Involvement of cholinergic receptors in the different stages of memory measured in the modified elevated plus maze test in mice

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1 Pharmacological Reports 12, 64, 66 8 ISSN Copyright 12 by Institute of Pharmacology Polish cademy 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, arbara udzyñska, Gra yna ia³a Chair, Department of Pharmacology and Pharmacodynamics, Medical University of Lublin, ChodŸki 4, PL -93 Lublin, Poland Correspondence: Marta Kruk-S³omka, marta.kruk@umlub.pl bstract: ackground 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 (.35 and.175 mg/kg, free base, sc), reduced TL on the second day of the experiment (TL2), thus improving memory. In turn, scopolamine, an antagonist of cholinergic receptors (.3 and 1. mg/kg, ip), significantly increased TL2 values, impairing cognition. Subsequently, we evaluated the influence of, a non-selective antagonist of nicotinic cholinergic receptors (nchrs) and of, an 4 2 partial nchrs agonist, on memory-related behaviors induced by nicotine and scopolamine. cute injections of (.5 and 1. mg/kg, ip) and (.5 and 1. mg/kg, ip), prior to the injections of nicotine (.35 mg/kg) or scopolamine (1. mg/kg), significantly suppressed nicotine-induced memory improvement or scopolamineinduced memory impairment. Conclusion: Our studies indicate that the cholinergic system plays a crucial role in memory processes. Pharmacological manipulation 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,,, memory and learning, modified elevated plus maze bbreviations: Ch acetylcholine, D lzheimer s disease, D dopamine, G -aminobutyric acid, mchrs muscarinic cholinergic receptors, mepm modified elevated plus maze, nchrs nicotinic cholinergic receptors, NMD N-methyl-D-aspartate, TL transfer latency, VT ventral tegmental area Introduction fair number of studies imply some role of the cholinergic system in cognitive functions, specifically in attention and memory encoding. Spatial memory is one 66 Pharmacological Reports, 12, 64, 66 8

2 Cholinergic receptors in different stages of memory Marta Kruk-S³omka et al. of the most essential forms of higher cognitive processes, demanding a certain capacity to record environmental and spatial orientation data. Thus, spatial memory formation represents a simple form of episodic-like memory in rodents that engages both the basal cholinergic system and its target structures [17, 18, 24]. It is known that acetylcholine (Ch) is a neurotransmitter essential for these cognitive functions. Previous studies concluded a strong correlation between synaptic Ch levels and cognitive function improvement, providing evidence that acetylcholinesterase (an Ch breaking down enzyme), lead to increased Ch levels in the brain, especially in cortical and hippocampal brain regions, i.e., the two major areas involved in cognitive processes. ccordingly, many acetylcholinesterase inhibitors improve performance levels in several cognitive models, involving humans or rodents [34]. Muscarinic (mchrs) and nicotinic (nchrs) receptors, i.e., the two main classes of cholinergic receptors that mediate Ch actions and play a crucial role in memory processing, are localized in the human brain [32, 64]. Out of them, it is the nchrs which are mainly involved in memory processes. The characteristic structure of nchrs includes a ring of five subunits, arranged around a ligand-gated excitatory ion channel. To date, 12 individual subunits of nchrs ( 2 ) and ( 2 4) have been identified. The two main neuronal categories, defined by their function and pharmacology, include heterologous pentamers, formed from the combinations of - and -subunits, and homologous pentamers, formed from one subunit type, 7, 8 or 9 [13, 58]. Out of all the central nchrs subtypes, both the 4 2 combination and 7 subunits seem to play the most important role in memory-related responses [35]. growing body of evidence reveals a certain involvement of the 4 2 subtype of nchrs in cognitive processing [49]. Furthermore, metanicotine, a selective 4 2 nchrs agonist, enhances reference and working memory in the radial arm maze performance in rats with ibotenic acid forebrain lesions and also improves passive avoidance retention in rats with scopolamine-induced amnesia [44, 45]. Consistent with the role of 7 nchrs in cognition, some deficits in olfactory working memory were also revealed in 7 knockout mice vs. wild-type controls [75]. dditionally, another research with the use of methyllycaconitine, a specific antagonist of 7 nchrs, infused into the ventral hippocampus, demonstrates that this type of nchrs is essential for working memory-related responses, measured in the radial arm maze test in rats [2, 3, 37]. n abnormally regulated cholinergic system, with a decline of cholinergic neurons in the basal forebrain, have been hypothesized as being responsible for the cognitive symptoms of neuropsychiatric disorders, e.g., lzheimer s disease (D) [18, 32]. The first clinical trials in patients, suffering from D, revealed no changes in the number, the structure or the function of mchrs, while significantly decreased levels of 4 2 nchrs subtypes were observed, especially in the cortex and the hippocampus [18, 29, 32, 41]. That reported decline may be appropriate for the cognitive deficits that characterize the condition. Therefore, an administration of drugs, which improve cholinergic signaling, is able to enhance cognitive functions [29, 43]. What is more, literature data suggest that agonists and partial agonists of various subtypes of nchrs are probably an interesting therapeutic target for the treatment of various central nervous system disorders, such as D [2, 29, 43]. ased on the above-mentioned data, much effort has been undertaken toward investigating memoryrelated effects of the two