum of 10 days. The use of animals in this study was the progression and severity of this decline, albeit only formally approved by the Ethics Committee of the Faculty of temporarily (Giacobini, 2000; Racchi et al., 2004). The beneficial Military Health Sciences, Czech Republic. All procedures effects of ChEIs on cognition have been demonstrated several involving animals were performed in accordance with current times in the laboratory rat, using either lesion- or drug- legislation. induced behavioural impairment models (Cheng and Tang, 1998; Poorheidari et al., 1998; Bejar et al., 1999). ChEIs usually Chemicals demonstrate a dose-dependent beneficial effect within a limited dose range, resulting in an inverted U-shaped dose- QNB HCl was synthesized de novo at the University of Defence effect curve (Braida et al., 1996; Wang and Tang, 1998; Bejar (Faculty of Military Health Sciences, Department of Toxicolo- et al., 1999). Therapeutic efficacy typically declines when gy) and was of 90% purity (HPLC determination). Scopolamine cholinesterase inhibition exceeds 40%, due to both central and hydrobromide and the ChEIs tacrine (9-amino-1,2,3,4-tetra- peripheral side effects of cholinergic over-stimulation. Fur- hydroacridine hydrochloride), rivastigmine ((S)-N-ethyl-3- thermore, the effective dose of ChEI varies considerably [(dimethyl-amino)ethyl]-N-methyl-phenylcarbamate hydro- between these studies, which could in part be attributed to gentartarate) and donepezil (1-benzyl-4-[(5,6-dimethox-1- the variable doses of cognitive-impairing agents and the indanon)-2-yl] methylpiperidine hydrochloride) were pur- behavioural methods used (Bejar et al., 1999). chased from Sigma–Aldrich Ltd. (Czech Republic) as well as A tropane alkaloid, scopolamine, is extensively used in the other chemicals for assessment of brain cholinesterase preclinical testing of new cognitive enhancers, as a standard inhibition [Tris–HCl buffer, acetylthiocholine, 5,50-dithiobis- model of 'cholinergic' amnesia in experimental animals (2-nitrobenzoic acid)]. Cholinergic antagonists and ChEIs (D'Hooge and De Deyn, 2001; De-Mello and Carobrez, 2002; were diluted in normal saline (0.9% natrium chloride, B. Gacar et al., 2011; Falsafi et al., 2012). Scopolamine affects Braun Medical Ltd., Czech Republic) immediately before cholinergic neurotransmission via the blockade of muscarinic administration to the experimental animals. (M) receptors, resulting in the failure of cognitive functions; the reversal of this cognitive impairment is predictive of the Inhibition of brain cholinesterase beneficial pharmaco-dynamic effects of cognitive enhancers (Lenz et al., 2012). As an alternative to scopolamine, the The doses of ChEIs were chosen on the basis of previous cholinergic antagonist 3-quinuclidinyl benzilate (QNB) has studies (Kirkby et al., 1996; Kosasa et al., 1999; Bruins Slot et al., also been used as a model of 'cholinergic' amnesia in rats 2003); tacrine at 10 mg kg 1, rivastigmine at 1.2 mg kg 1 and (Krejcova et al., 2004; Kunesova et al., 2008) and was used as an donepezil at 2.65 mg kg 1.Thein vivo inhibitory potential of amnesic agent in this current study. Similar to scopolamine, these doses was verified by evaluating the inhibition of brain QNB acts as a non-selective competitive antagonist of M cholinesterase in the rats after i.p. administration. Rats were receptors (Kinney et al., 1995; Haga et al., 2012). The doses of administered with the ChEIs at the same dose and via the both anticholinergic drugs have been established in previous same route as those rats utilized for the behavioural testing studies, producing an amnesic effect on reference memory in and euthanized by CO2 15 min after administration of rats performing several behavioural tests, including the the drug. The brains were removed immediately and multiple T-maze, elevated T-maze, water maze and passive homogenized using a homogenizator Ultra-Turrax T25 Basic avoidance tasks (De-Mello and Carobrez, 2002; Krejcova et al., (IKA® – WERKE, Germany) in Tris–HCl buffer (0.02 mol/l, pH 2004; Kunesova et al., 2008; Gacar et al., 2011). 7.6, 1:10) and the cholinesterase activity was determined by The current study assesses the potential beneficial effects standard spectrophotometric methods (Ellman et al., 1961), of a single dose of the three ChEIs tacrine, rivastigmine and using acetylthiocholine as a substrate (Tris–HCl buffer, donepezil on QNB-induced cholinergic depletion in male N = 0.1 mol/l, pH 7.6). The Helios Alpha spectrophotometer Wistar rats performing the multiple T-maze, a behavioural was used for determination of absorbance at 436 nm and the test of spatial learning and memory (Kunesova et al., 2008; cholinesterase activity was expressed as mkat/kg (mmol Sharma et al., 2010; Falsafi et al., 2012; Sase et al., 2012). The substrate hydrolyzed/kg wet tissue within 1 s). Untreated ChEIs were administered 15 min after the dose of QNB, and control values for brain cholinesterase activity were obtained their efficacy was evaluated 30 min and 24 h after the from rats administered with saline instead of ChEI (saline administration of QNB. control). journal of applied biomedicine 12 (2014) 211–217 213

