Brain Research Bulletin 152 (2019) 52–62
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Differential effects of post-training scopolamine on spatial and non-spatial learning tasks in mice David Thonnard, Zsuzsanna Callaerts-Vegh, Rudi D’Hooge
T
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Laboratory of Biological Psychology, University of Leuven, Belgium
ARTICLE INFO
ABSTRACT
Keywords: Scopolamine Odour discrimination Visual discrimination Spatial learning Post-training effects Reversal learning
Muscarinic antagonist scopolamine has been extensively used to model amnesia in lab rodents, but most studies have focused on the effects of pre-training scopolamine administration. Here, we examined post-training scopolamine administration in C57BL/6JRj mice. Learning was assessed in three different procedures: odour discrimination in a digging paradigm, visual discrimination in a touchscreen-based setup, and spatial learning in the Morris water maze. Scopolamine administration affected performance in the odour discrimination task. More specifically, scopolamine decreased perseverance, which facilitated reversal learning. Similar results were obtained in the visual discrimination task, but scopolamine did not affect performance in the spatial learning task. It is unlikely that these results can be explained by non-memory-related cognitive effects (e.g., attention), noncognitive behaviours (e.g., locomotor activity) or peripheral side-effects (e.g., mydriasis). They likely relate to the various neuropharmacological actions of scopolamine.
1. Introduction
acquisition of new information differently from post-acquisition processes (Hasselmo and McGaughy, 2004; Meeter et al., 2004). Posttraining administration of scopolamine was shown to impair performance in various spatial and non-spatial tasks (Doguc et al., 2012; Nedaei et al., 2016; Shi et al., 2013). Notably in respect to the present study, there are some indications that post-acquisition effects of cholinergic drugs might depend on the form of memory and learning investigated. Post-training administration of scopolamine into the perirhinal cortex actually facilitated performance in an object recognition task (Winters, 2006), whereas post-training intraseptal injection of the cholinergic agonist carbachol impaired delayed non-matching-tosample (Bunce et al., 2004). It has also been shown that post-training scopolamine enhances or blocks post-acquisition memory processes, depending on the delay between acquisition and retention testing (Popović et al., 2015). In the present study, we applied the same systemic, post-training dose of scopolamine in three different, non-spatial and spatial, complex learning tasks. We chose to use a relatively high dose of intraperitoneally (i.p.) administered scopolamine (5 mg/kg), because it was shown that post-training scopolamine influences memory performance only at doses of more than 4 mg/kg (Blake et al., 2011; Roldán et al., 1997). The impact of post-training scopolamine was assessed in an odour discrimination task, visual discrimination task and two spatial learning tasks, and included the reversal learning condition in all these
Declarative memory refers to the ability to encode, consolidate and retrieve episodic and semantic information (Baddeley, 1995; Bauer, 2013; Roediger et al., 2008; Ullman, 2016). Although rats and mice lack many declarative functions, rodent studies have been instrumental in elucidating the underlying neurobiological mechanisms of encoding, consolidation and retrieval (Ben-Yakov et al., 2015; Squire et al., 2015). The cholinergic hypothesis proposes a prominent role of acetylcholine in complex learning and memory, and continues to be the theoretical basis of experimental drug research for the treatment of declarative memory dysfunction (Hasselmo and McGaughy, 2004; Klinkenberg and Blokland, 2010; Volpato and Holzgrabe, 2018). Cholinergic neurotransmission is the neurochemical backbone of a network involved in higher cognition (or its rodent equivalent), which includes entorhinal cortex, dentate gyrus, CA1, CA3 and the medial septum. Much of the work supporting the cholinergic hypothesis is based on human and animal research that used the nonselective muscarinic antagonist scopolamine (hyoscine) to mimic cholinergic, neurocognitive impairment (Klinkenberg and Blokland, 2010). Pre-training cholinergic modulation has been shown to influence acquisition in spatial and non-spatial rodent tasks (Lee et al., 2018), but the effects of post-acquisition cholinergic modulation are not so well established. Some authors suggested that acetylcholine influences
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Corresponding author at: Biological Psychology, University of Leuven (KU Leuven), Tiensestraat 102, 3000, Leuven, Belgium. E-mail address:
[email protected] (R. D’Hooge).
https://doi.org/10.1016/j.brainresbull.2019.07.012 Received 17 May 2019; Received in revised form 2 July 2019; Accepted 9 July 2019 Available online 11 July 2019 0361-9230/ © 2019 Published by Elsevier Inc.
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Table 1 Odour discrimination protocol. After completing the shaping phase, animals performed three odour discrimination sessions and one reversal (one session per day). Of the two presented odours, only one was rewarded. The rewarded odour was randomized between animals. After criterion was reached in the third discrimination session (i.e., eight successful consecutive trials), animals were injected with 0, 1 or 5 mg/kg scopolamine. On the next day, reward contingencies were reversed, i.e. the previously rewarded odour became unrewarded and vice versa. Session
Odours
Acute scopolamine administration
Odour discrimination I Odour discrimination II Odour discrimination III Reversal
Paprika vs celery salt Parsley vs marjoram Oregano vs rosemary Rosemary vs oregano
– – 0, 1 or 5 mg/kg; after discrimination –
tasks to assess flexibility across cognitive domains. We had to use repeated injections in some of these tasks, but given the half-life of scopolamine (8 h, Malamed, 2018), carryover to the next acquisition session seemed unlikely. It was also unlikely that the observed effects could be attributed to the effects of scopolamine on non-memory-related cognitive effects (e.g., attention), non-cognitive behaviours (e.g., anxiety, locomotor activity) or peripheral side-effects (e.g., mydriasis), since scopolamine was injected after training (Klinkenberg and Blokland, 2010).
to the starting position of the animal (Fig. 1A). The platforms were separated by a plastic intersection and each platform had a circular opening where a plastic digging cup was installed. The digging cup contained bedding material (fine wood chips), and one of the six odours (paprika, celery, parsley, marjoram, oregano, rosemary; 0.01%). Small pieces of breakfast cereal, hidden in the bedding material, were used as reward associated with a certain target odour. By adding powdered cereal to the bedding material (0.1%), we prevented that animals would choose a digging cup based on the odour of the buried reward, rather than the target odours. Mice were placed on scheduled feeding during the entire test to keep their body weights at 85–95% of their freefeeding weight. Animals were shaped to dig in non-scented bedding material to search for the reward, until robust digging behaviour was established. The second shaping phase consisted of 7 daily session of 4 trials, where only one digging cup was filled with bedding material and a reward, while the other one remained empty. The position of the filled cup (left or right) was randomized between trials. To start a trial, the animal was placed in the box at the far side of the platforms facing the wall. Time to find and eat the reward was recorded. When the animal found the reward, it was placed back in its home cage, the setup was briefly cleaned with ethanol (70%), and the cup was filled again. Each animal completed all trials before the next animal started. After shaping was completed, animals were trained for three days in an odour discrimination protocol, followed by one day of reversal (see Table 1). During odour discrimination training (phases OD I – III), both digging cups were used and mixed with a different odour, but only one of the two odours was rewarded. Every day, a new pair of odours was used. Each animal performed trials until it approached and dug in the correct cup 8 times in a row (success criterion). During ODI and II, no drug treatment was given (see Table 1). After completing training phase OD III (reaching criterion and finishing the session), mice were i.p. injected with scopolamine or saline post-training, and placed back in their home cage. During reversal, 24 h later, animals were again presented the same odours as during ODIII, but now the rule was reversed, i.e. the previously rewarded odour became unrewarded and vice versa. Digging in the non-rewarded cup was scored as an error and the animal was allowed to correct (dig in rewarded cup). The rewarded odours and location (left or right) were counterbalanced over mice within each of the three treatment groups (i.e., control, 1 or 5 mg/kg scopolamine). Maximum trial duration was 10 min, criterion was eight consecutive successful trials. Dependent variables were number of trials to reach criterion, the total number of errors, and the average trial time of the last 8 trials.