cholinergic system affecting substances (nicotine and scopolamine) in mice. dditionally, assuming the reported data that 4 2 and 7 ligands may represent a new generation of compounds to treat memory disorders, such as D, we aimed to investigate and compare the influences, exerted on the memory and learning processes in mice, by, a non-selective nicotinic receptor antagonist, and by, a partial 4 2 nchr agonist, which has recently been approved as a pharmacological aid to cease the smoking habit [14, 33]. In our experiments, we used the recently developed modified elevated plus maze (mepm) memory animal model. The mepm test is a simple method that evaluates spatial memory and, moreover, this memory model allows investigating different stages of memory processes [3, 31, 54, 7]. We wanted to ascertain whether memory acquisition or consolidation processes were by those drugs in any way affected. The experiments may contribute to a better understanding of the cholinergic neuronal mechanisms, important for the modulation of memory and learning processes. The knowledge about the mechanisms of action of cholinergic ligands (not only and ) enables a development of more effective pharmacotherapies for human memory disorders associated with cholinergic pathways. Pharmacological Reports, 12, 64,

3 Materials and Methods nimals Experiments were carried out on naive male Swiss mice (Farm of Laboratory nimals, Warszawa, Poland) weighing 3 g. The animals were maintained under standard laboratory conditions (12-h light/dark cycle, room temperature 21 ± 1 C) with free access to tap water and laboratory chow (acutil, Motycz, Poland) in their home cages, and were adapted to the laboratory conditions for at least one week. Each experimental group consisted of 7 animals. ll behavioral experiments were performed between 8: and 15: h, being conducted, according to the National Institute of Health Guidelines for the Care and Use of Laboratory nimals and to the European Community Council Directive for the Care and Use of laboratory animals of 24 November 1986 (86/69/EEC), and approved by the local ethics committee. Drugs The following compounds were tested: ( )-nicotine hydrogen tartrate (.35,.175 and.35 mg/kg reported in freebase nicotine weight; scopolamine (.1,.3 and 1. mg/kg) and hydrochloride (.5, 1. and 2. mg/kg) (all from Sigma, St. Louis, MO, US) and (.5, 1. and 2. mg/kg) (CP , gift of Pfizer Inc, Groton, US). ll compounds were dissolved in solution (.9% NaCl). Except for nicotine, drug doses refer to the salt form. The ph of the nicotine solution was adjusted to 7.. Fresh drug solutions were prepared on each day of experimentation. ll agents were administered subcutaneously (sc) or intraperitoneally (ip) at a volume of ml/kg. Control groups received injections at the same volume and via the same route of administration. Experimental procedures arms ( cm), opposite to each other. The whole apparatus was made of dark Plexiglas and elevated to the height of 5 cm above the floor. dditionally, the mepm test was conducted under red, dim light. The mepm test has a spatial component. The principle of this test is based upon the aversion of rodents to open spaces and heights. The animals prefer enclosed, protected areas of the maze and they must remember the configuration of open and enclosed arms. The mice were individually placed at the end of the open arm, facing away from the central platform. Each group was twice submitted to the same procedure (with a 24 h interval between trials). During the first trial (pretest), the time that each mouse took to move from the open arm to either of the enclosed arms was recorded (TL1). If the mouse failed to enter the enclosed arm within 9 s, it was placed at an enclosed arm and permitted to explore the plus maze for additional 6 s. In those cases, TL1 value was recorded as 9 s. In the subsequent trial (retention) 24 h later, the test was performed in the same manner as the first trial and TL recorded as TL2. If the mouse did not enter the enclosed arm within 9 s on the second day, the test was stopped and TL2 was recorded as 9 s. We used TL2 values as an index of memory and learning effects. Memory improvement was characterized by reduction in the time necessary for the mouse to move from the open arms to either of the enclosed arms on the second day, relatively to the control group. Impairments in memory and learning were characterized by increases in these measures. The mepm test allows examining different memory stages, according to the time of drug treatment. Drug administration before the first trial (before pretest) should interfere with the acquisition of information, while the administration immediately after the first trial (after pretest) should exert an effect on the process of consolidation. In the reported experiments, drugs were administered 3 min before pretest or immediately after pretest and the effect of each compound on both acquisition and consolidation of memory was investigated. Memory-related behavior Memory-related responses were measured by the modified elevated plus maze (mepm) test [4, 5, 12, 3, 31, 36, 46, 54, 69, 7]. The applied experimental apparatus was shaped as a plus sign and consisted of a central platform (5 5 cm), two open arms (5 3 cm) and two enclosed Locomotor activity Locomotion was recorded individually in round actometer cages (Multiserv, Lublin, Poland; 32 cm in diameter, two light beams) kept in a sound-attenuated experimental room. Two photocell beams, located across the axis, automatically measured animal s movements. 68 Pharmacological Reports, 12, 64, 66 8