T-maze test Table 1 – Inhibition (%) of brain cholinesterase in vivo, 15 min after i.p. administration of cholinesterase inhibi- W The multiple T-maze consisted of several segments measuring tors tacrine, rivastigmine and donepezil. Mean S.E.M. 12 cm 20 cm 11 cm (L W H) with seven different choice Treatment/dose Cholinesterase Inhibition points, enabling possible right or left exits. The rats were activity (mkat kg 1) (%)

released from a starting position and the time taken to reach Saline 1 ml kg 1 170.6 7.48 – the goal compartment (containing a reward of several food Tacrine 10 mg kg 1 102.02 10.43 40.2 pellets) was measured. The distance between the starting Rivastigmine 1.2 mg kg 1 100.99 12.88 40.8 position and the goal compartment was 185 cm (Kunesova Donepezil 2.65 mg kg 1 99.29 11.82 41.8 et al., 2008). The rats were food-deprived, receiving 75% of common daily food intake and were trained once daily, with the aim being to pass through the maze in less than 3 s without entering the wrong arm. The maze was cleaned with 70% Table 2 – Number of error entries in rats performing the ethanol between trials to avoid odour cues. Well-trained rats multiple T-maze during training, 30 min and 24 h test W were able to perform the maze within the set time and without (mean S.E.M., n = 8). There were no significant effects in 30 min (H = 7.74, p = 0.17) and 24 h (H = 10.21, any errors after 30 consecutive days of training (excluding (5, 48) (5, 48) p = 0.07). weekends). Any initial inter-individual differences in the learning rate between rapid- and slow-learning rats were Treatment Mean number of error entries fully compensated for by the long-term training. Training 30 min 24 h On the day of the experiment, and 24 h after the last Saline + saline 0 0 0 training session, the rats were randomly divided into 6 groups QNB + saline 0 0.38 0.18 0.38 0.74 containing 8 individuals each. The anticholinergic drug QNB Scopolamine + saline 0 1.75 0.82 0 was administered to animals via an i.p. injection at a dose of QNB + tacrine 0 0.13 0.13 0 2.0 mg kg 1 (in a standardized volume of 1 ml kg 1) 30 min QNB + rivastigmine 0 0.63 0.32 0 before the test. Treated groups received a dose of either tacrine QNB + donepezil 0 0.63 0.32 0 (10.0 mg kg 1), rivastigmine (1.2 mg kg 1) or donepezil (2.65 mg kg 1) 15 min after administration of the QNB. A positive control group received QNB followed by saline in place persist up to the 24 h time point (U =1,Z = 3.25, p < 0.001). The of a ChEI and a second positive control group received mean number of wrong arm entries (error entries) was higher scopolamine at a dose of 0.3 mg kg 1 instead of QNB, followed in the scopolamine-treated group than in the QNB-treated by saline in place of a ChEI. The final group received normal group at the 30 min time point but this difference did not reach saline (1 ml kg 1) 15 and 30 min before the test, in place of both statistical significance (U = 23.5, Z = 0.89, p = 0.38) (Table 2). the anticholinergic drug and the ChEI. The performance of rats No error entries were observed in the scopolamine-treated rats running the maze was evaluated at 30 min and 24 h after the after 24 h. Subjectively, there was a mild hyperactivity QNB (or scopolamine) injection; the time taken to pass through observed in the QNB-treated groups several minutes post the maze and the number of entries into the wrong arms of the administration, which was not present in the scopolamine- maze were recorded. treated group. When treated with ChEIs, the performance of rats improved Statistical analysis in both the rivastigmine- and donepezil-treated groups (Fig. 2); there was no significant difference between the saline controls Statistical analysis was performed using Statistica software '98 [(Fig._1)TD$IG] Edition. Data were analyzed using non-parametric tests – Kruskal–Wallis test with post hoc or Mann–Whitney U-test. All values are presented as mean S.E.M. (standard error of the mean). Differences were considered at the significance level 2a = 0.05.