2. Methods 2.1. Animals and drug administration 10-12-week-old C57BL/6JRj female mice were obtained from Janvier Labs (Le Genest-Saint-Isle, FR). As in much of our previous work, we included only female animals to reduce variability due to male territorial fighting. Female mice are much easier to house socially (Van Loo et al., 2003), whereas territorial fighting and differences in social hierarchy in socially housed males has been shown to impact behavioural readouts (Arakawa, 2018). Estrous cycles were not monitored during the experiments. Although hormonal levels vary during the estrous cycle (Shaikh, 1971), recent meta-analyses that monitored behavioural, morphological, physiological, and molecular traits in over 500 studies, failed to find differences in trait variability between male and female mice (Becker et al., 2016; Prendergast et al., 2014). Mice were housed in temperature-controlled rooms (22 ± 1 °C) with a 12 -h light/dark cycle (lights on at 8am). Animals had ad libitum access to food and water unless specified otherwise. They were randomly assigned to either a scopolamine group (1 mg/kg SCOP1, or 5 mg/kg SCOP5) or vehicle group (control). Four independent batches of animals were tested: Batch 1 (odour discrimination) contained 16 animals assigned to three groups (controls, n = 5; SCOP1, n = 5; SCOP5, n = 6), Batch 2 (visual discrimination learning) included 15 animals divided in two groups (controls, n = 8; SCOP5; n = 7), Batch 3 and 4 (spatial learning tests) included 40 animals (controls, n = 20; SCOP5, n = 20). Scopolamine hydrobromide (Tocris Biosciences, Bristol, UK) was dissolved in saline (0.1 and 0.5 mg/ml), and aliquots were stored at −20 °C until use. Mice were injected i.p. at 1% body weight volumes, resulting in doses of 1 mg/kg (SCOP1) or 5 mg/kg (SCOP5). Control animals received comparable saline injections. Scopolamine was administered immediately (within 5 min) after odour discrimination session OD III (see protocol below), or after each visual discrimination or spatial acquisition session. All experiments were approved by the Animal Ethics Committee of the University of Leuven, in accordance with EU Directive 2010/63/EU.
2.3. Visual discrimination and reversal learning This form of learning was examined in 8 parallel operant cages containing touch-sensitive screens in which mice learned to discriminate between two visual images (Camden Instruments LTD, Loughborough, Leics., UK ; Mar et al., 2013) (Fig. 2A). Strawberryflavoured milk was provided as reward for correct choices. The experiment consisted of an initial shaping phase, followed by visual pairwise discrimination (PD) or reversal learning (RL). Every PD or RL
2.2. Odour discrimination and reversal learning This form of learning was assessed in a digging paradigm modified from Brown and colleagues (Birrell and Brown, 2000; McAlonan and Brown, 2003). The setup consisted of a rectangular grey plastic box (43 x 28 cm) with two metal platforms positioned side by side, opposite 53
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daily session was limited in number of correct trials (maximum 30) or time (maximum 60 min). The animals were stepwise shaped to touch a correct image, starting with touch training (one stimulus presented left or right; reward provided after a fixed amount of time or after a correct touch), must touch training (similar to touch training but a reward was provided only when the animal touched the correct screen) and must initiate training (every trial required a nose poke in the reward tray to be initiated). Promotion to the next stage was allowed when animals reached criterion of completing 30 trials in 60 min. After completing the must initiate training, animals were subjected to the final punish incorrect training, where a punishment (i.e., 5 s time-out with house lights on) was presented when the blank screen was touched. After reaching criterion (24/30 trials correct in 60 min for 2 consecutive days), animals moved on to the PD stage where two visual stimuli were presented, of which only one was rewarded. When the non-rewarded stimulus was chosen, a time-out (5 s house lights on) was introduced, followed by a correction trial. In this correction trial, the same left-right stimulus configuration was shown. Correction trials were presented until the right choice was made. After reaching criterion (24/30 trials correct in 60 min for 2 consecutive days), reward contingencies were reversed to assess flexibility (RL). Data was acquired with Abet II software. Dependent variables were total number of sessions, % correct (correction trials not included), and perseveration index PI, with PI = correction trials / incorrect trials (Brigman et al., 2008; Piiponniemi et al., 2017). Correction trials were not included in the calculation of % correct.
this compared to classic ANOVA (Boisgontier and Cheval, 2016). Additionally, we have set the cut-off for number of sessions to include in the statistical analysis and visualisation arbitrarily to 50%, i.e. when 50% of the animals reached criterion. Statistical analyses and plotting were done in R (R Core Team, 2018). P-values smaller than 0.05 were considered statistically significant, reported effect sizes are generalized eta squared (gη2, Bakeman, 2005) and data are presented as means ± SEM. 3. Results 3.1. Effect of post-training scopolamine on odour discrimination learning Animals were assigned randomly to either saline or scopolamine treatment after ODIII. To rule out a priori group effects, we analysed performance on ODI and ODII (Session) using repeated measures ANOVA (RM-ANOVA) with trials to criterion as dependent variable. This analysis revealed no significant interaction of Group x Session (F2,13 = 2.09, p = 0.16, gη2 = 0.15) or main effects of Session (F1,13 = 1.10, p = 0.31, gη2 = 0.04) or Group (F2,13 = 1.28, p = 0.31, 2 gη = 0.08). These results indicate that possible effects of scopolamine on reversal learning cannot be attributed to a priori differences between groups. Furthermore, a one-way ANOVA of trials to criterion for OD III with odours as between-subject factor indicated that performance during OD III was independent of the type of odours being used (F1,14 = 0.00, p = 0.95, gη2 < 0.01). To analyse the effect of scopolamine on RL, we compared the number of trials to criterion for OD III and RL using RM-ANOVA. We observed a marginal interaction effect Group x Session (F2,13 = 3.16, p = 0.08, gη2 = 0.18), and a significant main effect of Session (F1,13 = 6.88, p = 0.02, gη2 = 0.20), but the effect of Group was not significant (F2,13 = 1.41, p = 0.28, gη2 = 0.10). We conducted post-hoc comparisons (Tukey corrections) using specific contrasts in order to compare performance of each of the 3 groups during RL with the average performance during discrimination (as groups were only assigned after discrimination and no differences were found during the first two discrimination sessions). Both the control group (t21.6 = 2.76, p = 0.01) as well as the SCOP1 group (t21.6 = 2.10, p = 0.04) needed significantly more trials to reach criterion during reversal, compared to the average performance during discrimination. However, there was no statistical difference in trials to criterion during discrimination versus reversal for SCOP5 mice (t22 = 0.52, p = 0.61) (Fig. 1B ). RM-ANOVA of the total number of errors during OD III and RL resulted in a marginally significant interaction effect Group x Session (F2,13 = 2.90, p = 0.09, gη2 = 0.20), and a significant main effect of Session (F1,13 = 10.01, p < 0.01, gη2 = 0.31), but the effect of Group was not significant (F2,13 = 2.83, p = 0.10, gη2 = 0.16). Post-hoc comparisons using specific contrasts indicated that both the control group (t19 = 3.51, p < 0.01) as well as the SCOP1 group (t19 = 2.9, p < 0.01) committed more errors during reversal. However, there was no difference in committed errors between the discrimination and reversal session for SCOP5 animals (t19.7 = 0.42, p = 0.68) (Fig. 1C). Finally, we analysed the average time of the 8 last trials during OD III and RL. RM-ANOVA indicated no significant effect of the interaction effect Group x Session (F2,13 = 0.44, p = 0.65, gη2 = 0.009), nor for the main effect Session (F1,13 = 0.47, p = 0.50, gη2 = 0.005) or Group (F2,13 = 0.04, p = 0.97, gη2 = 0.005) (Fig. 1D). In summary, animals needed more trials when subjected to reversal learning, and made more errors, possibly due to a certain level of perseverance. In contrast, administration of high dose scopolamine (SCOP5) after ODIII, but not low dose (SCOP1), interfered with