4 Cholinergic receptors in different stages of memory Marta Kruk-S³omka et al. Treatment Memory-related behavior The first experiment was designed to estimate the influence of nicotine, scopolamine, and on memory-related responses in mice, using the mepm test. Nicotine (.35,.175 and.35 mg/kg, sc), scopolamine (.1,.3 and 1. mg/kg, ip), (.5, 1. and 2. mg/kg, ip), (.5, 1. and 2. mg/kg, ip) and were administered 3 min before the first trial or immediately after the first trial. The second set of experiments was designed to investigate and compare the influence of and on memory-related responses, induced by acute nicotine or scopolamine administration. Mecamylamine (.5 and 1. mg/kg, ip), (.5 and 1. mg/kg, ip) and were for that purpose administered 15 min prior to nicotine (.35 mg/kg, sc) or scopolamine (1. mg/kg, ip) and the mice were tested after 3 min and re-tested after 24 h. Experimental doses and procedures were chosen accordingly to those, frequently used in literature, including our previous study [4 7, 39, 54]. Locomotor activity Horizontal locomotor activity was measured 3 min after injection of nicotine (.35,.175 and.35 mg/ kg, sc), scopolamine (.1,.3 and 1. mg/kg, ip), (.5, 1. and 2. mg/kg, ip), (.5, 1. and 2. mg/kg, ip) and for the control group. Locomotor activity, i.e., the number of photocell beam breaks, was automatically recorded for 3 min. Statistics The data were expressed as the means ± SEM. For the mepm test, we measured TL, i.e., the time necessary for the mice to move from the open arm to either of the enclosed arms. For the locomotor activity, we measured the number of photocell beam breaks. Statistical analyses were performed using one- or two-way analyses of variance (NOV) for the factors of pretreatment, treatment and pretreatment/treatment interaction. Post-hoc comparison of means was carried out with the Tukey s test for multiple comparisons, when appropriate. The data were considered statistically significant at a confidence limit of p <.5. Results Memory-related behavior cross all experiments, the time (in s) that each mouse took to move from the open arm to either of the enclosed arms on the first trial (pre-test), i.e., TL1, did not significantly differ between groups (data not presented). Influence of nicotine, scopolamine,, on memory-related processes in the mepm test in mice One-way NOV analyses revealed that the acute sc doses of nicotine (.35,.175 and.35 mg/kg) caused a statistically significant effect on the TL2 val ** ** mg/kg nicotine * ** mg/kg nicotine Fig. 1. Effects of acute nicotine or injection on the transfer latency to the enclosed arm in the acquisition trial () or consolidation trial (), using the mepm test in mice. Nicotine (.35,.175 and.35 mg/kg; sc) or were administered 3 min before the first trial () or immediately after the first trial (); n = 7 9; the data are shown as the means ± SEM; * p <.5; ** p <.1 vs. control group; Tukey s test Pharmacological Reports, 12, 64,

5 ues [F (3, 3) = 6.114; p =.23], with respect to memory acquisition during the retention trial. The posthoc Tukey s test confirmed that mice treated with nicotine, at the dose.35 and.175 mg/kg, significantly decreased the TL2 values, as compared to the treated mice, indicating that nicotine improves of memory and learning processes (p <.1) (Fig. 1). Correspondingly, for the memory consolidation during retention trial, the acute sc doses of nicotine (.35;.175 and.35 mg/kg) significantly decreased the TL2 values as compared to the -treated mice ([F (3, 27) = 4.245; p =.14], one-way NOV). Indeed, the post-hoc Tukey s test revealed statistically significant effect (p <.5 for nicotine.35 mg/kg; p <.1 for nicotine.175 mg/kg) (Fig. 1), indicating that nicotine, at these doses used, also improved this stage of memory and learning processes. For both the acquisition and consolidation trials, the highest dose of nicotine (.35 mg/kg) did not cause any effect in this paradigm. The active doses of.35 and.175 m/kg of nicotine were chosen for the next experiments involving the use of and. Moreover, one-way NOV analyses revealed that the acute ip doses of scopolamine (.1,.3 and 1. mg/kg) caused a statistically significant effect on the TL2 values [F (3, 35 = 8.35; p =.3], with respect to memory acquisition during the retention trial. Indeed, treatment with scopolamine (.3 and 1. mg/kg) significantly increased the TL2 values in mice, in relation to the -treated control group * *** mg/kg scopolamine mg/kg ** mg/kg scopolamine mg/kg Fig. 2. Effects of acute scopolamine or injection on the transfer latency to the enclosed arm in the acquisition trial () or consolidation trial (), using the mepm test in mice. Scopolamine (.1,.3 and 1. mg/kg; ip) or were administered 3 min before the first trial () or immediately after the first trial (); n = 9 ; the data are shown as the means ± SEM; * p <.5; ** p <.1; *** p <.1 vs. control group; Tukey s test Fig. 3. Effects of acute or injection on the transfer latency to the enclosed arm in the acquisition trial () or consolidation trial () using the mepm test in mice. Mecamylamine (.5, 1. and 2. mg/kg; ip) or were administered 3 min before the first trial () or immediately after the first trial (); n = 8 9; the data are shown as the means ± SEM 7 Pharmacological Reports, 12, 64, 66 8

