Pharmacological Reports Copyright © 2012 2012, 64, 10661080 by Institute of Pharmacology ISSN 1734-1140 Polish Academy of Sciences
Involvement of cholinergic receptors in the different stages of memory measured in the modified elevated plus maze test in mice
Marta Kruk-S³omka, Barbara Budzyñska, Gra¿yna Bia³a
Chair, Department of Pharmacology and Pharmacodynamics, Medical University of Lublin, Chodki 4A, PL 20-093 Lublin, Poland
Correspondence: Marta Kruk-S³omka, e-mail: [email protected]
Abstract: Background and Methods: Several lines of evidence support a strong relationship between cholinergic pathways and memory. The aim of our experiments was to examine the mechanisms involved in the formation of different memory stages, to evaluate the impact of substances, which affect the cholinergic system in mice, with an employment of the modified elevated plus maze (mEPM) test. This test allows examining different processes of memory (acquisition, consolidation and retrieval), depending on the time of drug treatment. The time period, necessary for mice to move from the opened arm to the enclosed arm (i.e., transfer latency, TL) was used as an index of memory. Results: Our findings revealed that in both memory acquisition and consolidation, nicotine, an agonist of cholinergic receptors (0.035 and 0.175 mg/kg, free base, sc), reduced TL on the second day of the experiment (TL2), thus improving memory. In turn, sco- polamine, an antagonist of cholinergic receptors (0.3 and 1.0 mg/kg, ip), significantly increased TL2 values, impairing cognition. Subsequently, we evaluated the influence of mecamylamine, a non-selective antagonist of nicotinic cholinergic receptors (nAChRs) and of varenicline, an a4b2 partial nAChRs agonist, on memory-related behaviors induced by nicotine and scopolamine. Acute injections of mecamylamine (0.5 and 1.0 mg/kg, ip) and varenicline (0.5 and 1.0 mg/kg, ip), prior to the injections of nicotine (0.035 mg/kg) or scopolamine (1.0 mg/kg), significantly suppressed nicotine-induced memory improvement or scopolamine- induced memory impairment. Conclusion: Our studies indicate that the cholinergic system plays a crucial role in memory processes. Pharmacological manipula- tion of cholinergic transmission can be the base to develop more effective pharmacotherapies for these memory disturbances in which cholinergic receptors are involved.
Key words: nicotine, scopolamine, mecamylamine, varenicline, memory and learning, modified elevated plus maze
Abbreviations: ACh – acetylcholine, AD – Alzheimer’s dis- Introduction ease, DA – dopamine, GABA – g-aminobutyric acid, mAChRs – muscarinic cholinergic receptors, mEPM – modified elevated plus maze, nAChRs – nicotinic cholinergic receptors, NMDA – A fair number of studies imply some role of the cho- N-methyl-D-aspartate, TL – transfer latency, VTA – ventral linergic system in cognitive functions, specifically in tegmental area attention and memory encoding. Spatial memory is one
1066 Pharmacological Reports, 2012, 64, 10661080 Cholinergic receptors in different stages of memory Marta Kruk-S³omka et al.
of the most essential forms of higher cognitive pro- is essential for working memory-related responses, cesses, demanding a certain capacity to record environ- measured in the radial arm maze test in rats [2, 3, 37]. mental and spatial orientation data. Thus, spatial mem- An abnormally regulated cholinergic system, with ory formation represents a simple form of episodic-like a decline of cholinergic neurons in the basal forebrain, memory in rodents that engages both the basal cho- have been hypothesized as being responsible for the linergic system and its target structures [17, 18, 24]. cognitive symptoms of neuropsychiatric disorders, It is known that acetylcholine (ACh) is a neuro- e.g., Alzheimer’s disease (AD) [18, 32]. The first transmitter essential for these cognitive functions. clinical trials in patients, suffering from AD, revealed Previous studies concluded a strong correlation be- no changes in the number, the structure or the func- tween synaptic ACh levels and cognitive function im- tion of mAChRs, while significantly decreased levels provement, providing evidence that acetylcholinester- of a4b2 nAChRs subtypes were observed, especially ase (an ACh breaking down enzyme), lead to in- in the cortex and the hippocampus [18, 29, 32, 41]. creased ACh levels in the brain, especially in cortical That reported decline may be appropriate for the cog- and hippocampal brain regions, i.e., the two major ar- nitive deficits that characterize the condition. There- eas involved in cognitive processes. Accordingly, fore, an administration of drugs, which improve cho- many acetylcholinesterase inhibitors improve per- linergic signaling, is able to enhance cognitive func- formance levels in several cognitive models, involv- tions [29, 43]. What is more, literature data suggest ing humans or rodents [34]. that agonists and partial agonists of various subtypes Muscarinic (mAChRs) and nicotinic (nAChRs) of nAChRs are probably an interesting therapeutic receptors, i.e., the two main classes of cholinergic target for the treatment of various central nervous sys- receptors that mediate ACh actions and play a crucial tem disorders, such as AD [2, 29, 43]. role in memory processing, are localized in the human Based on the above-mentioned data, much effort brain [32, 64]. Out of them, it is the nAChRs which has been undertaken toward investigating memory- are mainly involved in memory processes. The char- related effects of the two cholinergic system affecting acteristic structure of nAChRs includes a ring of five substances (nicotine and scopolamine) in mice. Addi- subunits, arranged around a ligand-gated excitatory tionally, assuming the reported data that a4b2 and a7 ion channel. To date, 12 individual subunits of ligands may represent a new generation of com- nAChRs (a2–a10) and (b2–b4) have been identified. pounds to treat memory disorders, such as AD, we The two main neuronal categories, defined by their aimed to investigate and compare the influences, function and pharmacology, include heterologous exerted on the memory and learning processes in pentamers, formed from the combinations of a- and mice, by mecamylamine, a non-selective nicotinic re- b-subunits, and homologous pentamers, formed from ceptor antagonist, and by varenicline, a partial a4b2 one subunit type, a7, a8 or a9 [13, 58]. Out of all the nAChR agonist, which has recently been approved as central nAChRs subtypes, both the a4b2 combination a pharmacological aid to cease the smoking habit [14, and a7 subunits seem to play the most important role 33]. In our experiments, we used the recently devel- in memory-related responses [35]. A growing body of oped modified elevated plus maze (mEPM) memory evidence reveals a certain involvement of the a4b2 animal model. The mEPM test is a simple method that subtype of nAChRs in cognitive processing [49]. Fur- evaluates spatial memory and, moreover, this memory thermore, metanicotine, a selective a4b2 nAChRs model allows investigating different stages of mem- agonist, enhances reference and working memory in ory processes [30, 31, 54, 70]. We wanted to ascertain the radial arm maze performance in rats with ibotenic whether memory acquisition or consolidation pro- acid forebrain lesions and also improves passive cesses were by those drugs in any way affected. avoidance retention in rats with scopolamine-induced The experiments may contribute to a better under- amnesia [44, 45]. Consistent with the role of a7 standing of the cholinergic neuronal mechanisms, im- nAChRs in cognition, some deficits in olfactory portant for the modulation of memory and learning working memory were also revealed in a7 knockout processes. The knowledge about the mechanisms of mice vs. wild-type controls [75]. Additionally, another action of cholinergic ligands (not only mecamylamine research with the use of methyllycaconitine, a specific and varenicline) enables a development of more ef- antagonist of a7 nAChRs, infused into the ventral fective pharmacotherapies for human memory disor- hippocampus, demonstrates that this type of nAChRs ders associated with cholinergic pathways.