2.4. Spatial and reversal learning Spatial learning ability was evaluated in the Morris water maze (MWM, Fig. 4A) as previously described (D’Hooge et al., 2005). In brief, animals had to find a hidden escape platform (15 cm diameter) in a circular pool (150 cm diameter) filled with opacified water (26 ± 0.5 °C; Acusol ™ OP301 Opacifier, The Dow Chemical Company, Horgen, CH). During both spatial acquisition and reversal learning, mice performed 4 trials per day (random starting position) with a maximum trial duration of 2 min. After 10 days of spatial learning, the platform was moved to the opposite location for 5 days of reversal learning. To test spatial reference memory, the platform was removed and probe trials (100 s) were performed on day 6, 11, and 16. Swim tracks were recorded using Ethovision® tracking software (Noldus™, Wageningen, NL). Escape latency to find the hidden platform, mean distance to target, velocity, path length and time spent in quadrants were examined as dependent variables. To investigate the effect of overtraining, a second MWM experiment was conducted with a different batch of animals. In this experiment, mice performed 4 trails per day for 4 consecutive days (acquisition), after which the platform was moved to the opposite location (reversal learning, 4 trials in 1 day). 2.5. Statistical analysis Group means were compared with two-sided Welch t-tests (Derrick and White, 2016). Odour discrimination and Morris water maze data were analysed with repeated measures ANOVA. Post-hoc comparisons were conducted with Tukey’s HSD tests. Departure from sphericity, indicated by Mauchly’s test, was corrected with Greenhouse-Geisser’s method. Averaged touchscreen data were analysed with classic repeated measures ANOVA, but times-series were analysed using linear mixed effect models. Given the design requires mice to reach criterions, datasets are often incomplete (i.e., the number of sessions differs between animals). Linear mixed effect models are more suited to deal with
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Fig. 1. Odour discrimination. (A) After completing training, mice initiated discrimination sessions were 2 digging cups were presented. Each cup contained bedding material that was mixed with a specific odour. Only one cup (odour) was rewarded. After reaching criterion (8 successful consecutive trials), reward contingencies were reversed. (B) Trials to criterion. Both the control group as well as the 1 mg/kg scopolamine group need significantly more trials to reach criterion during reversal compared to OD III (p < 0.05), while there was no difference between ODIII and reversal for the 5 mg/kg scopolamine group. (C) Error trials. Control mice and low-dose scopolaminetreated animals performed more error trials during reversal compared to ODIII (p < 0.01), which was not the case for high-dose scopolamine-treated mice. (C) Average trial time. There was no difference in trial time between groups (p > 0.05). * p < 0.05, ** p < 0.01.
2 = 0.01), a non-significant main effect of Group (F1,13 = 0.05, p = 0.83, gη2 = 0.00) and a significant main effect of Schedule (F1,13 = 34, p < 0.0001, gη2 = 0.57) (Fig. 2D). To examine this more in detail, we analysed these data over sessions using linear mixed-effects models (LME) for RM-ANOVA. Because the number of animals varies over sessions (i.e., when criterion is met during PD, animals move on to the RL phase), we chose to limit the number of sessions to be included in these analyses to the point where 50% of the subjects reached criterion. During PD, 50% of the animals reached criterion within 11 sessions. During RL, 50% of the subjects reached criterion after 13 sessions. RM-ANOVA using LME on percentage correct during PD revealed no effect of interaction Group x Session (F10,87.7 = 0.51, p = 0.88), no main effect of Group (F1,10.4 = 1.50, p = 0.25) and a marginal main effect of Session (F10,87.7 = 1.72, p = 0.09) (Fig. 3A ). Analysis of percentage correct during RL indicated a marginal effect of interaction Group x Session (F12,131.8 = 1.71, p = 0.07), no main effect of Group (F1,11.4 = 0.76, p = 0.40) and a significant main effect of Session (F12,131.8 = 12.57, p < 0.0001). Posthoc analysis revealed marginal differences between groups on session 2 (t77.1 = 2.0, p = 0.05) and 11 (t86.5 = 1.83, p = 0.07) (Fig. 3B). Analysis of the perseveration index during PD revealed a significant interaction Group x Session (F10,90.3 = 2.37, p = 0.02), no main effect of Group (F1,13 = 0.40, p = 0.54) and a marginal main effect of Session (F10,90.3 = 1.94, p = 0.05). Post-hoc analysis revealed significant differences between groups on session 1 (t87.1 = 2.68, p = 0.009) and 10 (t98.3 = 2.88, p = 0.005) (Fig. 3C). Analysis of the perseveration index during RL showed a significant interaction Group x Session (F12,131.4 = 2.79, p = 0.002), no main effect of Group (F1,10.7 = 0.32,
perseverance and resulted in a lower number of trials to criterion and lower number of errors.
gη
3.2. Effect of post-training scopolamine on visual discrimination learning Visual discrimination was evaluated in a touchscreen experiment. A Welch Two Sample t-test showed there was no difference in total sessions between controls and SCOP5 animals during shaping (t10.9 = 1.05, p = 0.32), indicating that groups initiated PD sessions at a similar level. After completing shaping, mice continued with PD sessions, where scopolamine administration was initiated. RM-ANOVA on total sessions with Schedule (PD versus RL) as within and Group as between factor revealed a marginal interaction effect Group x Schedule (F1,13 = 3.73, p = 0.08, gη2 = 0.07) and non-significant main effects of Group (F1,13 = 0.75, p = 0.40, gη2 = 0.04) and Schedule (F1,13 = 2.41, p = 0.14, gη2 = 0.05). Post-hoc comparisons showed that control mice needed significantly more sessions to reach criterion during RL compared to PD (t13 = 2.55, p = 0.02), while this was not the case for mice treated with scopolamine (t13 = 0.26, p = 0.80). Compared to control animals, SCOP5 mice needed marginally more sessions to reach criterion during PD (t21.6 = 1.75, p = 0.09), but not during RL (t21.6 = 0.27, p = 0.79) (Fig. 2B ). RM-ANOVA on percentage correct resulted in a non-significant interaction effect GroupSchedule (F1,13 = 0.28, p = 0.61, gη2 = 0.01), a non-significant main effect of Group (F1,13 = 0.56, p = 0.47, gη2 = 0.02) and a significant main effect of Schedule (F1,13 = 68.01, p < 0.0001, gη2 = 0.73) (Fig. 2C). Analysis of the perseveration index indicated a non-significant interaction effect Group-Schedule (F1,13 = 0.15, p = 0.70,
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Fig. 2. Visual discrimination. (A) During pairwise discrimination (PD), two stimuli were presented simultaneously. Touching the correct stimulus resulted in a liquid food reward. After reaching criterion, reward contingencies were reversed. (B) Session to criterion. Control mice needed more sessions to reach criterion during reversal compared to PD (p < 0.5), while there was no difference between PD and reversal for scopolamine-treated mice. (C) Percentage correct. Mice performed better during PD compared to reversal (p < 0.001). (D) Perseveration index. The perseveration index was overall higher during reversal compared to PD (p < 0.001). * p < 0.05, *** p < 0.001.
p = 0.58) and a significant main effect of Session (F12,131.4 = 9.55, p < 0.0001). Post-hoc analysis showed a significant difference between groups on session 1 (t100.5 = 3.5, p < 0.001) (Fig. 3D). In summary, animals learned to discriminate between 2 visual stimuli and choose the correct one after 11 sessions. Scopolamine had no effect on visual discrimination. When the rule was reversed, control animals made initially more mistakes and needed more trials to criterion. In addition, control animals showed significant perseveration to the original rule setting. In contrast, scopolamine treated animals made initially less mistakes and adapted quicker to the new rule (lower perseveration score).