Results

The in vivo inhibitory potential of selected doses of ChEIs are shown in Table 1. All ChEIs were able to cause approximately 40% inhibition of the brain cholinesterase activity. In the multiple T-maze, the cholinergic antagonist QNB signiﬁcantly impaired the behavioural response of rats. QNB- – S1 treated rats showed prolonged passage times (H(5, 48) = 28.8, Fig. 1 Effect of cholinergic antagonists QNB (2.0 mg kg ) S p = 0.006; Fig. 1) as well as an increased incidence of wrong and scopolamine (0.3 mg kg 1) on passage times (s) entries at both the 30 min and 24 h time points (Table 2). The through the multiple T-maze in 30 min trial and 24 h trial. effect of scopolamine was equivalent to that of QNB at the Mean W S.E.M. * Statistically significant from the saline 30 min time point (U = 24, Z = 0.84, p = 0.44), but did not control. 214 journal of applied biomedicine 12 (2014) 211–217 [(Fig._2)TD$IG]

In the present study, scopolamine has been compared with another cholinergic antagonist QNB, and the effects of both drugs on the behavioural responses of rats performing the multiple T-maze test were compared. Both anticholinergic drugs significantly decreased the acquired behavioural response of rats to the multiple T-maze. The QNB-induced effect was almost identical to the scopolamine-induced effect at the 30 min time point. In contrast to scopolamine-treated rats, after 24 h the prolonged passage times remained evident in the QNB-treated control group. Incidence of error entries was more frequent in the scopolamine-treated rats compared to the QNB-treated group at the 30 min time point and error entries only occurred in the QNB- Fig. 2 – Passage times (s) of rats performing the multiple T- treated group after 24 h. This fact, along with the persistently maze at training trial, 30 min trial and 24 h trial. Treatment prolonged passage times through the maze, indicates that the provided 30 min (saline/QNB) and 15 min (saline/ChEI) QNB-induced cholinergic depletion is longer-lasting. In prac- before 30 min trial. Experimental groups: control (saline tice, this means that the long-term affects of QNB in these fi + saline), QNB (QNB + saline), rivastigmine (QNB models enables the observation of potential bene cial effects + rivastigmine), donepezil (QNB + donepezil), tacrine (QNB of inhibitors over longer periods of time in comparison with + tacrine). Mean W S.E.M. * Statistically significant from the standard scopolamine treatment. According to the cholinergic saline control. hypothesis (Contestabile, 2011), the behavioural effect of cholinergic antagonists is primarily caused by blockade of M1 receptors, which are abundant in areas of the brain considered to be involved in learning and memory (e.g. and both of these ChEIs-treated groups 30 min after QNB hippocampus, cortex). Using a radio-labelled form of QNB, administration (H(5, 48) = 28.80, p1 = 0.34, p2 = 0.20). The group washout from these target areas of rat brain was not apparent treated with tacrine showed no significant improvement and during the first 24 h (Gibson et al., 1991). Moreover, the the performance of these rats was somewhat impaired at the washout of QNB occurred at a very slow rate, with only 50% < 30 min time point (H(5, 48) = 28.80, p 0.001). Furthermore, loss of activity over 3 weeks; this could be connected to the tacrine-treated rats demonstrated mild cholinergic symptoms significant behavioural deficit observed after 24 h in this study. such as mastication, ataxia and mild tremor; these were not Furthermore, contribution from the nicotinic (N) receptors and quantified but occurred consistently in all of these rats and possibly other systems such as the N-methyl-D-aspartate with similar intensity. These signs were observed during the receptors, also known to be involved in memory formation test 15 min after the tacrine injection and diminished after (Bymaster et al., 1993; Kinney et al., 1995; Falsafi et al., 2012), approximately 30 min. At the 24 h time point, the performance may also play a role in the longer-lasting effects of QNB. of rats was improved in all three ChEI-treated groups including tacrine, in comparison to the positive QNB-treated controls The effect of ChEIs on the QNB-induced behavioural