Pharmacological Reports, 2012, 64, 10661080 1067 Materials and Methods arms (5 × 30 × 15 cm), opposite to each other. The whole apparatus was made of dark Plexiglas and ele- vated to the height of 50 cm above the floor. Addition- Animals ally, the mEPM test was conducted under red, dim light. The mEPM test has a spatial component. The prin- Experiments were carried out on naive male Swiss ciple of this test is based upon the aversion of rodents mice (Farm of Laboratory Animals, Warszawa, Po- to open spaces and heights. The animals prefer en- land) weighing 20–30 g. The animals were maintained closed, protected areas of the maze and they must re- under standard laboratory conditions (12-h light/dark member the configuration of open and enclosed arms. cycle, room temperature 21 ± 1°C) with free access to The mice were individually placed at the end of the tap water and laboratory chow (Bacutil, Motycz, Po- open arm, facing away from the central platform. Each land) in their home cages, and were adapted to the group was twice submitted to the same procedure (with laboratory conditions for at least one week. Each ex- a 24 h interval between trials). During the first trial perimental group consisted of 7–10 animals. All be- (pretest), the time that each mouse took to move from havioral experiments were performed between 8:00 the open arm to either of the enclosed arms was re- and 15:00 h, being conducted, according to the Na- corded (TL1). If the mouse failed to enter the enclosed tional Institute of Health Guidelines for the Care and arm within 90 s, it was placed at an enclosed arm and Use of Laboratory Animals and to the European Com- permitted to explore the plus maze for additional 60 s. munity Council Directive for the Care and Use of labo- In those cases, TL1 value was recorded as 90 s. In the ratory animals of 24 November 1986 (86/609/EEC), subsequent trial (retention) 24 h later, the test was per- and approved by the local ethics committee. formed in the same manner as the first trial and TL recorded as TL2. If the mouse did not enter the Drugs enclosed arm within 90 s on the second day, the test was stopped and TL2 was recorded as 90 s. The following compounds were tested: (–)-nicotine hy- We used TL2 values as an index of memory and drogen tartrate (0.035, 0.175 and 0.35 mg/kg reported learning effects. Memory improvement was charac- in freebase nicotine weight; scopolamine (0.1, 0.3 and terized by reduction in the time necessary for the 1.0 mg/kg) and mecamylamine hydrochloride (0.5, 1.0 mouse to move from the open arms to either of the en- and 2.0 mg/kg) (all from Sigma, St. Louis, MO, USA) closed arms on the second day, relatively to the con- and varenicline (0.5, 1.0 and 2.0 mg/kg) (CP-526555, trol group. Impairments in memory and learning were gift of Pfizer Inc, Groton, USA). All compounds were characterized by increases in these measures. dissolved in saline solution (0.9% NaCl). Except for The mEPM test allows examining different memory nicotine, drug doses refer to the salt form. The pH of stages, according to the time of drug treatment. Drug ad- the nicotine solution was adjusted to 7.0. Fresh drug ministration before the first trial (before pretest) should solutions were prepared on each day of experimenta- interfere with the acquisition of information, while the tion. All agents were administered subcutaneously (sc) administration immediately after the first trial (after pre- or intraperitoneally (ip) at a volume of 10 ml/kg. Con- test) should exert an effect on the process of consolida- trol groups received saline injections at the same vol- tion. In the reported experiments, drugs were adminis- ume and via the same route of administration. tered 30 min before pretest or immediately after pretest and the effect of each compound on both acquisition and Experimental procedures consolidation of memory was investigated.
Memory-related behavior Locomotor activity
Memory-related responses were measured by the Locomotion was recorded individually in round acto- modified elevated plus maze (mEPM) test [4, 5, 12, meter cages (Multiserv, Lublin, Poland; 32 cm in di- 30, 31, 36, 46, 54, 69, 70]. ameter, two light beams) kept in a sound-attenuated The applied experimental apparatus was shaped experimental room. Two photocell beams, located as a “plus” sign and consisted of a central platform (5 × across the axis, automatically measured animal’s 5 cm), two open arms (5 × 30 cm) and two enclosed movements.
1068 Pharmacological Reports, 2012, 64, 10661080 Cholinergic receptors in different stages of memory Marta Kruk-S³omka et al.