RM-ANOVA of path length during acquisition showed a marginal interaction effect Group x Session (F4.9,102.3 = 2.28, p = 0.05, 2 gη = 0.08), no main effect of Group (F1,21 = 0.06, p = 0.81, 2 gη < 0.001) and a significant main effect of Session (F4.9,102.3 = 54.51, p < 0.0001, gη2 = 0.67). Post-hoc comparisons revealed slight differences on session 2 (control: M = 769.7, SD = 607.3; SCOP5: M = 896.6, SD = 701.2; t175.6 = 1.84, p = 0.07) and session 3 (control: m = 693.9, sd = 628.8; SCOP5: m = 456.6, sd = 400.7; t175.6 = 1.84, p < 0.05). RM-ANOVA of path length during RL revealed no effect of interaction Group x Session (F1.7,36 = 0.43, p = 0.62, gη2 = 0.01), no main effect of Group (F1,21 = 0.33, p = 0.57, gη2 = 0.008), and a significant main effect of Session (F1.7,36 = 19.5, p < 0.0001, gη2 = 0.31). Analysis of velocity during acquisition indicated a significant interaction Group x Session (F6.5,136.1 = 3.64, p = 0.002, gη2 = 0.03), no main effect of Group (F1,21 = 1.68, p = 0.21, gη2 = 0.06) and a significant main effect of Session (F6.5,136.1 = 2.69, p = 0.01, gη2 = 0.03). Post-hoc comparisons revealed significant group differences on session 5 (control: m = 13.7, sd = 6.2; SCOP5: m = 17.3, sd = 4.5; t33.2 = 2.21, p = 0.03), session 7 (control: m = 13, sd = 6.3; SCOP5: m = 17, sd = 3.8, t33.2 = 2.54, p = 0.02) and session 8 (control: m = 13.2, sd = 6.5; SCOp5: m = 16.9, sd = 5.5, t33.2 = 2.06, p = 0.05). RM-ANOVA of velocity during RL revealed no interaction effect Group x Session (F3.2,68.1 = 0.19, p = 0.91, gη2 = 0.001), no main effect of Group (F1,21 = 2.79, p = 0.11, gη2 = 0.10) and a significant main effect of Session (F3.2,68.1 = 4.45, p = 0.005, gη2 = 0.03) (Fig. 4C).
3.3. Effect of scopolamine on spatial learning and reversal in the Morris water maze Spatial learning was assessed in the Morris water maze. Scopolamine or vehicle was administered daily after the last swim trial. RM-ANOVA of latency to platform revealed no interaction Group x Session (F4.3,91.1 = 1.04, p = 0.40, gη2 = 0.01), no main effect of Group (F1,21 = 1.53, p = 0.23, gη2 = 0.05) and a significant main effect of Session (F4.3,91.1 = 41.59, p < 0.0001, gη2 = 0.36). For RL, there was no interaction Group x Session (F2.5,53.3 = 2.05, p = 0.13, gη2 = 0.04), no main effect of Group (F1,21 = 0.86, p = 0.36, gη2 = 0.02) and a significant main effect of Session (F2.5,53.3 = 20.92, p < 0.0001, 2 gη = 0.28) (Fig. 4B).
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Fig. 3. Visual discrimination, detailed analysis. (A) Percentage correct during pairwise discrimination (PD). There was no difference between groups (p > 0.05). (B) Percentage correct during reversal. There was no difference between groups, but performance increased significantly over sessions (p < 0.001). (C) Perseveration index during PD. There was a significant interaction (p < 0.05). (D) Perseveration index during reversal. There was a significant interaction (p < 0.01) and a significant main effect of Sessions (p < 0.001). Post-hoc analysis revealed a difference between groups on Session 1 (p < .001). *** p < 0.001.
Given the dependence of latency on velocity and the aforementioned group differences in swim speed, we have analysed mean distance to target, a parameter which is less dependent on velocity. RM-ANOVA of mean distance to target during acquisition indicated no interaction effect Group x Session (F3.6,75.9 = 1.05, p = 0.38, gη2 = 0.02), no main effect of Group (F1,21 = 1.12, p = 0.30, gη2 = 0.03) and a significant main effect of Session (F3.6,75.9 = 22.94, p < 0.0001, gη2 = 0.35). Analysis of mean distance to target during RL revealed no interaction Group x Session (F2.9,60.6 = 0.45, p = 0.71, gη2 = 0.009), no main effect of Group (F1,21 = 0.59, p = 0.45, gη2 = 0.02), and a significant main effect of Session (F2.9,60.6 = 26.46, p < 0.0001, gη2 = 0.36) (Fig. 4D). Quadrant preference in probe trials was analysed per group. For the control group, a RM-ANOVA revealed significant interaction Quadrant x Probe (F2.4,21.7 = 5.03, p = 0.01, gη2 = 0.24) and significant main effects of Quadrant (F1.7,15.4 = 6.09, p = 0.01, gη2 = 0.23) and Probe (F1.7,15.5 = 12.65, p < 0.001, gη2 < 0.001). Specific contrast testing indicated that during probe 1, there was no preference for the target quadrant compared to other quadrants (t77.2 = 0.99, p = 0.32). During probe 2 (t77.2 = 3.61, p < 0.001) and probe 3 (t77.2 = 5.57, p < 0.0001), there was a significant preference for the target quadrant and opposite quadrant, respectively (Fig. 4E). For the SCOP5 mice, a RM-ANOVA indicated significant interaction Quadrant x Probe (F3,33.3 = 13.04, p < 0.0001, gη2 = 0.45), a significant main effect of Quadrant (F2,21.6 = 16.27, p < 0.0001, gη2 = 0.30) and no effect of Probe (F1,11 = 1.31, p = 0.28, gη2 = 0.002). Specific contrasts showed that there was a marginal preference for the target quadrant over other quadrants during probe 1 (t98.3 = 1.90, p = 0.06), a significant preference during probe 2 (t98.3 = 7.19, p < 0.0001), and a significant
preference for the opposite quadrant over other quadrants during probe 3 (t98.3 = 7.66, p < 0.0001) (Fig. 4F). In summary, vehicle treated mice learned to localize a hidden platform, which was not affected by administration of 5 mg/kg scopolamine after training sessions. However, we did observe a significant effect on swim velocity after scopolamine administration. For mean distance to target, which is less sensitive to velocity, there was no difference between groups during acquisition or reversal. With a separate batch of animals, we repeated the MWM, but included only 4 spatial acquisition sessions to investigate if overtraining might have affected the outcome in the first experiment. After 4 acquisition sessions (A1-A4), the animals were subjected to one reversal session. RM-ANOVA of latency to platform during acquisition revealed no interaction Group x Session (F1,14 = 0.07, p = 0.79, gη2 = 0.003), no main effect of Group (F2.1,28.8 = 0.41, p = 0.67, gη2 = 0.01) and a significant main effect of Session (F2.1,28.8 = 17.0, p < 0.0001, 2 gη = 0.36). Analysis of latency to platform during RL indicated there was no difference between groups (t59.9 = 0.45, p = 0.66) (Fig. 5A). Performance during reversal was further investigated by comparing performance of each trial. RM-ANOVA revealed no interaction Group x Trial (F2.5,34.5 = 1.41, p = 0.26, gη2 = 0.05), no main effect of Group (F1,14 = 0.11, p = 0.75, gη2 = 0.004) and a significant main effect of Trial (F2.5,34.5 = 1.41, p < 0.01, gη2 = 0.17) (Fig. 5B). RM-ANOVA of path length during acquisition revealed no interaction Group x Session (F2.7,37.6 = 0.61, p = 0.60, gη2 = 0.03), no main effect of Group (F1,14 = 2.24, p = 0.16, gη2 = 0.06) and a significant main effect of Session (F2.7,37.6 = 25.5, p < 0.0001, gη2 = 0.52). RMANOVA analysis of velocity during acquisition indicated no interaction
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Fig. 4. Spatial learning. (A) Morris water maze setup. Mice had to swim and locate the hidden platform (original target: full circle, reversal target: dotted circle). There were 10 days of acquisition (A1-10) and 5 days of reversal (R15), each with 4 swim trials per day. Reference memory was assessed in 3 probe trials. (B) Escape latency. There was no difference between groups during acquisition or reversal. (C) Swim speed. There was a significant interaction effect during acquisition (p < 0.05) and there were significant main effects of Session during acquisition as well as during reversal (p < 0.05). (D) Mean distance to target. There was no difference between groups during acquisition or reversal. (E) Probe data controls. Mice showed a significant preference for the quadrant containing the platform during probe 2 and 3, but not during probe 1 (p < 0.001). (F) Probe data scopolamine group. There was significant preference for the quadrant containing the platform during probe 2 and 3, but not during probe 1 (p < 0.001). * p < 0.05, ** p < 0.01, *** p < 0.001.