(H(3, 32) = 15.72, p = 0.0013). There were no error entries made by impairment the ChEIs-treated rats at the 24 h time point (Table 2). The curative effect of ChEIs on QNB-induced behavioural impairment in rats performing the multiple T-maze test has Discussion been evaluated in this study. When administered therapeutically 15 min after the QNB, some of the ChEIs (rivastigmine and The inhibitory potential of ChEIs donepezil) were able to ameliorate the behavioural impairment, although the performance of these ChEIs-treated The chosen doses of ChEIs inhibited the cholinesterase activity groups did not reach the performance of the saline control in rat brains by approximately 40%, which is considered the group. Treatment with tacrine induced additional deteriora- theoretical limit above which the therapeutic efficacy of such tion of the behavioural response, which was more pronounced drugs is usually decreased (Bejar et al., 1999). Nevertheless, the than the impairment induced by QNB alone. The observed doses of ChEIs might be responsible for inhibition exceeding deficit, demonstrated as prolonged passage time, was obvi- this 40% threshold due to a dilution effect (Hallak and ously non-cognitive; tacrine-treated rats showed typical signs Giacobini, 1989) and for the related dose-dependent side of cholinergic over-stimulation such as a mild tremor and effects (Dronfield et al., 2000) observed in tacrine-treated rats ataxia, indicating that the impairment was caused by the side in this study. effects of the drug and that the prolonged passage time was due to decreased mobility. Surprisingly, tacrine-treated rats The effect of QNB and scopolamine on the T-maze performance showed the lowest number of error entries among the experimental groups; this low number of errors was probably Scopolamine treatment is a standard approach used to test secondary to decreased exploratory activity caused by cholin- cognitive enhancers and other psychoactive compounds in ergic over-stimulation. Moreover, tacrine also causes activa- pharmacological studies (Falsafi et al., 2012; Lenz et al., 2012). tion of N and M receptors (with the exception of cholinesterase journal of applied biomedicine 12 (2014) 211–217 215

inhibition) and stimulation of ACh release (Wagstaff and due to cholinergic over-stimulation. The beneficial effects of McTavish, 1994), which may contribute to overall cholinergic all three of the tested inhibitors, including tacrine, were excess. Tacrine is considered to be less affective at inhibiting evident in the reduction of long-lasting QNB-induced beha- brain AChE, but more potent at the periphery where it inhibits vioural deficits. mainly plasma butyrylcholinesterase (Cheng and Tang, 1998). Therapeutic doses of tacrine used in behavioural studies Methodological consideration are usually relatively high (up to 30.0 mg kg 1; Kirkby et al., 1996); this may be related to low bioavailability or rapid The T-maze test required the rats to learn a complex route metabolism of the drug (Cheng and Tang, 1998; Bejar et al., with several choice points and a consistent correct path from 1999). Cheng and Tang (1998) observed behavioural improve- trial to trial. Performing animals learn to identify the hidden ment in rats treated orally with 8 mg kg 1 of tacrine, which food reward and their learning ability is observable as a correlates to less than 10% of cortical and hippocampal decreased passage time through the maze and also as a cholinesterase inhibition. Bejar et al. (1999) found beneficial decreased incidence of incorrect entries. Thus the perfor- effects of tacrine at a high dose of 12.5 mg kg 1 whereas Bruins mance of well-trained rats in the T-maze could be attributed to Slot et al. (2003) found no effect of tacrine at a dose of spatial reference memory. Accordingly, the T-maze and other 10 mg kg 1 on scopolamine-induced behavioural deficits. tests of spatial navigation such as the water maze have been Whereas tacrine failed to reverse QNB-induced impair- extensively used as a paradigm in behavioural studies ments at the 30 min time point, rivastigmine (1.2 mg kg 1) and investigating the cognitive functions of experimental animals donepezil (2.65 mg kg 