Treatment Results
Memory-related behavior Memory-related behavior The first experiment was designed to estimate the in- Across all experiments, the time (in s) that each mouse fluence of nicotine, scopolamine, mecamylamine and took to move from the open arm to either of the en- varenicline on memory-related responses in mice, closed arms on the first trial (pre-test), i.e., TL1, did not using the mEPM test. Nicotine (0.035, 0.175 and significantly differ between groups (data not presented). 0.35 mg/kg, sc), scopolamine (0.1, 0.3 and 1.0 mg/kg, ip), mecamylamine (0.5, 1.0 and 2.0 mg/kg, ip), vare- nicline (0.5, 1.0 and 2.0 mg/kg, ip) and saline were Influence of nicotine, scopolamine, mecamylamine, administered 30 min before the first trial or immedi- varenicline on memory-related processes in the mEPM test in mice ately after the first trial. The second set of experi- ments was designed to investigate and compare the One-way ANOVA analyses revealed that the acute sc influence of mecamylamine and varenicline on doses of nicotine (0.035, 0.175 and 0.35 mg/kg) memory-related responses, induced by acute nicotine caused a statistically significant effect on the TL2 val- or scopolamine administration. Mecamylamine (0.5 and 1.0 mg/kg, ip), varenicline (0.5 and 1.0 mg/kg, ip) and saline were for that purpose administered 15 min 80 A prior to nicotine (0.035 mg/kg, sc) or scopolamine 70 (1.0 mg/kg, ip) and the mice were tested after 30 min 60 and re-tested after 24 h. Experimental doses and procedures were chosen 50 accordingly to those, frequently used in literature, in- 40 cluding our previous study [4–7, 39, 54]. 30 20 ** ** 10
Locomotor activity transfer latency (TL2) [s] 0 saline 0.035 0.175 0.35 mg/kg Horizontal locomotor activity was measured 30 min af- nicotine ter injection of nicotine (0.035, 0.175 and 0.35 mg/ kg, sc), scopolamine (0.1, 0.3 and 1.0 mg/kg, ip), meca- mylamine (0.5, 1.0 and 2.0 mg/kg, ip), varenicline (0.5, B 1.0 and 2.0 mg/kg, ip) and saline for the control group. 80 Locomotor activity, i.e., the number of photocell beam 70 breaks, was automatically recorded for 30 min. 60 50 Statistics 40 30 The data were expressed as the means ± SEM. For the * 20 ** mEPM test, we measured TL, i.e., the time necessary
transfer latency (TL2)10 [s] for the mice to move from the open arm to either of 0 the enclosed arms. For the locomotor activity, we saline 0.035 0.175 0.35 mg/kg measured the number of photocell beam breaks. nicotine Statistical analyses were performed using one- or two-way analyses of variance (ANOVA) for the fac- tors of pretreatment, treatment and pretreatment/treat- Fig. 1. Effects of acute nicotine or saline injection on the transfer ment interaction. Post-hoc comparison of means was latency to the enclosed arm in the acquisition trial (A) or consolida- tion trial (B), using the mEPM test in mice. Nicotine (0.035, 0.175 and carried out with the Tukey’s test for multiple compari- 0.35 mg/kg; sc) or saline were administered 30 min before the first sons, when appropriate. The data were considered sta- trial (A) or immediately after the first trial (B); n = 79; the data are shown as the means ± SEM; * p < 0.05; ** p < 0.01 vs. saline control tistically significant at a confidence limit of p < 0.05. group; Tukeys test
Pharmacological Reports, 2012, 64, 10661080 1069 ues [F (3, 30) = 6.114; p = 0.0023], with respect to at these doses used, also improved this stage of memory memory acquisition during the retention trial. The post- and learning processes. For both the acquisition and hoc Tukey’s test confirmed that mice treated with nico- consolidation trials, the highest dose of nicotine tine, at the dose 0.035 and 0.175 mg/kg, significantly (0.35 mg/kg) did not cause any effect in this paradigm. decreased the TL2 values, as compared to the saline- The active doses of 0.035 and 0.175 m/kg of nico- treated mice, indicating that nicotine improves of mem- tine were chosen for the next experiments involving ory and learning processes (p < 0.01) (Fig. 1A). Corre- the use of mecamylamine and varenicline. spondingly, for the memory consolidation during reten- Moreover, one-way ANOVA analyses revealed that tion trial, the acute sc doses of nicotine (0.035; 0.175 the acute ip doses of scopolamine (0.1, 0.3 and and 0.35 mg/kg) significantly decreased the TL2 values 1.0 mg/kg) caused a statistically significant effect on as compared to the saline-treated mice ([F (3, 27) = the TL2 values [F (3, 35 = 8.305; p = 0.0003], with 4.245; p = 0.0140], one-way ANOVA). Indeed, the respect to memory acquisition during the retention post-hoc Tukey’s test revealed statistically significant trial. Indeed, treatment with scopolamine (0.3 and effect (p < 0.05 for nicotine 0.035 mg/kg; p < 0.01 for 1.0 mg/kg) significantly increased the TL2 values in nicotine 0.175 mg/kg) (Fig. 1B), indicating that nicotine, mice, in relation to the saline-treated control group
80 A *** 80 A 70 70
60 * 60 50 50 40 40
30 30 20 20 transfer latency (TL2) [s]
transfer latency (TL2)10 [s] 10 0 0 saline 0.1 0.3 1.0 mg/kg saline 0.5 1.0 2.0 mg/kg mecamylamine scopolamine
80 B 80 B ** 70 70
60 60 50 50 40 40 30 30 20 20 transfer latency (TL2) [s] transfer latency (TL2)10 [s] 10 0 0 saline 0.1 0.3 1.0 mg/kg saline 0.5 1.0 2.0 mg/kg scopolamine mecamylamine
Fig. 2. Effects of acute scopolamine or saline injection on the transfer la- Fig. 3. Effects of acute mecamylamine or saline injection on the trans- tency to the enclosed arm in the acquisition trial (A) or consolidation trial fer latency to the enclosed arm in the acquisition trial (A) or consoli- (B), using the mEPM test in mice. Scopolamine (0.1, 0.3 and 1.0 mg/kg; ip) dation trial (B) using the mEPM test in mice. Mecamylamine (0.5, 1.0 or saline were administered 30 min before the first trial (A) or immediately and 2.0 mg/kg; ip) or saline were administered 30 min before the first after the first trial (B); n = 910; the data are shown as the means ± SEM; trial (A) or immediately after the first trial (B); n = 89; the data are * p < 0.05; ** p < 0.01; *** p < 0.001 vs. saline control group; Tukeys test shown as the means ± SEM
1070 Pharmacological Reports, 2012, 64, 10661080 Cholinergic receptors in different stages of memory Marta Kruk-S³omka et al.