Group x Session (F1.8,25.4 = 1.26, p = 0.30, gη2 = 0.03), and no main effects of Group (F1,14 = 0.32, p = 0.58, gη2 = 0.02) and Session (F1.8,25.4 = 2.10, p = 0.15, gη2 = 0.04). During RL, there was also no difference in velocity between groups (t60.4 = 0.02, p = 0.99). Analogous to the first MWM experiment, we analysed the mean distance to target. During acquisition, RM-ANOVA indicated no interaction Group x Session (F2.9,40.2 = 0.16, p = 0.92, gη2 = 0.004), no main effect of Group (F1,14 = 2.69, p = 0.12, gη2 = 0.11) and a significant main effect of Session (F2.9,40.2 = 10.63, p < 0.0001, gη2 = 0.20). During RL, there was also no difference in distance to target between controls and SCOP5 animals (t59 = 1.2, p = 0.25) (Fig. 5C). Given the
already present difference between groups on A4, we conducted a RMANOVA of A4/R data. This analysis indicated no interaction Group x Session (F1,14 = 1.49, p = 0.24, gη2 = 0.06), no main effect of Group (F1,14 = 0.11, p = 0.74, gη2 = 0.003) and a significant main effect of Session (F1,14 = 6.04, p = 0.03, gη2 = 0.21). Detailed analysis of RL data over trials failed to show interaction Group x Trial (F2.5,34.4 = 0.55, p = 0.62, gη2 = 0.03), no main effect of Group (F1,14 = 1.71, p = 0.21, 2 gη = 0.03) and a significant main effect of Trial (F2.5,34.4 = 4.98, p < 0.01, gη2 = 0.21) (Fig. 5D). In summary, using a shorter training protocol, we observed that both groups displayed significant perseverance when the platform
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Fig. 5. Spatial learning with limited number of acquisition sessions. (A) Escape latency. There was no difference between groups during acquisition or reversal (p > 0.05). (B) Trial analysis of escape latency during the reversal session. There was no difference between groups (p > 0.05). (C) Mean distance to target. There was no difference between groups during acquisition or reversal (p > 0.05). RM-ANOVA analysis of A4/R data returned a non-significant interaction effect (p > 0.05). (D) Trial analysis of mean distance to target during reversal. There was no difference between groups (p 0 > 0.05).
high-dose treatment. These results are in line with previous reports indicating that for post-training treatment, only high doses (i.e., > 4 mg/kg) affect later performance (Blake et al., 2011; Roldán et al., 1997). To test whether our differences could be explained by motivational aspects, we analysed duration of the last eight trials (i.e., the trials that led to criterion). Given the lack of differences in trial duration, it seems unlikely that effects of scopolamine were caused by differences in motivation. Although the effect of high-dose scopolamine on odour detection/ sensitivity has not been investigated yet, it has been shown that lowdose scopolamine (0.10-0.20 mg/kg) did not affect odour detection performance in rats (Doty et al., 2003). Furthermore, since scopolamine was applied post-training in current study, it is unlikely that results are due to variations in odour detection. Studies examining the effect of scopolamine treatment on odour discrimination in rodents have predominantly focused on pre-training interventions (De Rosa and Hasselmo, 2000; Ravel et al., 1992). However, few studies reported post-training effects of scopolamine treatment as well. Carballo-Márquez and colleagues showed that bilateral infusion of scopolamine in the prelimbic area immediately after training produced severe amnesia in an odour discrimination task (Carballo-Márquez et al., 2007). Miranda et al. reported that low-dose scopolamine disrupted memory consolidation in an odour habituation task, but results were dose and odour dependent (Miranda et al., 2009). In this study, we used different odours for odour discrimination as well. However, analysis showed that there was no difference between odours, i.e. mice were able to discriminate between different odours, regardless which odour was rewarded. In the second experiment, we investigated the effect of post-training scopolamine administration on visual discrimination. In this
location was changed, evidenced by an increase in latency to find the platform. In contrast, there was little perseverance during reversal for mean distance to target in both groups. 4. Discussion Scopolamine, a non-selective muscarinic antagonist, is one of the most frequently used drugs to induce experimental amnesia (Klinkenberg and Blokland, 2010). The role of cholinergic neurotransmission in learning and memory has been extensively explored ever since the discovery of its amnestic effects (Kopelman, 1986). M1 (and M5) receptors are predominantly situated in brain regions important for memory and learning, including neocortex, hippocampus and amygdala. It has been suggested that cholinergic transmission has different effects on acquisition/encoding and post-acquisition processes (Hasselmo and McGaughy, 2004; Meeter et al., 2004). In this study, we investigated the effect of post-training scopolamine administration in different cognitive tasks. Post-training scopolamine was first administered during an odour discrimination digging protocol. Results show that low-dose scopolamine (1 mg/kg) did not affect memory consolidation, as evidenced by the observation that SCOP1 mice were as able to reach criterion during reversal as the control group. The fact that all mice needed more time to reach criterion during reversal indicated that previously learned information interfered with the acquisition of new information. However, a high-dose of scopolamine (5 mg/kg) eliminated the difference in numbers of trials needed to reach criterion between discrimination and reversal, suggesting that old information did not interfere with the acquisition of new information. We observed that low-dose scopolamine resulted in more error trials (similar to controls), compared to 59
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experiment, scopolamine was injected after every discrimination session. Results show that scopolamine-treated mice needed more sessions than control animals to reach criterion during pairwise discrimination. This indicates that, since scopolamine was applied post-training, it interfered with consolidation and/or retrieval of previously learned information. Detailed analysis of percentage correct showed there was no difference between both groups during pairwise discrimination. The absence of a difference in percentage correct while there is a clear difference in total sessions can be explained by the inclusion criterion of data in the more detailed linear mixed-effect models analysis. Indeed, due the methodological design where animals shift to the reversal phase after reaching criterion in pairwise discrimination, the number of animals per session varies, making the statistical analysis problematic, especially towards the end of the discrimination phase. Because of this, we decided to only include sessions in the analysis up until the point where 50% of the subjects reached criterion. While there was no difference in percentage correct during pairwise discrimination, control mice performed slightly better during reversal learning. Though this difference is not reflected in the total number of sessions needed to reach criterion, it might indicate that impaired