6 Cholinergic receptors in different stages of memory Marta Kruk-S³omka et al. (p <.5 for scopolamine.3 mg/kg; p <.1 for scopolamine 1. mg/kg, Tukey s test) (Fig. 2), indicating that scopolamine, at these doses used, impaired the acquisition of memory and learning. Respectively, it can be seen from Figure 2 that for memory consolidation during the retention trial, administration of the acute ip dose of scopolamine (.1,.3 and 1. mg/kg) significantly increased the TL2 values [F (3, 36) = 4.498; p =.88, one-way NOV], as compared to the -treated mice. Indeed, the post-hoc Tukey s test revealed statistically significant effect for scopolamine at the dose of 1. mg/kg (p <.1) (Fig. 2), indicating that scopolamine, at this dose used, also impaired this stage of memory and learning processes. The active dose of 1. mg/kg of scopolamine was then chosen for the subsequent experiments dealing with the use of and. Much evidence indicates that for both acquisition [F (3, 29) = 2.239; p =.49, one-way NOV] and consolidation trials [F (3, 3) =.3178; p =.8124, one-way NOV], at any dose tested (.5, 1. and 2. mg/kg), did not significantly alter the TL2 values in the mepm test (Fig. 3 and Fig. 3). The inactive doses of.5 and 1. mg/kg of were then chosen for the next experiments with nicotine and scopolamine. Similarly, for both acquisition [F (3, 29) =.788; p =.5143, one-way NOV] and consolidation trials [F (3, 28) = 1.624; p =.62), one-way NOV], at any dose tested (.5, 1. and 2. mg/kg), did not significantly alter the TL2 values in the EPM test (Fig. 4 and Fig. 4). The inactive doses of.5 and 1. mg/kg of were then chosen for the subsequent experiments with nicotine and scopolamine mg/kg mg/kg Fig. 4. Effects of acute or injection on the transfer latency to the enclosed arm in the acquisition trial () or consolidation trial () using the mepm test in mice. Varenicline (.5, 1. and 2. mg/kg; ip) or were administered 3 min before the first trial () or immediately after the first trial (); n = 8 9; the data are shown as the means ± SEM Influence of on memory-related responses induced by nicotine in the mepm in mice Finally, we examined the effects of combined administration of and nicotine. Two-way NOV analyses revealed that a statistically significant effect was caused by pretreatment [F (2, 46) = 9.5; p =.4] and nicotine treatment [F (1, 46) = 39.78; p <.1]; however, there was no interaction between pretreatment and nicotine treatment [F (2, 46) =.11; p <.9981], for the memory acquisition during the retention trial. Nevertheless, (.5 or 1. mg/kg, ip) prevented memory improvement after acute administration of.35 mg/kg nicotine, resulting in an increased TL2 value (p <.5 for.5 mg/kg and p <.1 for 1. mg/kg; Tukey s test) (Fig. 5). Similarly, for memory consolidation during the retention trial, two-way NOV revealed that there was a statistically significant effect caused by nicotine treatment [F (1, 46) = 7.358; p =.94], while there was no effect caused by pretreatment [F (2, 46) = 1.871; p =.1655] or an interaction between pretreatment and nicotine treatment [F (2, 46) = 1.632; p =.67]. However, (1. mg/kg) prevented memory improvement after treatment with.35 mg/kg nicotine, resulting in an increased TL2 value (p <.1; Tukey s test) (Fig. 5). Pharmacological Reports, 12, 64,

7 ^^ (.35 mg/kg) ^ (.35 mg/kg) mg/kg + * ** mg/kg (.35 mg/kg) ** (.35 mg/kg) that there was a statistically significant effect caused by scopolamine treatment [F (1, 47) = 4.487; p =.395] and an interaction between pretreatment and scopolamine treatment [F (2, 47) = 4.718; p =.136], while there was no effect caused by pretreatment [F (2, 47) =.8871; p =.9153], with respect to memory acquisition during retention trial. Nevertheless, (.5 and 1. mg/kg, ip) prevented memory impairment after acute administration of 1. mg/kg scopolamine, resulting in a decreased TL2 value (p <.5 for.5 mg/kg and 1. mg/kg; Tukey s test) (Fig. 6). Similarly, for memory consolidation during retention trial, two-way NOV analyses revealed that there was a statistically significant effect caused by pretreatment [F (2, 47) = 5.31; p =.5] and an interaction between pretreatment and scopolamine treatment [F (2, 47) = 6.958; p =.23], while there was no effect caused by scopolamine treatment [F (1, 47) = 1.55; p =.226]. Indeed, the post-hoc Tukey s test revealed that (.5 and 1. mg/kg, ip) prevented memory impairment of memory provoked by acute injection of 1. mg/kg scopolamine, resulting in a decreased TL2 value (p <.1 for.5 mg/kg and p <.1 for 1. mg/kg) (Fig. 6). Influence of on memory-related responses induced by nicotine in the mepm in mice Fig. 5. Influence of on the memory-related response induced by an acute nicotine administration in the acquisition trial () or consolidation trial () using the mepm test in mice. Mecamylamine (.5 and 1. mg/kg, ip) or were administered 15 min prior to or nicotine (.35 mg/kg, sc) injection, 3 min before the first trial () or immediately after the first trial (); n = 8 9; the data are shown as the means ± SEM; ^ p <.5; ^^ p <.1 vs. -treated group and * p <.5; ** p <.1 vs. nicotine-treated group; Tukey s test Influence of on memory-related responses induced by scopolamine in the mepm in mice Moreover, in the experiments in which the combined administration of and scopolamine was performed, two-way NOV analyses revealed n interesting effect was also observed in the next experiments in which the combined administration of and nicotine was performed. For memory acquisition during retention trial, two-way NOV analyses revealed that there was a statistically significant effect caused by pretreatment [F (2, 42) = 4.175; p =.222], however, there was no effect caused by nicotine treatment [F (1, 42) = 3.77; p =.867] and there was no interaction between pretreatment and nicotine treatment [F (2, 42) =.7665; p =.47]. Nevertheless, (.5 and 1. mg/kg, ip) significantly prevented memory improvement after.35 mg/kg of nicotine, resulting in an increased TL2 value (p <.5 for.5 mg/kg and p <.1 for 1. mg/kg; Tukey s test) (Fig. 7). 72 Pharmacological Reports, 12, 64, 66 8