1) were effective with no observable (Lohninger et al., 2001; Zheng et al., 2009; Sase et al., 2012). cholinergic side effects at the tested doses. Rivastigmine was Experimental manipulation and disruption of spatial memory subjectively the most effective of the ChEIs tested in this study. in behavioural tests via cholinergic antagonists has been Contrary to tacrine, rivastigmine is more selective for brain performed using mainly scopolamine (Bymaster et al., 1993; cholinesterase than the peripheral compartment (Weinstock von Linstow Roloff et al., 2007; Klinkenberg and Blokland, 2010; et al., 1994; Racchi et al., 2004) and therefore a lower dose is Doguc et al., 2012), less frequently using QNB (Mezey et al., necessary to reach an appropriate level of brain AChE 1999; Krejcova et al., 2004; Kunesova et al., 2008) or other drugs. inhibition. Rivastigmine produces a similar inhibition to However, cholinergic antagonists potentially interact with tacrine in the cortex but is more effective in hippocampus several different systems. Besides the cholinergic transmis- (Bejar et al., 1999), which could be beneficial for spatial sion directly connected with cognition, these drugs can also navigation. Rivastigmine at a dose of 1.5 mg kg 1 effectively interact with non-cognitive processes such as sensory and reverses the spatial memory deficit induced by 0.5 mg kg 1 motor functions or motivation (Hodges et al., 2009). These scopolamine, which corresponds to almost 50% AChE inhibi- side-effects could parallel the mnemonic function of ACh and tion in the cortex (Bejar et al., 1999). may eventually lead to impairment in behavioural tests Another tested compound, donepezil, is a centrally-acting (Hodges et al., 2009; Stuchlik et al., 2012). Thus, it is sometimes ChEI which selectively inhibits AChE (Kosasa et al., 1999; Ogura difficult to distinguish direct cognitive impairment from the et al., 2000; Racchi et al., 2004). Moreover, donepezil also drug side-effects, and these are confounding factors that elevates ACh levels via stimulation of catecholamine release partially contribute to inter-laboratory variability (Hodges (Giacobini et al., 1996). Efficacy of donepezil was found at doses et al., 2009). ranging from 1 to 3 mg kg 1 (Kirkby et al., 1996; Cheng and We assumed that the impairment in performances of the T- Tang, 1998; Poorheidari et al., 1998; Spowart-Manning and van maze was possibly secondary, related to motivational deficits der Staay, 2004), whereas van der Staay and Bouger (2005) induced by anticholinergic drugs rather than the direct found that donepezil at doses ranging from 0.1 to 1.0 mg kg 1 impairment of the spatial navigation in both the QNB- and had no effect on scopolamine-induced behavioural deficits in scopolamine-treated rats. No sensorimotor or motivational rats performing spatial discrimination tasks. Additional side effects of QNB at the same dose were found either neuroprotective effects of donepezil are probably mediated affecting spatial navigation in the water maze or fear by activation of N receptors (Fujiki et al., 2005, 2008). conditioning in the passive avoidance test. However, the T- In general, ChEIs are often shown to be beneficial in the maze is based on a different principle of positive motivation. treatment of pharmacologically-induced cognitive deficits in The response of rats in the T-maze could be affected by a laboratory animals, although the results are rather inconsis- peripheral side effect of dry oral mucous membranes, which is tent between studies. The variability between the efficiencies mediated by reduced salivation through the M3 receptors in of different ChEIs depends mainly on their central/peripheral the salivary glands (Hodges et al., 2009). Xerostomia ('a dry inhibitory potential, selectivity for individual brain areas, mouth effect') causes difficulty in eating dry food (Vance, 1965) cholinesterase selectivity, chemical properties and, not least, and thus lessens positive motivation in the T-maze; Hodges on the doses of the both the ChEI and the cognitive deficit- et al. (2009) reported a reduced food intake in rats administered inducing agent used. It is evident that at least part of the with low doses of scopolamine, and