consolidation and/or retrieval during pairwise discrimination affected post-discrimination learning and flexibility. Analysis of the perseveration index, a measure for flexibility, showed a modest decrease in perseverance for scopolamine-treated mice during the initial reversal sessions, in line with previous results. Scopolamine has been reported to affect visual discrimination when applied pre-training in various studies (Andrews et al., 1992; Drinkenburg et al., 1995; Talpos et al., 2009). However, it has been argued that these results might be biased by peripheral side-effects such as scopolamine-induced mydriasis (Klinkenberg and Blokland, 2010). In current study, drug treatment was applied post-training, thus avoiding side-effect complications. To our knowledge, this is the first study to test the effect of post-training scopolamine treatment on visual discrimination. Finally, we tested the effect of post-training scopolamine administration on spatial learning in two Morris water maze experiments. In the first spatial learning experiment, there was a 10-days acquisition phase followed by a 5-days reversal. Scopolamine treatment was applied after every acquisition session. Results indicate that scopolamine-treatment had in general no effect on spatial learning performance. Although not statistically significant, latency to target data showed that scopolaminetreated mice performed slightly better towards the end of the acquisition. However, this difference can be explained by the observed difference in swim speed, with overall lower velocity for control animals. Indeed, analysis of mean distance to target, a measure less sensitive to swim speed, showed that there was no difference between both groups. Although both groups displayed a typical learning curve, analysis of probe data indicated that only during the second probe (i.e. after 10 days of acquisition) animals were able to clearly distinguish between the platform quadrant and others. However, this slower learning rate was present in both groups. A second Morris water maze experiment was conducted that included only 4 days of acquisition followed by 1 day of reversal. By limiting the number of acquisition days, we aimed to test the hypothesis that overtraining affected performance in the first experiment. Results showed that there was no difference between groups for latency to target, velocity or mean distance to target. Furthermore, the observed difference in velocity during the first spatial learning experiment was not present in the second test. Overall, these results are consistent with the first Morris water maze experiment and suggest that post-training scopolamine administration does not impair spatial learning. To our knowledge, only effects of pre-training scopolamine administration on spatial learning have been reported so far, with mixed results. For instance, some authors reported increased escape latencies and/or path lengths after pre-training scopolamine administration (Smith et al., 1994; Zhang et al., 2008). Others found no effect of scopolamine on escape latency in the Morris water maze (Berger-Sweeney et al., 1995;
Decker et al., 1990). The observed increase in velocity after scopolamine administration has been reported before (Riekkinen et al., 1990). However, it is not clear whether this is a direct consequence of scopolamine administration, since the compound was injected after training, 24 h before next session. In this study, scopolamine was administered at a relatively high dose (5 mg/kg). Previous reports indicated that post-training scopolamine administration affect memory processes at doses above 4 mg/kg (Blake et al., 2011). Present results in the odour discrimination task confirmed that only high dose (5 mg/kg), but not low dose (1 mg/kg) administration affected performance. Scopolamine is typically considered to be a anticholinergic drug, but its effects reported here should probably be attributed to its various neuropharmacological actions at high dose. Indeed, it has been reported that high doses of scopolamine (> 2 mg/kg i.p. or s.c.) inhibit 5-HT3 receptors (Lochner and Thompson, 2016), and influence interactions between muscarinic and glutamatergic activity (Deutsch et al., 1990; Doguc et al., 2012). Moreover, chronic application of scopolamine has been associated with various cellular changes, including altered neurofilaments expression (Lee et al., 2018), induced myelin basic protein degradation (Park et al., 2016), decreased hippocampal NR2A protein expression (Doguc et al., 2012), altered brain levels of nerve growth factor (Albrech et al., 1991), and increased muscarinic receptor expression (Marks et al., 1984). Cholinergic modulation of attentional processes should also be taken into consideration here. Various authors implemented the acetylcholine system in attentional processes (e.g., Sarter et al., 2003; Schmitz and Duncan, 2018). Cholinergic processes in prefrontal, parietal and somatosensory areas (i.e., areas primarily involved in attentional processes) have been proposed to modulate top-down control of attention (Klinkenberg et al., 2011). However, Klinkenberg and Blokland (2010) emphasized that the reported effects of scopolamine on attention are ambiguous, due to differences in methodology (e.g., animal strains, experimental procedures, application method, targeted region). Moreover, given the 8 -h half-life of scopolamine (Malamed, 2018) and its post-training administration, it seems unlikely that the results of this study can be reduced to attention-related processes, or for that matter, to previously reported non-cognitive effects (e.g., anxiety, locomotor activity) or peripheral side-effects (e.g., mydriasis, drymouth effect) of scopolamine (Gholamreza et al., 2002; Hodges et al., 2009; Jones and Higgins, 1995). In conclusion, post-training administration of scopolamine affected odour discrimination in a digging task and touchscreen-based visual discrimination. In both experiments, scopolamine administration decreased perseverance during the reversal learning phase, compared to controls. In contrast, administration of scopolamine did not affect spatial and reversal learning in the Morris water maze. We argue that it is unlikely that the observed effects can be reduced to non-mnemonic central effects or peripheral side-effects of scopolamine. Acknowledgement DT is a doctoral student of the Flemish science and technological development fund Agentschap Innoveren & Ondernemen (IWT Flanders). This study was also financed by a C1 grant of the University Research Council to RDH. References Albrech, J., Carman-Krzan, M., Fabrazzo, M., Wise, B.C., 1991. Chronic treatment with scopolamine and physostigmine changes nerve growth factor (NGF) receptor density and NGF content in rat brain. Brain Res. 542, 233–240. https://doi.org/10.1016/ 0006-8993(91)91572-I. Andrews, J.S., Grützner, M., Stephens, D.N., 1992. Effects of cholinergic and non-cholinergic drugs on visual discrimination and delayed visual discrimination performance in rats. Psychopharmacology (Berl.) 106, 523–530. https://doi.org/10.1007/ BF02244825. Arakawa, H., 2018. Ethological approach to social isolation effects in behavioral studies of laboratory rodents. Behav. Brain Res. 341, 98–108. https://doi.org/10.1016/j.bbr.