8 Cholinergic receptors in different stages of memory Marta Kruk-S³omka et al. tra nsfer latency (TL2) [s] ^^^ * * ^^^ * ** + (1. mg/kg) mg/kg + (1. mg/kg) + (.35 mg/kg) mg/kg + (.35 mg/kg) ^^ (1. mg/kg) mg/kg + ** *** (1. mg/kg) ^ (.35 mg/kg) mg/kg + * (.35 mg/kg) Fig. 6. Influence of on the memory-related response provoked by an acute scopolamine administration in the acquisition trial () or consolidation trial () using the mepm test in mice. Mecamylamine (.5 and 1. mg/kg, ip) or were administered 15 min prior to or scopolamine (1. mg/kg, ip) injection, 3 min before the first trial () or immediately after the first trial (); n = 9 ; the data are shown as the means ± SEM; ^^ p <.1; ^^^ p <.1 vs. -treated group and * p <.5; ** p <.1; *** p <.1 vs. scopolamine-treated group; Tukey s test Fig. 7. Influence of on the memory-related response induced by an acute nicotine administration in the acquisition trial () or consolidation trial () using the mepm test in mice. Varenicline (.5 and 1. mg/kg, ip) or were administered 15 min prior to or nicotine (.35 mg/kg, sc) injection, 3 min before the first trial () or immediately after the first trial (); n = 8 9; the data are shown as the means ± SEM; ^ p <.5; ^^ p <.1 vs. treated group and * p <.5; ** p <.1 vs. nicotine-treated group; Tukey s test dditionally, for memory consolidation during the retention trial, two-way NOV analyses revealed that there was only a statistically significant effect caused by an interaction between pretreatment and nicotine treatment [F (2, 44) = 3.533; p =.378], while there was no effect caused by nicotine treatment [F (1, 44) = 1.957; p =.1689) or pretreatment [F (2, 44) =.3768; p =.6882]. However, (1. mg/kg, ip) prevented memory improvement after treatment with.35 mg/kg nicotine, resulting in an increased TL2 value (p <.5; Tukey s test) (Fig. 7). Pharmacological Reports, 12, 64,

9 ^^^ (1. mg/kg) ^^ (1. mg/kg) mg/kg + (1. mg/kg) Fig. 8. Influence of on the memory-related response provoked by an acute scopolamine administration in the acquisition trial () or consolidation trial () using the mepm test in mice. Varenicline (.5 and 1. mg/kg, ip) or were administered 15 min prior to or scopolamine (1. mg/kg, ip) injection, 3 min before the first trial () or immediately after the first trial (); n = 9 ; the data are shown as the means ± SEM; ^^ p <.1; ^^^ p <.1 vs. -treated group and * p <.5; ** p <.1 vs. scopolamine-treated group; Tukey s test * ** mg/kg (1. mg/kg) * significant effect caused by an interaction between pretreatment and scopolamine treatment [F (2, 44) = 3.268; p =.475], however, there was no effect caused by pretreatment [F (2, 44) =.364; p = 6969] or scopolamine treatment [F (1, 44) = 2.56; p =.16], with respect to memory acquisition during the retention trial. However, (1. mg/kg, ip) prevented memory impairment after treatment with 1. mg/kg of scopolamine, resulting in a decreased TL2 value (p <.1; Tukey s test) (Fig. 8). Furthermore, for memory consolidation during the retention trial, two-way NOV analyses revealed that there was a statistically significant effect caused by pretreatment [F (2, 42) = 8.; p =.11] and scopolamine treatment [F (1, 42) = 12.5; p =.], however, there was no interaction between pretreatment and scopolamine treatment [F (2, 42) = 2.664; p =.814]. Nevertheless, (.5 and 1. mg/kg) prevented memory impairment after treatment with 1. mg/kg scopolamine, resulting in a decreased TL2 value (p <.5; Tukey s test) (Fig. 8). Locomotor activity Influence of nicotine, scopolamine, and on the locomotor activity in mice One-way NOV analyses revealed that nicotine (.35,.175 and.35 mg/kg, sc), scopolamine (.1,.3 and 1. mg/kg, ip), (.5, 1. and 2. mg/kg, ip) and (.5, 1. and 2. mg/kg, ip), at the doses tested, caused statistically significant changes in the locomotor activity of mice: [F (12, 8) = 28.8; p <.1]. dditionally, Tukey s test indicated that only nicotine at the dose of.35 mg/kg decreased locomotor activity (p <.1), and scopolamine at the doses of.1,.3 and 1. increased locomotor activity (p <.1 for scopolamine.1 and p <.1 for scopolamine.3 and 1.), as compared with the control -injected group (Tab. 1). Influence of on memory-related responses induced by scopolamine in the mepm in mice n important effect was observed in the subsequent experiments in which the combined administration of and scopolamine was performed. Two-way NOV analyses revealed that there was a statistically Discussion Diverse reports in literature indicate that cholinergic pathways participate via cholinergic receptors in memory-related behavior [4, 5, 36, 41]. Cholinergic re- 74 Pharmacological Reports, 12, 64, 66 8

10 Cholinergic receptors in different stages of memory Marta Kruk-S³omka et al. Tab.1. Effects of nicotine, scopolamine, and on the locomotor activity (the data are shown as the means ± SEM, photocell beam breaks) of mice measured 3 min after injection for 3 min; ** p <.1; *** p <.1 vs. control group; Tukey s test Treatment n Means ± SEM Saline ± 37.4 Nicotine.35 mg/kg ± Nicotine.175 mg/kg ± 24.1 Nicotine.35 mg/kg ± 35.77** Scopolamine.1 mg/kg ± 114.7** Scopolamine.3 mg/kg ± 6.51*** Scopolamine 1. mg/kg ± 66.51*** Mecamylamine.5 mg/kg 4.88 ± Mecamylamine 1. mg/kg 498. ± 35.5 Mecamylamine 2. mg/kg 38. ± 3. Varenicline.5 mg/kg ± Varenicline 1. mg/kg 448. ± 37.5 Varenicline 2. mg/kg ± ceptor antagonists impair memory-related responses and therefore, are used as a pharmacological tool to model the cholinergic neurotransmission aspect of D. On the other hand, the drugs, which can affect nchrs, provide a promising new lead in the search for methods to ameliorate cognitive deficits [29, 53, 77]. The use of mchr and nchr ligands has been fairly extensive in studies on the role of cholinergic receptor subtypes in attention and memory processes [13, 53]. Following a number of reports from preclinical data, nicotine and other nchr agonists appear to enhance cognition in both normal and impaired rodents and non-human primates, what has been confirmed in a variety of animal models. However, some contradictory results have also been published. Some researchers indicate a certain improvement of memory and cognition in rodents after acute nicotine treatment [4, 5, 39, 41, 42], while others report no effects or present negative influences of nicotine