(p < 0.05 for scopolamine 0.3 mg/kg; p < 0.001 for A scopolamine 1.0 mg/kg, Tukey’s test) (Fig. 2A), indi- 80 cating that scopolamine, at these doses used, impaired 70 the acquisition of memory and learning. Respectively, 60 it can be seen from Figure 2B that for memory consoli- 50 dation during the retention trial, administration of the 40 acute ip dose of scopolamine (0.1, 0.3 and 1.0 mg/kg) 30 significantly increased the TL2 values [F (3, 36) = 4.498; 20 p = 0.0088, one-way ANOVA], as compared to the saline-treated mice. Indeed, the post-hoc Tukey’s test transfer latency (TL2) [s] 10 revealed statistically significant effect for scopolamine 0 saline 0.5 1.0 2.0 mg/kg at the dose of 1.0 mg/kg (p < 0.01) (Fig. 2B), indicating varenicline that scopolamine, at this dose used, also impaired this stage of memory and learning processes. The active dose of 1.0 mg/kg of scopolamine was 80 B then chosen for the subsequent experiments dealing with the use of mecamylamine and varenicline. 70 Much evidence indicates that for both acquisition 60 [F (3, 29) = 2.239; p = 0.1049, one-way ANOVA] and 50 consolidation trials [F (3, 30) = 0.3178; p = 0.8124, 40 one-way ANOVA], at any dose tested (0.5, 1.0 and 30 2.0 mg/kg), mecamylamine did not significantly alter 20 the TL2 values in the mEPM test (Fig. 3A and
transfer latency (TL2)10 [s] Fig. 3B). The inactive doses of 0.5 and 1.0 mg/kg of 0 mecamylamine were then chosen for the next experi- saline 0.5 1.0 2.0 mg/kg ments with nicotine and scopolamine. varenicline Similarly, for both acquisition [F (3, 29) = 0.7808; p = 0.5143, one-way ANOVA] and consolidation trials [F (3, 28) = 1.624; p = 0.2062), one-way ANOVA], at Fig. 4. Effects of acute varenicline or saline injection on the transfer latency to the enclosed arm in the acquisition trial (A) or consolida- any dose tested (0.5, 1.0 and 2.0 mg/kg), varenicline did tion trial (B) using the mEPM test in mice. Varenicline (0.5, 1.0 and not significantly alter the TL2 values in the EPM test 2.0 mg/kg; ip) or saline were administered 30 min before the first trial (A) or immediately after the first trial (B); n = 89; the data are shown (Fig. 4A and Fig. 4B). The inactive doses of 0.5 and as the means ± SEM 1.0 mg/kg of varenicline were then chosen for the sub- sequent experiments with nicotine and scopolamine.
Influence of mecamylamine on memory-related ing in an increased TL2 value (p < 0.05 for meca- responses induced by nicotine in the mEPM in mice mylamine 0.5 mg/kg and p < 0.01 for mecamylamine 1.0 mg/kg; Tukey’s test) (Fig. 5A). Finally, we examined the effects of combined admini- Similarly, for memory consolidation during the re- stration of mecamylamine and nicotine. Two-way tention trial, two-way ANOVArevealed that there was ANOVA analyses revealed that a statistically signifi- a statistically significant effect caused by nicotine cant effect was caused by mecamylamine pretreat- treatment [F (1, 46) = 7.358; p = 0.0094], while there ment [F (2, 46) = 9.205; p = 0.0004] and nicotine was no effect caused by mecamylamine pretreatment treatment [F (1, 46) = 39.780; p < 0.0001]; however, [F (2, 46) = 1.871; p = 0.1655] or an interaction be- there was no interaction between mecamylamine pre- tween mecamylamine pretreatment and nicotine treat- treatment and nicotine treatment [F (2, 46) = 0.011; ment [F (2, 46) = 1.632; p = 0.2067]. However, meca- p < 0.9981], for the memory acquisition during the re- mylamine (1.0 mg/kg) prevented memory improve- tention trial. Nevertheless, mecamylamine (0.5 or ment after treatment with 0.035 mg/kg nicotine, 1.0 mg/kg, ip) prevented memory improvement after resulting in an increased TL2 value (p < 0.01; Tukey’s acute administration of 0.035 mg/kg nicotine, result- test) (Fig. 5B).
Pharmacological Reports, 2012, 64, 10661080 1071 80 A that there was a statistically significant effect caused 70 by scopolamine treatment [F (1, 47) = 4.487; p = 0.0395] and an interaction between mecamylamine 60 pretreatment and scopolamine treatment [F (2, 47) = 50 4.718; p = 0.0136], while there was no effect caused 40 * ** by mecamylamine pretreatment [F (2, 47) = 0.08871; 30 p = 0.9153], with respect to memory acquisition dur- 20 ^^ ing retention trial. Nevertheless, mecamylamine (0.5 transfer latency (TL2)10 [s] and 1.0 mg/kg, ip) prevented memory impairment af- 0 ter acute administration of 1.0 mg/kg scopolamine, 0.5 1.0 0.5 1.0 mg/kg resulting in a decreased TL2 value (p < 0.05 for meca- mecamylamine mecamylamine saline
saline + saline + nicotine mylamine 0.5 mg/kg and 1.0 mg/kg; Tukey’s test) + saline
+ nicotine (0.035 mg/kg) (Fig. 6A).