in this study both the referred improvements is related primarily to the positive QNB- and scopolamine-treated rats usually refused the food effect of ChEIs on non-cognitive side effects. In the present reward when they reached the goal compartment of the maze. study, ChEIs administered as a single therapeutic dose When an effective ChEI was administered therapeutically, the improved the behavioural responses of rats in the multiple drug-induced state was attenuated and the rats improved T-maze test. The ChEIs rivastigmine and donepezil were their performance in the maze. Thus, the observed effect of effective but tacrine was ineffective at the 30 min time point, anticholinergic drugs in the T-maze is possibly due to 216 journal of applied biomedicine 12 (2014) 211–217

motivational side effects rather than the direct effect on Bruins Slot, L.A., Chopin, P., Colpaert, F.C., 2003. Tacrine- retrieval of spatial information as noted previously (Krejcova scopolamine interactions on state-dependent retrieval. Psychopharmacology (Berl.) 166, 33–39. et al., 2004; Kunesova et al., 2008). Moreover, QNB was found to Bymaster, F.P., Heath, I., Hendrix, J.C., Shannon, H.E., 1993. have no impact on the retrieval of reference memory in other Comparative behavioral and neurochemical activities of behavioural tests; this is similar to scopolamine, which is cholinergic antagonists in rats. J. Pharmacol. Exp. Ther. 267, thought to disrupt short-term and working memory rather 16–24. than reference memory (von Linstow Roloff et al., 2007; Cheng, D.H., Tang, X.C., 1998. Comparative studies of huperzine Klinkenberg and Blokland, 2010; Doguc et al., 2012). Neverthe- A E2020, and tacrine on behavior and cholinesterase – less, scopolamine data are rather inconsistent, sometimes also activities. Pharmacol. Biochem. Behav. 60, 377 386. Contestabile, A., 2011. The history of the cholinergic hypothesis. pointing a disruption of retrieval (Gacar et al., 2011). This might Behav. Brain Res. 221, 334–340. in part be due to the variable doses of scopolamine adminis- D'Hooge, R., De Deyn, P.P., 2001. Applications of the Morris water tered to animals in different behavioural studies, varying from maze in the study of learning and memory. Brain Res. Brain 0.2 to 2.0 mg kg (Bejar et al., 1999). The use of lower doses is Res. Rev. 36, 60–90. recommended due to the variable central and peripheral side De-Mello, N., Carobrez, A.P., 2002. Elevated T-maze as an animal effects seen with higher doses (Hodges et al., 2009). If there is a model of memory: effects of scopolamine. Behav. Pharmacol. – direct effect on retrieval in the T-maze, it could be potentially 13, 139 148. Doguc, D.K., Delibas, N., Vural, H., Altuntas, I., Sutcu, R., Sonmez, found by using a lower dose of QNB or scopolamine, without Y., 2012. Effects of chronic scopolamine administration on causing a dry mouth effect. spatial working memory and hippocampal receptors related In summary, ChEIs used in this study could not be directly to learning. Behav. Pharmacol. 23, 762–770. judged as cognitive enhancers (Poorheidari et al., 1998) on the Dronfield, S., Egan, K., Marsden, C.A., Green, A.R., 2000. basis of the data presented. Although they reversed QNB- Comparison of donepezil-, tacrine-, rivastigmine- and induced deficits in the T-maze task, there is no direct evidence metrifonate-induced central and peripheral cholinergically mediated responses in the rat. J. Psychopharmacol. 14, that the impairment was primarily related to cognitive 275–279. disruption. Nevertheless, the ChEIs could overcome the Ellman, G.L., Courtney, D.K., Andres Jr., V., Featherstone, R.M., QNB-induced state, though this reversal may be dependent 1961. A new and rapid determination of acetylcholinesterase on another mechanism. The ChEIs showed a curative effect on activity. Biochem. Pharmacol. 