administration on cognitive functions [43]. Furthermore, some data indicate that nchr antagonists are able to impair attention and memory-related responses [53, 63, 72]. In turn, a large body of evidence reveals that cognition impairment is caused by mchr antagonists, e.g., scopolamine. Scopolamine interferes with memory processes and cognitive functions, both in humans [19, 68] and in experimental animals [5]. growing amount of evidence indicates that scopolamine is able to disrupt memory responses, explored in many animal memory models, e.g., the passive avoidance test, the Y-maze task, the Morris water maze or the novel object recognition test [25, 5, 76, 77]. The results of the present study are in accordance with data in the literature, as well as with the observations in our previous research [4, 5, 36], showing a very clear difference between the nicotinergic and the muscarinic system with respect to spatial learning. Our studies have revealed that in the mepm test, an acute injection of nicotine, a specific agonist of nchrs, significantly improved spatial memory-related processes in mice, in the acquisition and consolidation trial. In contrast, an acute injection of scopolamine, a specific antagonist of mchrs, significantly impaired spatial learning in both memory stages. In the context of our present data, it should be noted that nicotine can affect both types of parameters, but in the applied modification of the test, especially the learning and memory capacity can be evaluated for the spatial configuration of the arms. Our previous experiment [4, 5, 36] as well as other, already published results, showed a high effectiveness of the mepm test in investigating the spatial learning in rodents, also in the context of pharmacological manipulations of the cholinergic systems [3, 31, 7]. Moreover, the mepm test allows investigating different processes of memory (acquisition, consolidation and retrieval) depending on drug treatment duration [61]. The attempted extensive investigations have suggested that cholinergic receptors are important for the complex mechanisms of cognitive processes, especially spatial learning, but the critical neural mechanisms, which are responsible for memory-response induced by cholinergic receptor ligands, are still not fully understood. In general, a stimulation of nicotinic receptors leads to spatial memory improvement, while their suppression brings spatial memory deficits [17]. Nicotine produces its pro-cognitive effects, acting at the diverse nchrs populations [1]. ecause nchrs are easily desensitized, nicotinic agonists provide both receptor stimulation and inhibition via receptor desensitization. s yet, there has been little firm evidence, showing which of nicotine s effects on cognitive-related responses is due to stimulation of nicotinic receptors and which is due to their desensitization. Some findings suggest that nicotine administration could result in increased receptor activity (i.e., upregulation of 4 2 and 7 nchr expression) in the central ner- Pharmacological Reports, 12, 64,

11 vous system, especially in the hippocampus [1,, 16, 58]. Other studies show that a chronic exposure to nicotine increases nicotine binding density, mainly to 4 2 nchr types, both in rodents and humans [48]. Considering the line of evidence for the crucial role of mesolimbic 4 2 nchrs, a deletion of either 4 or 2 subunit impairs the pharmacological and behavioral responses of nicotine [1,, 42, 49, 58, 6]. dditionally, a targeted expression of 2 subunits in the ventral tegmental area (VT) of 2 knockout mice reinstates nicotine-seeking behavior and release of dopamine (D), induced by nicotine [49]. Impaired spatial memory has been demonstrated in different animal models and in humans for a number of 4 2 antagonists, for instance, T-418 [9, 6] or dihydro- -erythroidine [17]. Since 4 2 and 7 nchr subtypes are generally thought to mediate nicotine s effects of enhancing cognition, much research has been focused on the design of compounds improving the selectivity of receptors vs. nicotine [11]. lthough nchrs play a permissive role in nicotineinduced enhancement of spatial memory, these effects of nicotine can also result from the release of several neurotransmitters in the brain. Nicotine, via activation of presynaptic nchrs, induces the release of Ch, a neurotransmitter which is essential for cognitive processes, D, that plays a crucial role in both rewarding and memory-related processes, -aminobutyric acid (G), serotonin, norepinephrine and glutamate, also essential for memory formation [43, 74]. The mechanisms underlying scopolamine s induced cognitive impairments, have not yet been investigated and would seem an important area for future research [19]. Scopolamine, a centrally acting anticholinergic drug, has been shown to antagonize mchrs (subtypes: M1 and M2). In particular, this drug is quite selective for M1 receptors, potentially indicating that impairments of cognitive-related responses are associated with the blockade of mchrs in the basal forebrain regions. Therefore, in rodents, the blockade of muscarinic receptors induce deficits in both place learning and strategy selection, e.g., in mepm. It has been proposed that these impairments of memory may result from an inability to inhibit non-efficient escape strategies [17]. Interestingly enough, scopolamine may affect not only mchrs, but also nchrs and so, memoryrelated effects of scopolamine are often interpreted in terms of some role of both types of ChRs in mnemonic processes [17, 53]. It should also be noted that, in our experiments, scopolamine in all used doses, significantly increased locomotion ability. Those results are, in general, consistent with previous studies in which scopolamine disrupted locomotor activity in both rats and mice [15, 26, 