(0.035 mg/kg) Similarly, for memory consolidation during reten- tion trial, two-way ANOVA analyses revealed that there was a statistically significant effect caused by mecamylamine pretreatment [F (2, 47) = 5.031; p = B 0.0105] and an interaction between mecamylamine
80 pretreatment and scopolamine treatment [F (2, 47) = 6.958; p = 0.0023], while there was no effect caused 70 by scopolamine treatment [F (1, 47) = 1.505; p = 60 0.2260]. Indeed, the post-hoc Tukey’s test revealed 50 ** that mecamylamine (0.5 and 1.0 mg/kg, ip) prevented 40 memory impairment of memory provoked by acute 30 ^ injection of 1.0 mg/kg scopolamine, resulting in 20 a decreased TL2 value (p < 0.01 for mecamylamine transfer latency (TL2) [s] 10 0.5 mg/kg and p < 0.001 for mecamylamine 0 1.0 mg/kg) (Fig. 6B). 0.5 1.0 0.5 1.0 mg/kg mecamylamine mecamylamine saline saline
+ saline + saline + nicotine
+ nicotine (0.035 mg/kg) Influence of varenicline on memory-related
(0.035 mg/kg) responses induced by nicotine in the mEPM in mice
An interesting effect was also observed in the next ex- Fig. 5. Influence of mecamylamine on the memory-related response periments in which the combined administration of induced by an acute nicotine administration in the acquisition trial (A) or consolidation trial (B) using the mEPM test in mice. Mecamylamine varenicline and nicotine was performed. For memory (0.5 and 1.0 mg/kg, ip) or saline were administered 15 min prior to acquisition during retention trial, two-way ANOVA saline or nicotine (0.035 mg/kg, sc) injection, 30 min before the first trial (A) or immediately after the first trial (B); n = 89; the data are analyses revealed that there was a statistically signifi- shown as the means ± SEM; ^ p < 0.05; ^^ p < 0.01 vs. cant effect caused by varenicline pretreatment saline-treated group and * p < 0.05; ** p < 0.01 vs. nicotine-treated group; Tukeys test [F (2, 42) = 4.175; p = 0.0222], however, there was no effect caused by nicotine treatment [F (1, 42) = 3.077; p = 0.0867] and there was no interaction between varenicline pretreatment and nicotine treatment Influence of mecamylamine on memory-related responses induced by scopolamine in the mEPM [F (2, 42) = 0.7665; p = 0.4710]. Nevertheless, vareni- in mice cline (0.5 and 1.0 mg/kg, ip) significantly prevented memory improvement after 0.035 mg/kg of nicotine, Moreover, in the experiments in which the combined resulting in an increased TL2 value (p < 0.05 for vare- administration of mecamylamine and scopolamine nicline 0.5 mg/kg and p < 0.01 for varenicline was performed, two-way ANOVA analyses revealed 1.0 mg/kg; Tukey’s test) (Fig. 7A).
1072 Pharmacological Reports, 2012, 64, 10661080 Cholinergic receptors in different stages of memory Marta Kruk-S³omka et al.
A A 80 ^^^ 80 70 70
60 60 * * 50 50 * ** 40 40 30 30 sfer latency (TL2) [s] n 20 20 ^^^ tra 10 transfer latency (TL2) [s] 10 0 0 0.5 1.0 0.5 1.0 mg/kg 0.5 1.0 0.5 1.0 mg/kg mecamylamine mecamylamine saline saline varenicline varenicline saline + saline + scopolamine saline + saline
+ saline + saline + nicotine (1.0 mg/kg) + nicotine
(1.0 mg/kg) (0.035 mg/kg) (0.035 mg/kg) + scopolamine
B B 80 ^^ 80 70 70
60 60 50 ** 50 40 * *** 40 30 30 ^ 20 20 transfer latency (TL2) [s] 10 transfer latency (TL2)10 [s] 0 0 0.5 1.0 0.5 1.0 mg/kg 0.5 1.0 0.5 1.0 mg/kg mecamylamine mecamylamine saline saline varenicline varenicline saline + saline + scopolamine saline + saline
+ saline + nicotine (1.0 mg/kg) + saline + nicotine (1.0 mg/kg) (0.035 mg/kg) (0.035 mg/kg) + scopolamine
Fig. 6. Influence of mecamylamine on the memory-related response Fig. 7. Influence of varenicline on the memory-related response in- provoked by an acute scopolamine administration in the acquisition duced by an acute nicotine administration in the acquisition trial trial (A) or consolidation trial (B) using the mEPM test in mice. Meca- (A) or consolidation trial (B) using the mEPM test in mice. Varenicline mylamine (0.5 and 1.0 mg/kg, ip) or saline were administered 15 min (0.5 and 1.0 mg/kg, ip) or saline were administered 15 min prior to prior to saline or scopolamine (1.0 mg/kg, ip) injection, 30 min before saline or nicotine (0.035 mg/kg, sc) injection, 30 min before the first the first trial (A) or immediately after the first trial (B); n = 910; the trial (A) or immediately after the first trial (B); n = 89; the data are data are shown as the means ± SEM; ^^ p < 0.01; ^^^ p < 0.001 vs. shown as the means ± SEM; ^ p < 0.05; ^^ p < 0.01 vs. saline- saline-treated group and * p < 0.05; ** p < 0.01; *** p < 0.001 vs. treated group and * p < 0.05; ** p < 0.01 vs. nicotine-treated group; scopolamine-treated group; Tukeys test Tukeys test
Additionally, for memory consolidation during the treatment [F (1, 44) = 1.957; p = 0.1689) or vareni- retention trial, two-way ANOVA analyses revealed cline pretreatment [F (2, 44) = 0.3768; p = 0.6882]. that there was only a statistically significant effect However, varenicline (1.0 mg/kg, ip) prevented mem- caused by an interaction between varenicline pretreat- ory improvement after treatment with 0.035 mg/kg ment and nicotine treatment [F (2, 44) = 3.533; p = nicotine, resulting in an increased TL2 value (p < 0.0378], while there was no effect caused by nicotine 0.05; Tukey’s test) (Fig. 7B).