7, 88–93. experimentally induced cholinergic hypofunction and thus Falsafi, S.K., Deli, A., Höger, H., Pollak, A., Lubec, G., 2012. could be useful in the treatment of AD (Cheng and Tang, 1998; Scopolamine administration modulates muscarinic, Poorheidari et al., 1998). Further investigation should be nicotinic and NMDA receptor systems. PLoS ONE 7, http://dx. doi.org/10.1371/journal.pone.0032082. focused on investigating potential effects of lower doses of Fujiki, M., Kobayashi, H., Uchida, S., Inoue, R., Ishii, K., 2005. these cognitive-impairing agents, to avoid side effects or by Neuroprotective effect of donepezil, a nicotinic choice of behavioural tests that do not confound the possible acetylcholine-receptor activator, on cerebral infarction in disruption of positive motivation as the T-maze test. rats. Brain Res. 1043, 236–241. Fujiki, M., Kubo, T., Kamida, T., Sugita, K., Hikawa, T., Abe, T., Ishii, K., Kobayashi, H., 2008. Neuroprotective and Conflicts of interests antiamnesic effect of donepezil, a nicotinic acetylcholine- receptor activator, on rats with concussive mild traumatic brain injury. J. Clin. Neurosci. 15, 791–796. fl The authors state no con icts of interests. Gacar, N., Mutlu, O., Utkan, T., Komsuoglu Celikyurt, I., Gocmez, S.S., Ulak, G., 2011. Beneficial effects of resveratrol on scopolamine but not mecamylamine induced memory Acknowledgements impairment in the passive avoidance and Morris water maze tests in rats. Pharmacol. Biochem. Behav. 99, 316–323. Giacobini, E., 2000. Cholinesterase inhibitors stabilize Alzheimer This work was supported by Grant Agency of the Czech disease. Neurochem. Res. 25, 1185–1190. Republic (no. P303/12/0611). The authors would like to thank Giacobini, E., Zhu, X.D., Williams, E., Sherman, K.A., 1996. The Mrs. E. Reslova for her skilful technical assistance, Dr. G. effect of the selective reversible acetylcholinesterase Krejcova Kunesova for her help with data evaluation and Dr. inhibitor E2020 on extracellular acetylcholine and biogenic M. Dobromylskyj and Dr. R.E. Raskin for their proofreading of amine levels in rat cortex. Neuropharmacology 35, 205–211. the English language. Gibson, R.E., Zeeberg, B.R., Melograna, J.M., Wang, T.F., Ruch, J., Braun, A., Reba, R.C., 1991. In vivo dissociation kinetics of [3H]quinuclidinyl benzilate: relationship to muscarinic references receptor concentration and in vitro kinetics. Brain Res. 553, 110–161. Haga, K., Kruse, A.C., Asada, H., Yurugi-Kobayashi, T., Shiroishi, M., Zhang, C., Weis, W.I., Okada, T., Kobilka, B.K., Haga, T., Bejar, C., Wang, R.H., Weinstock, M., 1999. Effect of rivastigmine Kobayashi, T., 2012. Structure of the human M2 muscarinic on scopolamine-induced memory impairment in rats. Eur. J. acetylcholine receptor bound to an antagonist. Nature 482, Pharmacol. 383, 231–240. 547–551. Braida, D., Paladini, E., Griffini, P., Lamperti, M., Maggi, A., Sala, Hallak, M., Giacobini, E., 1989. Physostigmine, tacrine and M., 1996. An inverted U-shaped curve for metrifonate: the effect of multiple doses on acetylcholine heptylphysostigmine on radial maze performance in rats: metabolism in rat brain. Neuropharmacology 28, 199–206. comparison with other cholinesterase inhibitors. Eur. J. Hodges Jr., D.B., Lindner, M.D., Hogan, J.B., Jones, K.M., Markus, E. Pharmacol. 302, 13–20. J., 2009. Scopolamine induced deficits in a battery of rat journal of applied biomedicine 12 (2014) 211–217 217

cognitive tests: comparisons of sensitivity and specificity. on scopolamine-induced deficits in the delayed matching to Behav. Pharmacol. 20, 237–251. position task in rats: a comparison with the cholinesterase Howland, R.H., 2011. Alternative drug therapies for dementia. J. inhibitor E2020. Psychopharmacology (Berl.) 135, 242–255. Psychosoc. Nurs. Ment. Health Serv. 49, 17–20. Racchi, M., Mazzucchelli, M., Porrello, E., Lanni, C., Govoni, S., Isensee, K., Petroianu, G., Stark, H., 2007. Pharmacological 2004. Acetylcholinesterase inhibitors: novel activities of old aspects of cognitive impairment: past, present and future of molecules. Pharmacol. Res. 50, 441–451. drugs in dementia. J. Appl. Biomed. 5, 57–70. Sase, S., Khan, D., Sialana, F., Höger, H., Russo-Schlaff, N., Lubec, Kinney, H.C., Panigrahy, A., Rava, L.A., White, W.F., 1995. 3- G., 2012. Modafinil improves performance in the multiple T- Dimensional distribution of [H3] quinuclidinyl benzilate Maze and modifies GluR1, GluR2, D2 and NR1 receptor binding to muscarinic cholinergic receptors in the complex levels in the C57BL/6J mouse. Amino Acids 43, developing human brain-stem. J. Comp. Neurol. 