60
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D. Thonnard, et al. 2017.12.022. Baddeley, A.D., 1995. The psychology of memory. Handbook of Memory Disorders. John Wiley & Sons, Oxford, England, pp. 3–25. Bakeman, R., 2005. Recommended effect size statistics for repeated measures designs. Behav. Res. Methods 37, 379–384. https://doi.org/10.3758/BF03192707. Bauer, P.J., 2013. Chapter 16 – Memory Development. In: Rubenstein, J., Rakic, P. (Eds.), Memory Development. Academic Press, Oxford, pp. 297–314. https://doi.org/10. 1016/B978-0-12-397267-5.00040-6. Becker, J.B., Prendergast, B.J., Liang, J.W., 2016. Female rats are not more variable than male rats: a meta-analysis of neuroscience studies. Biol. Sex Differ. 7. https://doi.org/ 10.1186/s13293-016-0087-5. Ben-Yakov, A., Dudai, Y., Mayford, M.R., 2015. Memory retrieval in mice and men. Cold Spring Harb. Perspect. Biol. 7, a021790. https://doi.org/10.1101/cshperspect. a021790. Berger-Sweeney, J., Arnold, A., Gabeau, D., Mills, J., 1995. Sex differences in learning and memory in mice: effects of sequence of testing and cholinergic blockade. Behav. Neurosci. 109, 859–873. https://doi.org/10.1037/0735-7044.109.5.859. Birrell, J.M., Brown, V.J., 2000. Medial frontal cortex mediates perceptual attentional set shifting in the rat. J. Neurosci. 20, 4320–4324. https://doi.org/10.1523/jneurosci. 20-11-04320.2000. Blake, M.G., Boccia, M.M., Krawczyk, M.C., Baratti, C.M., 2011. Scopolamine prevents retrograde memory interference between two different learning tasks. Physiol. Behav. 102, 332–337. https://doi.org/10.1016/j.physbeh.2010.11.026. Boisgontier, M.P., Cheval, B., 2016. The anova to mixed model transition. Neurosci. Biobehav. Rev. 68, 1004–1005. https://doi.org/10.1016/j.neubiorev.2016.05.034. Brigman, J.L., Feyder, M., Saksida, L.M., Bussey, T.J., Mishina, M., Holmes, A., 2008. Impaired discrimination learning in mice lacking the NMDA receptor NR2A subunit. Learn. Mem. 15, 50–54. https://doi.org/10.1101/lm.777308. Bunce, J.G., Sabolek, H.R., Chrobak, J.J., 2004. Timing of administration mediates the memory effects of intraseptal carbachol infusion. Neuroscience 127, 593–600. https://doi.org/10.1016/j.neuroscience.2004.05.039. Carballo-Márquez, A., Vale-Martínez, A., Guillazo-Blanch, G., Torras-Garcia, M., BoixTrelis, N., Martí-Nicolovius, M., 2007. Differential effects of muscarinic receptor blockade in prelimbic cortex on acquisition and memory formation of an odour-reward task. Learn. Mem. 14, 616–624. https://doi.org/10.1101/lm.597507. D’Hooge, R., Lullmann-Rauch, R., Beckers, T., Balschun, D., Schwake, M., Reiss, K., von Figura, K., Saftig, P., 2005. Neurocognitive and psychotiform behavioral alterations and enhanced hippocampal long-term potentiation in transgenic mice displaying neuropathological features of human alpha-mannosidosis. J. Neurosci. 25, 6539–6549. https://doi.org/10.1523/Jneurosci.0283-05.2005. De Rosa, E., Hasselmo, M.E., 2000. Muscarinic cholinergic neuromodulation reduces proactive interference between stored odour memories during associative learning in rats. Behav. Neurosci. 114, 32–41. https://doi.org/10.1037/0735-7044.114.1.32. Decker, M.W., Gill, T.M., McGaugh, J.L., 1990. Concurrent muscarinic and β-adrenergic blockade in rats impairs place-learning in a water maze and retention of inhibitory avoidance. Brain Res. 513, 81–85. https://doi.org/10.1016/0006-8993(90)91091-T. Derrick, B., White, P., 2016. Why Welch’s test is Type I error robust. Quant. Methods Psychol. 12, 30–38. https://doi.org/10.20982/tqmp.12.1.p030. Deutsch, S.I., Panchision, D.M., Rosse, R.B., Novitzki, M.R., Miller, L.P., Mastropaolo, J., 1990. Interaction of cholinergic and glutamatergic transmission in the hippocampus: an in vitro autoradiographic receptor analysis. Neurosci. Lett. 118, 124–127. https:// doi.org/10.1016/0304-3940(90)90264-A. Doguc, D.K., Delibas, N., Vural, H., Altuntas, I., Sutcu, R., Sonmez, Y., 2012. Effects of chronic scopolamine administration on spatial working memory and hippocampal receptors related to learning. Behav. Pharmacol. 23, 762–770. https://doi.org/10. 1097/FBP.0b013e32835a38af. Doty, R.L., Bagla, R., Misra, R., Mueller, E., Kerr, K.L., 2003. No influence of scopolamine hydrobromide on odour detection performance of rats. Chem. Senses 28, 761–765. https://doi.org/10.1093/chemse/bjg067. Drinkenburg, W., Sondag, H., Coenders, C.J.H., Andrews, J.S., Vossen, J.M.H., 1995. Effects of selective antagonism or depletion of the cholinergic system on visual-discrimination performance in rats. Behav. Pharmacol. 6, 695–702. Gholamreza, P., Pratt, J.A., Nima, D., 2002. Effects of low-dose scopolamine on locomotor activity: No dissociation between cognitive and non-cognitive effects. Neurosci. Res. Commun. 31, 165–174. https://doi.org/10.1002/nrc.10049. Hasselmo, M.E., McGaughy, J., 2004. High acetylcholine levels set circuit dynamics for attention and encoding and low acetylcholine levels set dynamics for consolidation. Progress in Brain Research. pp. 207–231. https://doi.org/10.1016/S0079-6123(03) 45015-2. Hodges, D.B., Lindner, M.D., Hogan, J.B., Jones, K.M., Markus, E.J., 2009. Scopolamine induced deficits in a battery of rat cognitive tests: comparisons of sensitivity and specificity. Behav. Pharmacol. 20, 237–251. https://doi.org/10.1097/FBP. 0b013e32832c70f5. Jones, D.N.C., Higgins, G.A., 1995. Effect of scopolamine on visual attention in rats. Psychopharmacology (Berl.) 120, 142–149. https://doi.org/10.1007/BF02246186. Klinkenberg, I., Blokland, A., 2010. The validity of scopolamine as a pharmacological model for cognitive impairment: a review of animal behavioral studies. Neurosci. Biobehav. Rev. 34, 1307–1350. https://doi.org/10.1016/j.neubiorev.2010.04.001. Klinkenberg, I., Sambeth, A., Blokland, A., 2011. Acetylcholine and attention. Behav. Brain Res. 221, 430–442. https://doi.org/10.1016/j.bbr.2010.11.033. Kopelman, M.D., 1986. The cholinergic neurotransmitter system in human memory and dementia: a review. Q. J. Exp. Psychol. Sect. A 38, 535–573. https://doi.org/10. 1080/14640748608401614. Lee, J.C., Park, J.H., Ahn, J.H., Park, J., Kim, I.H., Cho, J.H., Shin, B., Lee, T.K., Kim, H., Song, M., Cho, G.S., Kim, D.W., Kang, I.J., Kim, Y.M., Won, M.H., Choi, S.Y., 2018. Effects of chronic scopolamine treatment on cognitive impairment and neurofilament