Pharmacological Reports, 2012, 64, 10661080 1073 A significant effect caused by an interaction between 80 varenicline pretreatment and scopolamine treatment ^^^ 70 [F (2, 44) = 3.268; p = 0.0475], however, there was no 60 effect caused by varenicline pretreatment [F (2, 44) = 0.3640; p = 06969] or scopolamine treatment 50 ** [F (1, 44) = 2.506; p = 0.1206], with respect to mem- 40 ory acquisition during the retention trial. However, 30 varenicline (1.0 mg/kg, ip) prevented memory impair- 20 ment after treatment with 1.0 mg/kg of scopolamine, transfer latency (TL2) [s] 10 resulting in a decreased TL2 value (p < 0.01; Tukey’s 0 test) (Fig. 8A). 0.5 1.0 0.5 1.0 mg/kg Furthermore, for memory consolidation during the saline saline varenicline varenicline
+ saline + saline + scopolamine retention trial, two-way ANOVA analyses revealed (1.0 mg/kg) (1.0 mg/kg) that there was a statistically significant effect caused
+ scopolamine by varenicline pretreatment [F (2, 42) = 8.020; p = 0.0011] and scopolamine treatment [F (1, 42) = 12.50; p = 0.0010], however, there was no interaction be- tween varenicline pretreatment and scopolamine B treatment [F (2, 42) = 2.664; p = 0.0814]. Neverthe- 80 ^^ less, varenicline (0.5 and 1.0 mg/kg) prevented mem- 70 ory impairment after treatment with 1.0 mg/kg sco- 60 polamine, resulting in a decreased TL2 value (p < 50 * 0.05; Tukey’s test) (Fig. 8B). * 40 30 Locomotor activity 20 transfer latency (TL2) [s] 10 Influence of nicotine, scopolamine, mecamylamine 0 and varenicline on the locomotor activity in mice 0.5 1.0 0.5 1.0 mg/kg varenicline varenicline saline saline + saline One-way ANOVA analyses revealed that nicotine + saline + scopolamine (1.0 mg/kg) (0.035, 0.175 and 0.35 mg/kg, sc), scopolamine (0.1, (1.0 mg/kg) 0.3 and 1.0 mg/kg, ip), mecamylamine (0.5, 1.0 and 2.0 + scopolamine mg/kg, ip) and varenicline (0.5, 1.0 and 2.0 mg/kg, ip), at the doses tested, caused statistically significant changes in the locomotor activity of mice: [F (12, 108) Fig. 8. Influence of varenicline on the memory-related response pro- = 28.08; p < 0.0001]. Additionally, Tukey’s test indi- voked by an acute scopolamine administration in the acquisition trial (A) cated that only nicotine at the dose of 0.35 mg/kg de- or consolidation trial (B) using the mEPM test in mice. Varenicline (0.5 and 1.0 mg/kg, ip) or saline were administered 15 min prior to saline or creased locomotor activity (p < 0.01), and scopolamine scopolamine (1.0 mg/kg, ip) injection, 30 min before the first trial (A) or immediately after the first trial (B); n = 910; the data are shown as the at the doses of 0.1, 0.3 and 1.0 increased locomotor ac- means ± SEM; ^^ p < 0.01; ^^^ p < 0.001 vs. saline-treated group and tivity (p < 0.01 for scopolamine 0.1 and p < 0.001 for * p < 0.05; ** p < 0.01 vs. scopolamine-treated group; Tukeys test scopolamine 0.3 and 1.0), as compared with the control saline-injected group (Tab. 1).
Influence of varenicline on memory-related responses induced by scopolamine in the mEPM in mice Discussion An important effect was observed in the subsequent experiments in which the combined administration of Diverse reports in literature indicate that cholinergic varenicline and scopolamine was performed. Two-way pathways participate via cholinergic receptors in me- ANOVAanalyses revealed that there was a statistically mory-related behavior [4, 5, 36, 41]. Cholinergic re-
1074 Pharmacological Reports, 2012, 64, 10661080 Cholinergic receptors in different stages of memory Marta Kruk-S³omka et al.
Tab.1. Effects of nicotine, scopolamine, mecamylamine and vareni- cline on the locomotor activity (the data are shown as the means amount of evidence indicates that scopolamine is able ± SEM, photocell beam breaks) of mice measured 30 min after injec- to disrupt memory responses, explored in many ani- tion for 30 min; ** p < 0.01; *** p < 0.001 vs. saline control group; Tukeys test mal memory models, e.g., the passive avoidance test, the Y-maze task, the Morris water maze or the novel Treatment n Means ± SEM object recognition test [25, 50, 76, 77]. The results of the present study are in accordance Saline 10 588.87 ± 37.40 with data in the literature, as well as with the observa- Nicotine 0.035 mg/kg 8 551.22 ± 57.84 tions in our previous research [4, 5, 36], showing Nicotine 0.175 mg/kg 8 396.54 ± 24.01 a very clear difference between the nicotinergic and the Nicotine 0.35 mg/kg 8 261.10 ± 35.77** muscarinic system with respect to spatial learning. Our Scopolamine 0.1 mg/kg 10 894.80 ± 114.07** studies have revealed that in the mEPM test, an acute injection of nicotine, a specific agonist of nAChRs, sig- Scopolamine 0.3 mg/kg 8 1023.70 ± 60.51*** nificantly improved spatial memory-related processes Scopolamine 1.0 mg/kg 9 1174.00 ± 66.51*** in mice, in the acquisition and consolidation trial. In Mecamylamine 0.5 mg/kg 10 420.88 ± 27.14 contrast, an acute injection of scopolamine, a specific Mecamylamine 1.0 mg/kg 10 498.00 ± 35.50 antagonist of mAChRs, significantly impaired spatial Mecamylamine 2.0 mg/kg 10 380.00 ± 30.00 learning in both memory stages. Varenicline 0.5 mg/kg 10 505.12 ± 33.34 In the context of our present data, it should be noted that nicotine can affect both types of parame- Varenicline 1.0 mg/kg 10 448.00 ± 37.50 ters, but in the applied modification of the test, espe- Varenicline 2.0 mg/kg 10 352.86 ± 25.46 cially the learning and memory capacity can be evalu- ated for the spatial configuration of the arms. Our pre- vious experiment [4, 5, 36] as well as other, already ceptor antagonists impair memory-related responses and published results, showed a high effectiveness of the therefore, are used as a pharmacological tool to model mEPM test in investigating the spatial learning in ro- the cholinergic neurotransmission aspect of AD. On the dents, also in the context of pharmacological manipu- other hand, the drugs, which can affect nAChRs, pro- lations of the cholinergic systems [30, 31, 70]. Moreo- vide a promising new lead in the search for methods to ver, the mEPM test allows investigating different pro- ameliorate cognitive deficits [29, 53, 77]. cesses of memory (acquisition, consolidation and The use of mAChR and nAChR ligands has been retrieval) depending on drug treatment duration [61]. fairly extensive in studies on the role of cholinergic re- The attempted extensive investigations have suggested ceptor subtypes in attention and memory processes [13, that cholinergic receptors are important for the complex 53]. Following a number of reports from preclinical mechanisms of cognitive processes, especially spatial data, nicotine and other nAChR agonists appear to en- learning, but the critical neural mechanisms, which are re- hance cognition