59]. However, scopolamine-induced hypermotility, assessed by a specific motor activity task, represents a behavioral effect only, similar to that caused by many anticholinergic drugs that do not affect cognitive performance []. The increase in locomotor activity induced by scopolamine, does not correlate with the presented results, thus suggesting a clear scopolamine-induced memory impairment. Moreover, as we have already mentioned, scopolamine has widely been used as a model in memory impairment studies, due to the arguments that cognitive deficits, observed after its use, are directly related to decreased central cholinergic function [8]. Consistently with the findings, which indicate an involvement of nchrs ligands in responses to nicotine and scopolamine [41], we estimated and compared the effects of two nchr ligands, namely, a relatively non-selective nchr antagonist, and, a partial 4 2 nchr agonist, on the pro-cognitive effects of nicotine and on the amnestic effects of scopolamine. We found out that both and were able to attenuate the antiamnestic effect of nicotine and the amnestic effect of scopolamine in the acquisition and consolidation trials in the scope of the mepm test. It is important to note that, in the mepm test and with the used doses, neither nor caused any significant effect on the spatial memory by itself. The mechanisms by which and produce their pharmacological effects, especially when combined with nicotine or scopolamine, are not yet completely understood. Mecamylamine is widely used as an agent to help cease smoking in humans []. The mechanism of its action is not selective to one specific nchr subtype. It appears to inhibit preferentially 3, then 4, and finally 7 subunits and may exert a similar effect on 2 and 4 nchr subunits. Preclinical studies reveal that a systemic, nicotine-induced D release in the rat nucleus accumbens is blocked by in the VT. Following other data, blocks nicotine-induced enhancement of reference memory in the 16-arm radial maze in rodents [38, 39]. 76 Pharmacological Reports, 12, 64, 66 8

12 Cholinergic receptors in different stages of memory Marta Kruk-S³omka et al. lthough, can act as a nicotinicreceptor antagonist, perhaps its antagonistic influence on the amnestic effects of scopolamine may also be a result of action at both nchrs and mchrs. It is possible to speculate on interactions between nchrs and mchrs and on their influence on memory processes. Moreover, some findings demonstrate that scopolamine and synergize memoryrelated responses in passive avoidance test, when administered in combination prior to training, however, antagonizing each other when administered in combination after training [22]. These results suggest that central muscarinic and nicotinic cholinergic mechanisms have separable behavioral functions. In literature, scopolamine has been shown to increase Ch release, whereas decreases Ch release in the hippocampus [27]. dditionally, other data also suggest that centrally acting muscarinic and nicotinic antagonists have dissociable effects on memory processes in rodents [55]. Thus, a treatment with the combination of these drugs can modulate cholinergic neurotransmission and so, it could consequently improve cognition in our present experiments. Moreover, the reported findings are in agreement with literature data, showing results of the combined treatment with and scopolamine in the fivechoice serial reaction time task [23, 56]. Moreover, it has been suggested that many effects of, exerted on attention and cognition, can be mediated by actions of this drug as an N- methyl-d-aspartate (NMD) receptor antagonist. Some previous reports have suggested that dizocilpine, an NMD receptor antagonist, produces similar effects to those of [23]. ccording to cited data, some of s effects may be due to blockade of NMD receptors. Mecamylamine could also act by the central dopaminergic system [51]. It has been reported that antagonists of D, such as haloperidol, improve selective attention in animals. Haloperidol has also been shown to potentiate -induced deficits in radial arm maze performance [51]. Thus, it could, therefore, be possible that exerts similar behavioral effects to haloperidol, improving, in this case, attention processes. However, a more detailed knowledge on the influence of on memory and learning processes deserves further investigation. In turn, is widely known as a novel drug used for smoking cessation. Clinical trials indicate that is effective in reducing returns to smoking in humans [57, 65]. This drug attenuates the severity of tobacco withdrawal symptoms, reduces the subjective rewarding effects of cigarette smoking and improves cognitive performance in abstinent smokers [57, 73]. Preclinical studies, while examining pharmacological effects of, also demonstrate that inhibits nicotine self-administration in animals, partially generalizing to nicotine in the drug discrimination preclinical animal paradigm [65, 66] and reproducing behavioral effects of nicotine in some rodent models of cognition [67]. dditionally, it is of particular relevance that reverses withdrawal-related learning and memory impairment in rodents [62] and in abstinent smokers [57]. Interestingly enough, given the interest in nchrs subtypes as targets for novel therapeutics for disorders other than nicotine addiction, recent studies on have focused on exploring its positive effects on alcohol addiction, mood, cognition, attention and memory-related responses [28]. It can be found in preclinical literature that is able to reduce self-administration of ethanol, but not sucrose, and to decrease voluntary consumption of ethanol, but not water in rats [71]. Clinical data regarding the effects of on cognition in normal smokers and in smokers in schizophrenia are also described [65, 66]. Especially