expression in the mouse hippocampus. Mol. Med. Rep. 17, 1625–1632. https://doi. org/10.3892/mmr.2017.8082. Lochner, M., Thompson, A.J., 2016. The muscarinic antagonists scopolamine and atropine are competitive antagonists at 5-HT3 receptors. Neuropharmacology 108, 220–228. https://doi.org/10.1016/j.neuropharm.2016.04.027. Malamed, S.F., 2018. Pharmacology. Chapter 25 In: Malamed, S.F. (Ed.), Sedation (Sixth Edition). Mosby, pp. 319–358. https://doi.org/10.1016/B978-0-323-40053-4. 00025-1. Mar, A.C., Horner, A.E., Nilsson, S.R.O., Alsio, J., Kent, B.A., Kim, C.H., Holmes, A., Saksida, L.M., Bussey, T.J., 2013. The touchscreen operant platform for assessing executive function in rats and mice. Nat. Protoc. 8, 1985–2005. https://doi.org/10. 1038/nprot.2013.123. Marks, M.J., O’Connor, M.F., Artman, L.D., Burch, J.B., Collins, A.C., 1984. Chronic scopolamine treatment and brain cholinergic function. Pharmacol. Biochem. Behav. 20, 771–777. https://doi.org/10.1016/0091-3057(84)90198-9. McAlonan, K., Brown, V.J., 2003. Orbital prefrontal cortex mediates reversal learning and not attentional set shifting in the rat. Behav. Brain Res. 146, 97–103. https://doi.org/ 10.1016/j.bbr.2003.09.019. Meeter, M., Murre, J.M.J., Talamini, L.M., 2004. Mode shifting between storage and recall based on novelty detection in oscillating hippocampal circuits. Hippocampus 14, 722–741. https://doi.org/10.1002/hipo.10214. Miranda, M.I., Ortiz-Godina, F., García, D., 2009. Differential involvement of cholinergic and beta-adrenergic systems during acquisition, consolidation, and retrieval of longterm memory of social and neutral odours. Behav. Brain Res. 202, 19–25. https://doi. org/10.1016/j.bbr.2009.03.008. Nedaei, S.E., Rezayof, A., Pourmotabbed, A., Nasehi, M., Zarrindast, M.R., 2016. Activation of endocannabinoid system in the rat basolateral amygdala improved scopolamine-induced memory consolidation impairment. Behav. Brain Res. 311, 183–191. https://doi.org/10.1016/j.bbr.2016.05.043. Park, J.H., Choi, H.Y., Cho, Jeong Hwi, Kim, I.H., Lee, T.K., Lee, J.C., Won, M.H., Chen, B.H., Shin, B.N., Ahn, J.H., Tae, H.J., Choi, J.H., Chung, J.Y., Lee, C.H., Cho, Jun Hwi, Kang, I.J., Kim, J.D., 2016. Effects of chronic scopolamine treatment on cognitive impairments and myelin basic protein expression in the mouse Hippocampus. J. Mol. Neurosci. 59, 579–589. https://doi.org/10.1007/s12031-016-0780-1. Piiponniemi, T.O., Bragge, T., Vauhkonen, E.E., Vartiainen, P., Puoliväli, J.T., Sweeney, P.J., Kopanitsa, M.V., 2017. Acquisition and reversal of visual discrimination learning in APPSwDI/Nos2−/− (CVN) mice. Neurosci. Lett. 650, 126–133. https://doi.org/ 10.1016/J.NEULET.2017.04.049. Popović, M., Giménez de Béjar, V., Popović, N., Caballero-Bleda, M., 2015. Time course of scopolamine effect on memory consolidation and forgetting in rats. Neurobiol. Learn. Mem. 118, 49–54. https://doi.org/10.1016/j.nlm.2014.11.006. Prendergast, B.J., Onishi, K.G., Zucker, I., 2014. Female mice liberated for inclusion in neuroscience and biomedical research. Neurosci. Biobehav. Rev. 40, 1–5. https://doi. org/10.1016/j.neubiorev.2014.01.001. R Core Team, 2018. R: a Language and Environment for Statistical Computing. R Core Team https://doi.org/3-900051-14-3. Ravel, N., Vigouroux, M., Elaagouby, A., Gervais, R., 1992. Scopolamine impairs delayed matching in an olfactory task in rats. Psychopharmacology (Berl.) 109, 439–443. https://doi.org/10.1007/BF02247720. Riekkinen, P., Sirviö, J., Valjakka, A., Pitkanen, A., Partanen, J., Riekkinen, P., 1990. The effects of concurrent manipulations of cholinergic and noradrenergic systems on neocortical EEG and spatial learning. Behav. Neural Biol. 54, 204–210. https://doi. org/10.1016/0163-1047(90)91436-F. Roediger, H.L., Zaromb, F.M., Goode, M.K., 2008. In: Byrne, J.H.B.T.-L., M.A.C.R (Eds.), 1.02 - A Typology of Memory Terms. Academic Press, Oxford, pp. 11–24. https://doi. org/10.1016/B978-012370509-9.00047-4. Roldán, G., Bolaños-Badillo, E., González-Sánchez, H., Quirarte, G.L., Prado-Alcalá, R.A., 1997. Selective M1 muscarinic receptor antagonists disrupt memory consolidation of inhibitory avoidance in rats. Neurosci. Lett. 230, 93–96. https://doi.org/10.1016/ S0304-3940(97)00489-8. Sarter, M., Bruno, J.P., Givens, B., 2003. Attentional functions of cortical cholinergic inputs: What does it mean for learning and memory? Neurobiol. Learn. Mem. 245–256. https://doi.org/10.1016/S1074-7427(03)00070-4. Schmitz, T.W., Duncan, J., 2018. Normalization and the cholinergic microcircuit: a unified basis for attention. Trends Cogn. Sci. 22, 422–437. https://doi.org/10.1016/j. tics.2018.02.011. Shaikh, A.A., 1971. Estrone and estradiol levels in the ovarian venous blood from rats during the estrous cycle and pregnancy. Biol. Reprod. 5, 297–307. https://doi.org/ 10.1093/biolreprod/5.3.297. Shi, Z., Chen, L., Li, S., Chen, S., Sun, X., Sun, L., Li, Y., Zeng, J., He, Y., Liu, X., 2013. Chronic scopolamine-injection-induced cognitive deficit on reward-directed instrumental learning in rat is associated with CREB signaling activity in the cerebral cortex and dorsal hippocampus. Psychopharmacology (Berl.) 230, 245–260. https://doi. org/10.1007/s00213-013-3149-y. Smith, C.P.S., Hunter, A.J., Bennett, G.W., 1994. Effects of (R)-α-methylhistamine and scopolamine on spatial learning in the rat assessed using a water maze. Psychopharmacology (Berl.) 114, 651–656. https://doi.org/10.1007/BF02244997. Squire, L.R., Genzel, L., Wixted, J.T., Morris, R.G., 2015. Memory consolidation. Cold Spring Harb. Perspect. Biol. 7https://doi.org/10.1101/cshperspect.a021766. a021766–a021766. Talpos, J.C., Winters, B.D., Dias, R., Saksida, L.M., Bussey, T.J., 2009. A novel touchscreen-automated paired-associate learning (PAL) task sensitive to pharmacological manipulation of the hippocampus: a translational rodent model of cognitive impairments in neurodegenerative disease. Psychopharmacology (Berl.) 205, 157–168. https://doi.org/10.1007/s00213-009-1526-3. Ullman, M.T., 2016. Chapter 76 In: Hickok, G., Small, S.L.B.T.-N. of L (Eds.), The
61
Brain Research Bulletin 152 (2019) 52–62
D. Thonnard, et al. Declarative/Procedural Model: A Neurobiological Model of Language Learning, Knowledge, and Use. Academic Press, San Diego, pp. 953–968. https://doi.org/10. 1016/B978-0-12-407794-2.00076-6. Van Loo, P.L.P., Van Zutphen, L.F.M., Baumans, V., 2003. Male management: coping with aggression problems in male laboratory mice. Lab. Anim. 37, 300–313. https://doi. org/10.1258/002367703322389870. Volpato, D., Holzgrabe, U., 2018. Designing hybrids targeting the cholinergic system by modulating the muscarinic and nicotinic receptors: a concept to treat alzheimer’s
disease. Molecules 23, 3230. https://doi.org/10.3390/molecules23123230. Winters, B.D., 2006. Paradoxical facilitation of object recognition memory after infusion of scopolamine into perirhinal cortex: implications for cholinergic system function. J. Neurosci. 26, 9520–9529. https://doi.org/10.1523/jneurosci.2319-06.2006. Zhang, W.W., Song, M.K., Cui, Y.Y., Wang, H., Zhu, L., Niu, Y.Y., Yang, L.M., Lu, Y., Chen, H.Z., 2008. Differential neuropsychopharmacological influences of naturally occurring tropane alkaloids anisodamine versus scopolamine. Neurosci. Lett. 443, 241–245. https://doi.org/10.1016/j.neulet.2008.07.048.
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