in both normal and impaired rodents sponsible for memory-response induced by cholinergic and non-human primates, what has been confirmed in receptor ligands, are still not fully understood. a variety of animal models. However, some contradic- In general, a stimulation of nicotinic receptors leads tory results have also been published. Some research- to spatial memory improvement, while their suppres- ers indicate a certain improvement of memory and cog- sion brings spatial memory deficits [17]. Nicotine pro- nition in rodents after acute nicotine treatment [4, 5, duces its pro-cognitive effects, acting at the diverse 39, 41, 42], while others report no effects or present nAChRs populations [1]. Because nAChRs are easily negative influences of nicotine administration on cog- desensitized, nicotinic agonists provide both receptor nitive functions [43]. Furthermore, some data indicate stimulation and inhibition via receptor desensitiza- that nAChR antagonists are able to impair attention tion. As yet, there has been little firm evidence, show- and memory-related responses [53, 63, 72]. ing which of nicotine’s effects on cognitive-related re- In turn, a large body of evidence reveals that cogni- sponses is due to stimulation of nicotinic receptors tion impairment is caused by mAChR antagonists, and which is due to their desensitization. Some find- e.g., scopolamine. Scopolamine interferes with mem- ings suggest that nicotine administration could result ory processes and cognitive functions, both in humans in increased receptor activity (i.e., upregulation of [19, 68] and in experimental animals [50]. A growing a4b2 and a7 nAChR expression) in the central ner-
Pharmacological Reports, 2012, 64, 10661080 1075 vous system, especially in the hippocampus [1, 10, It should also be noted that, in our experiments, 16, 58]. Other studies show that a chronic exposure to scopolamine in all used doses, significantly increased nicotine increases nicotine binding density, mainly to locomotion ability. Those results are, in general, con- a4b2 nAChR types, both in rodents and humans [48]. sistent with previous studies in which scopolamine Considering the line of evidence for the crucial role of disrupted locomotor activity in both rats and mice mesolimbic a4b2 nAChRs, a deletion of either a4 or [15, 26, 59]. However, scopolamine-induced hyper- b2 subunit impairs the pharmacological and behav- motility, assessed by a specific motor activity task, ioral responses of nicotine [1, 10, 42, 49, 58, 60]. Ad- represents a behavioral effect only, similar to that ditionally, a targeted expression of b2 subunits in the caused by many anticholinergic drugs that do not af- ventral tegmental area (VTA) of b2 knockout mice re- fect cognitive performance [10]. The increase in loco- instates nicotine-seeking behavior and release of do- motor activity induced by scopolamine, does not cor- pamine (DA), induced by nicotine [49]. Impaired spa- relate with the presented results, thus suggesting tial memory has been demonstrated in different ani- a clear scopolamine-induced memory impairment. mal models and in humans for a number of a4b2 Moreover, as we have already mentioned, scopola- antagonists, for instance, ABT-418 [9, 60] or dihydro- b-erythroidine [17]. mine has widely been used as a model in memory im- Since a4b2 and a7 nAChR subtypes are generally pairment studies, due to the arguments that cognitive thought to mediate nicotine’s effects of enhancing cog- deficits, observed after its use, are directly related to nition, much research has been focused on the design of decreased central cholinergic function [8]. compounds improving the selectivity of receptors vs. Consistently with the findings, which indicate an nicotine [11]. involvement of nAChRs ligands in responses to nico- Although nAChRs play a permissive role in nicotine- tine and scopolamine [41], we estimated and com- induced enhancement of spatial memory, these effects of pared the effects of two nAChR ligands, namely me- nicotine can also result from the release of several neu- camylamine, a relatively non-selective nAChR an- rotransmitters in the brain. Nicotine, via activation of tagonist, and varenicline, a partial a4b2 nAChR presynaptic nAChRs, induces the release of ACh, a neu- agonist, on the pro-cognitive effects of nicotine and rotransmitter which is essential for cognitive processes, on the amnestic effects of scopolamine. We found out DA, that plays a crucial role in both rewarding and that both mecamylamine and varenicline were able to memory-related processes, g-aminobutyric acid (GABA), attenuate the antiamnestic effect of nicotine and the serotonin, norepinephrine and glutamate, also essential amnestic effect of scopolamine in the acquisition and for memory formation [43, 74]. consolidation trials in the scope of the mEPM test. It The mechanisms underlying scopolamine’s in- is important to note that, in the mEPM test and with duced cognitive impairments, have not yet been in- the used doses, neither mecamylamine nor varenicline vestigated and would seem an important area for fu- caused any significant effect on the spatial memory ture research [19]. Scopolamine, a centrally acting an- by itself. ticholinergic drug, has been shown to antagonize The mechanisms by which mecamylamine and mAChRs (subtypes: M1 and M2). In particular, this varenicline produce their pharmacological effects, es- drug is quite selective for M1 receptors, potentially pecially when combined with nicotine or scopola- indicating that impairments of cognitive-related re- mine, are not yet completely understood. sponses are associated with the blockade of mAChRs Mecamylamine is widely used as an agent to help in the basal forebrain regions. Therefore, in rodents, the blockade of muscarinic receptors induce deficits cease smoking in humans [20]. The mechanism of its in both place learning and strategy selection, e.g., in action is not selective to one specific nAChR subtype. mEPM. It has been proposed that these impairments It appears to inhibit preferentially a3, then a4, and fi- of memory may result from an inability to inhibit nally a7 subunits and may exert a similar effect on b2 non-efficient escape strategies [17]. and b4 nAChR subunits. Preclinical studies reveal Interestingly enough, scopolamine may affect not that a systemic, nicotine-induced DA release in the rat only mAChRs, but also nAChRs and so, memory- nucleus accumbens is blocked by mecamylamine in related effects of scopolamine are often interpreted in the VTA. Following other data, mecamylamine blocks terms of some role of both types of AChRs in mne- nicotine-induced enhancement of reference memory monic processes [17, 53]. in the 16-arm radial maze in rodents [38, 39].