interesting is the influence of on memory-related processes. The results from our experiment are in accordance with the previously cited findings [62]. In our study, we demonstrated preventing nicotine-induced improvement and scopolamine-induced impairment in memory in the acquisition and consolidation trials in mice. Regarding the observed effects of, a neurobiological mechanism can be proposed. Varenicline is a partial agonist of 4 2 nchr and a full agonist of 7 nchr [52]. It is known that both receptors exist in large numbers in the hippocampus [48] and the amygdala [1]. stimulation of 4 2 and 7 nchr in these brain areas, that are involved in cognition, increases D release, which is responsible for reinforcement, associated with nicotine addiction, being also important in memory-related responses [47]. In turn, nicotine abstinence reduces D release and thus leads to the withdrawal syndrome development. cting as a partial 4 2 nchr agonist, competitively inhibits the ability of nicotine to bind to the receptor and thus reduces the reinforcement. Moreover, has high affinity for 4 2 nchrs Pharmacological Reports, 12, 64,

13 but lower efficacy, when compared with other nchr agonists, such as nicotine and Ch [16, 52, 65, 66]. This evidence suggests that and nicotine have a common neural mechanism for cognitive effects. Thus, these results may support the possibility that may be acting at 4 2 nchrs to ameliorate scopolamine-induced learning deficits. However, 4 2 nchrs are rapidly desensitized [21], suggesting that the lower affinity 7 nchrs is also critical for cognition and learning [4]. Varenicline binds to 7 receptors and has a high level of efficacy at these receptors [52]. s such, this compound, also acting as an 7 agonist, may increase the sensitivity to the memory-impairing effects of scopolamine. Varenicline is probably an interesting pharmacological target. However, the interactions of with scopolamine-induced response have not yet been thoroughly investigated. Thus, further research is still necessary to better understand the mechanisms influencing the actual efficacy of. Conclusion While providing further perspectives for discussion and proposing possible neuronal mechanisms implicated in memory, our study shows that a combined administration of nchr ligands, and, prevents memory improvement induced by nicotine and memory impairment induced by scopolamine in acquisition and consolidation trials. Thus, our data support the hypothesis that the cholinergic system can be involved in different memory stages, mainly via the 4 2 nchr subtype. These results may help in the understanding of the processes, which underlie cognition, and may also provide a firm basis to design effective treatment protocols not only for cases of addiction but also for cognitive dysfunctions, such as, for example, lzheimer s disease. cknowledgments: The authors would like to thank Pfizer Inc. (Groton, US) for the generous gift of. This work was supported by the Statutory Funds of the Medical University of Lublin (DS 23/), and received no special grant from any funding agency in the public, commercial or not-for-profit sectors. ll authors declare that they have no conflict of interest to disclose. References: 1. ddy N, Nakijama, Levin ED: Nicotinic mechanisms of memory: Effects of acute local DH E and ML infusions in the basolateral amygdala. Cognit rain Res, 3, 16, ancroft, Levin ED: Ventral hippocampal 4 2 nicotinic receptors and chronic nicotine effects on memory. Neuropharmacology,, 39, ettany JH, Levin ED: Ventral hippocampal 7 nicotinic receptor blockade and chronic nicotine effects on memory performance in the radial-arm maze. Pharmacol iochem ehav, 1, 7, iala G, Kruk M: Cannabinoid receptor ligands suppress memory-related effects of nicotine in the elevated plus maze test in mice. ehav rain Res, 8, 192, iala G, Kruk M: Influence of bupropion and calcium channel antagonists on the nicotine-induced memoryrelated response of mice in the elevated plus maze. Pharmacol Rep, 9, 61, iala G, Staniak N: Varenicline and attenuate locomotor sensitization and cross-sensitization induced by nicotine and morphine in mice. Pharmacol iochem ehav,, 96, iala G, Staniak N, udzynska : Effects of and on the acquisition, expression, and reinstatement of nicotine-conditioned place preference by drug priming in rats. Naunyn-Schmiedeberg s rch Pharmacol,, 381, rown RW, eale KS, Frye J: Mecamylamine blocks enhancement of reference memory but not working memory produced by post-training injection of nicotine in rats tested on the radial arm maze. ehav rain Res, 2, 134, lokland : cetylcholine: a neurotransmitter for learning and memory? rain Res rain Rev, 1995, 21, uccafusco JJ: Neuronal nicotinic receptor subtypes: Defining therapeutic targets. Mol Interv, 4, 4, uccafusco JJ, Jackson WJ, Terry Jr. V, Marsh KC, Decker MW, rneric SP: Improvement in performance of a delayed matching-to-sample task by monkeys following T-418: novel cholinergic channel activator for memory enhancement. Psychopharmacology, 1995, 1, ushnell PJ: Effects of scopolamine on locomotor activity and metabolic rate in mice. Pharmacol iochem ehav, 1987, 26, Celikyurt IK, kar FY, Ulak G, Mutlu O, Erden F: Effects of risperidone on learning and memory in naive and MK-81-treated rats. Pharmacology, 11, 87, Corringerr PJ, Le NN, Changeux JP: Nicotinic receptors at the amino acid level. nnu Rev Pharmacol Toxicol,, 4, Crunelle CL, Miller ML, ooij J, van den rink W: The nicotinic acetylcholine receptor partial agonist and the treatment of drug dependence: review. Eur Neuropsychopharmacol,,, Cunha GM, Canas PM, Melo CS, Hockemeyer J, Müller CE, Oliveira CR, Cunha R: denosine 2 receptor blockade prevents memory dysfunction caused 78 Pharmacological Reports, 12, 64, 66 8

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