362, 350–367. 2285–2292. Kirkby, D.L., Jones, D.N., Barnes, J.C., Higgins, G.A., 1996. Effects Sharma, S., Rakoczy, S., Brown-Borg, H., 2010. Assessment of of anticholinesterase drugs tacrine and E2020, the 5-HT(3) spatial memory in mice. Life Sci. 87, 521–536. antagonist ondansetron, and the H(3) antagonist Spowart-Manning, L., van der Staay, F.J., 2004. The T-maze thioperamide, in models of cognition and cholinergic continuous alternation task for assessing the effects of function. Behav. Pharmacol. 7, 513–525. putative cognition enhancers in the mouse. Behav. Brain Res. Klinkenberg, I., Blokland, A., 2010. The validity of scopolamine 151, 37–46. as a pharmacological model for cognitive impairment: a Stuchlik, A., Petrasek, T., Hatalova, H., Rambousek, L., review of animal behavioral studies. Neurosci. Biobehav. Nekovarova, T., Vales, K., 2012. Behavioral tests for Rev. 34, 1307–1350. evaluation of information processing and cognitive deficits Kosasa, T., Kuriya, Y., Matsui, K., Yamanishi, Y., 1999. Effect of in rodent animal models of neuropsychiatric disorders. In: donepezil hydrochloride (E2020) on basal concentration of Burne, T.H.J. (Ed.), Schizophrenia in the 21st Century. InTech, extracellular acetylcholine in the hippocampus of rats. Eur. J. Rijeka, pp. 153–180. Pharmacol. 380, 101–107. Vance, W.B., 1965. Observations on the role of salivary Krejcova, G., Patocka, J., Slaninova, J., 2004. Effect of humanin secretions in the regulation of food and fluid intake in the analogues on experimentally induced impairment of spatial white rat. Psychol. Monogr. 79, 1–22. memory in rats. J. Pept. Sci. 10, 636–639. van der Staay, F.J., Bouger, P.C., 2005. Effects of the Kunesova, G., Hlavacek, J., Patocka, J., Evangelou, A., Zikos, C., cholinesterase inhibitors donepezil and metrifonate on Benaki, D., Paravatou-Petsotas, M., Pelecanou, M., Livaniou, scopolamine-induced impairments in the spatial cone field E., Slaninova, J., 2008. The multiple T-maze in vivo testing of orientation task in rats. Behav. Brain Res. 156, 1–10. the neuroprotective effect of humanin analogues. Peptides von Linstow Roloff, E., Harbaran, D., Micheau, J., Platt, B., Riedel, 29, 1982–1987. G., 2007. Dissociation of cholinergic function in spatial and Lenz, R.A., Baker, J.D., Locke, Ch., Rueter, L.E., Mohler, E.G., procedural learning in rats. Neuroscience 146, 875–889. Wesnes, K., bi-Saab, W., Saltarelli, M.D., 2012. The Weinstock, M., Razin, M., Chorev, M., Enz, A., 1994. scopolamine model as a pharmacodynamic marker in early Pharmacological evaluation of phenyl-carbamates as CNS drug development. Psychopharmacology 220, 97–107. selective acetylcholinesterase inhibitors. J. Neural Transm. Lohninger, S., Strasser, A., Bubna-Littitz, H., 2001. The effect of L- Suppl. 43, 219–225. carnitine on T-maze learning ability in aged rats. Arch. Wagstaff, A.J., McTavish, D., 1994. Tacrine. Drug Aging 4, Gerontol. Geriatr. 32, 245–253. 510–540. Mezey, S.Z., Szekely, A.D., Bourne, R.C., Kabai, P., Csillag, A., Wang, T., Tang, X.C., 1998. Reversal of scopolamine-induced 1999. Changes in binding to muscarinic and nicotinic deficits in radial maze performance by ()-huperzine A: cholinergic receptors in the chick telencephalon, following comparison with E2020 and tacrine. Eur. J. Pharmacol. 349, passive avoidance learning. Neurosci. Lett. 270, 75–78. 137–142. Ogura, H., Kosasa, T., Kuriya, Y., Yamanishi, Y., 2000. Donepezil, Wynn, Z.J., Cummings, J.L., 2004. Cholinesterase inhibitor a centrally acting acetylcholinesterase inhibitor, alleviates therapies and neuropsychiatric manifestations of learning deficits in hypocholinergic models in rats. Methods Alzheimer's disease. Dement. Geriatr. Cogn. Disord. 17, Find. Exp. Clin. Pharmacol. 22, 89–95. 100–108. Pohanka, M., 2012. Acetylcholinesterase inhibitors: a patent Zheng, J.F., Patil, S.S., Chen, W.Q., An, W., He, J.Q., Höger, H., review (2008–present). Expert Opin. Ther. Pat. 22, 871–886. Lubec, G., 2009. Hippocampal protein levels related to spatial Poorheidari, G., Stanhope, K.J., Pratt, J.A., 1998. Effects of the memory are different in the Barnes maze and in the multiple potassium channel blockers, apamin and 4-aminopyridine, T-maze. J. Proteome Res. 8, 4479–4486.