1076 Pharmacological Reports, 2012, 64, 10661080 Cholinergic receptors in different stages of memory Marta Kruk-S³omka et al.
Although, mecamylamine can act as a nicotinic- cate that varenicline is effective in reducing returns to receptor antagonist, perhaps its antagonistic influence smoking in humans [57, 65]. This drug attenuates the on the amnestic effects of scopolamine may also be severity of tobacco withdrawal symptoms, reduces the a result of action at both nAChRs and mAChRs. It is subjective rewarding effects of cigarette smoking and possible to speculate on interactions between nAChRs improves cognitive performance in abstinent smokers and mAChRs and on their influence on memory pro- [57, 73]. Preclinical studies, while examining phar- cesses. Moreover, some findings demonstrate that macological effects of varenicline, also demonstrate scopolamine and mecamylamine synergize memory- that varenicline inhibits nicotine self-administration related responses in passive avoidance test, when ad- in animals, partially generalizing to nicotine in the ministered in combination prior to training, however, drug discrimination preclinical animal paradigm [65, antagonizing each other when administered in combi- 66] and reproducing behavioral effects of nicotine in nation after training [22]. These results suggest that some rodent models of cognition [67]. Additionally, it central muscarinic and nicotinic cholinergic mecha- is of particular relevance that varenicline reverses nisms have separable behavioral functions. In litera- withdrawal-related learning and memory impairment ture, scopolamine has been shown to increase ACh re- in rodents [62] and in abstinent smokers [57]. Inter- lease, whereas mecamylamine decreases ACh release estingly enough, given the interest in nAChRs sub- in the hippocampus [27]. Additionally, other data also types as targets for novel therapeutics for disorders suggest that centrally acting muscarinic and nicotinic other than nicotine addiction, recent studies on vare- antagonists have dissociable effects on memory pro- nicline have focused on exploring its positive effects cesses in rodents [55]. Thus, a treatment with the on alcohol addiction, mood, cognition, attention and combination of these drugs can modulate cholinergic memory-related responses [28]. It can be found in neurotransmission and so, it could consequently im- preclinical literature that varenicline is able to reduce prove cognition in our present experiments. Moreo- self-administration of ethanol, but not sucrose, and to ver, the reported findings are in agreement with litera- decrease voluntary consumption of ethanol, but not ture data, showing results of the combined treatment water in rats [71]. Clinical data regarding the effects with mecamylamine and scopolamine in the five- of varenicline on cognition in normal smokers and in choice serial reaction time task [23, 56]. smokers in schizophrenia are also described [65, 66]. Moreover, it has been suggested that many effects Especially interesting is the influence of vareni- of mecamylamine, exerted on attention and cognition, cline on memory-related processes. The results from can be mediated by actions of this drug as an N- our experiment are in accordance with the previously methyl-D-aspartate (NMDA) receptor antagonist. Some cited findings [62]. In our study, we demonstrated previous reports have suggested that dizocilpine, an varenicline preventing nicotine-induced improvement NMDA receptor antagonist, produces similar effects and scopolamine-induced impairment in memory in to those of mecamylamine [23]. According to cited the acquisition and consolidation trials in mice. Re- data, some of mecamylamine’s effects may be due to garding the observed effects of varenicline, a neuro- blockade of NMDA receptors. biological mechanism can be proposed. Varenicline is Mecamylamine could also act by the central dopa- a partial agonist of a4b2 nAChR and a full agonist of minergic system [51]. It has been reported that an- a7 nAChR [52]. It is known that both receptors exist tagonists of DA, such as haloperidol, improve selec- in large numbers in the hippocampus [48] and the tive attention in animals. Haloperidol has also been amygdala [1]. A stimulation of a4b2 and a7 nAChR shown to potentiate mecamylamine-induced deficits in these brain areas, that are involved in cognition, in- in radial arm maze performance [51]. Thus, it could, creases DA release, which is responsible for rein- therefore, be possible that mecamylamine exerts simi- forcement, associated with nicotine addiction, being lar behavioral effects to haloperidol, improving, in also important in memory-related responses [47]. In this case, attention processes. turn, nicotine abstinence reduces DA release and thus However, a more detailed knowledge on the influ- leads to the withdrawal syndrome development. ence of mecamylamine on memory and learning pro- Acting as a partial a4b2 nAChR agonist, varenicline cesses deserves further investigation. competitively inhibits the ability of nicotine to bind to In turn, varenicline is widely known as a novel the receptor and thus reduces the reinforcement. More- drug used for smoking cessation. Clinical trials indi- over, varenicline has high affinity for a4b2 nAChRs
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