Research Article - Journal of Biochemistry and Biotechnology (2020) Volume 3, Issue 1
Scopolamine disrupts the formation of the conjunctive contextual representation and context-shock associative memory in a two-process contextual fear conditioning paradigm
- *Corresponding Author:
- Weihai Chen
PhD, Faculty of Psychology, Southwest University Tianshen Road 2, Beibei District Chongqing, China
E-mail: [email protected]
Accepted on January 23, 2020
Citation: Chen W, Li CX. Scopolamine disrupts the formation of the conjunctive contextual representation and context-shock associative memory in a two-process contextual fear conditioning paradigm. J Biochem Biotech 2020;3(1):1-6.
The article entitled “Scopolamine disrupts the formation of the conjunctive contextual representation and context-shock associative memory in a two-process contextual fear conditioning paradigm” has been accepted for publication in Journal of Biochemistry and Biotechnology considering the statements providing in the article as personal opinion of author which was found not having any conflict or biasness towards anything. As the article was a perspective one, information provided by the author was considered as an opinion to be expressed through an publication. Publisher took an decision to make the article online solely based on the reviewer’s suggestion which considered the article not but an personel opinion of the author. However it is considered that the author has some personal concerns and issues, therefore being retracted from the journal.
Contextual fear conditioning, Scopolamine, Muscarinic cholinergic neurotransmission.
Cholinergic neurotransmission is essential for many forms of learning, especially the hippocampus-dependent learning, such as contextual fear conditioning and spatial learning. It has been demonstrated that muscarinic receptor antagonists (scopolamine or atropine) impaired learning and memory in humans, monkeys, rabbits and rodents . The impairment of learning and memory by muscarinic-cholinergic antagonists is related to the disruption of hippocampal theta rhythm, which produces the attenuation of hippocampal long-term potentiation (LTP) . Thus, it was reported that amnesia produced by muscarinic-cholinergic antagonism is similar to that produced by hippocampal lesions .
Anagnostaras et al. reported that pre-training 1.0 mg/kg of scopolamine disrupted the acquisition of contextual fear, but not tone fear conditioning. Whereas, post-training scopolamine failed to make retrograde amnesia of contextual fear . However, these studies exploited a classical contextual fear conditioning paradigm, that is, a rodent was placed in a context and a few minutes later received an unsignal electrical footshock. Subsequently, the animal was measured freezing in the original shocking context. Freezing, as a natural defensive response, was recorded as an index of fear memory. Actually, classical contextual fear conditioning composes two processes: (1) the independent features that make up the context are first integrated into a unitary conjunctive representation, and then (2) context-shock association, namely, the configural context representation is implicated with the footshock . Thus, the effect of scopolamine on context-shock association during contextual fear conditioning may be obscured by the typical training paradigm, in which the acquisition of context representation and context-shock association take place almost simultaneously within a single training session. To address this issue, context-shock association should be temporally detached, as some previous studies recommended using a two-process training paradigm for contextual fear conditioning . If a rat is placed into a context and gets an immediate shock, nearly no fear will be observed in the subsequent test. However, if the animal is pre-exposed to the context in which it will receive an immediate footshock in the next day, the animal will exhibit strong freezing . It suggests that context pre-exposure facilitates the contextual fear conditioning due to that the rat acquires a conjunctive representation of the context. Thus the two-process training paradigm allows us to separate contextual fear conditioning into two processes: (1) Pre-exposure to the context, and (2) immediate shock.
The present study used the two-process paradigm to investigate the effect of scopolamine on contextual fear conditioning. According to Brown, administration of 0.5 mg/kg scopolamine prior to the pre-exposure session induced impairments in contextual freezing in mice . However, there is no evidence to determine whether cholinergic neurotransmission is involved in the acquisition and consolidation of the context-shock associative memory. In the present study, we employed a twoprocess paradigm to investigate the contribution that cholinergic neurotransmission makes to each process of the contextual fear conditioning, and we used scopolamine to temporarily block cholinergic neurotransmission in each process. The study will address the question that which processes that cholinergic neurotransmission contributes to contextual fear conditioning: acquisition of context representation or context-shock associative learning.
Materials and Methods
Common to all experiments
Subjects and housing: Male Sprague-Dawley rats weighting 200-250 g were purchased from Experiment Animal Center, Research Institute of Surgery, Third Military Medical University, Chongqing, China. They were initially housed in pairs in transparent cages (47 cm × 32 cm × 21 cm) with corncob granule for bedding in a colony on a 12-hour light/dark cycle (lighting on at 08:00). The rats were allowed freely access to food and water in their home cages. All animals were handled daily (1 min per rat) for 7 days prior to the start of experiments to be acclimated to experimenters. All animal experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996 and all animal procedures were approved by the animal care and use committee at Southwest University, China.
Drugs and choice of doses: Scopolamine-HBr (SCOP, Aladdin Industrial Corporation, Shanghai, China) was prepared fresh in 0.9% saline (vehicle) to obtain a concentration of 0.1 mg/ml, which was injected intraperitoneally (i. p.) in a volume of 1.0 ml/kg or equivalent saline injections were given.
Contexts: Two contexts (Context A and Context B) were used in the present study. Context A is a standard rodent conditioning chambers (30.1 cm × 24.7 cm × 23.3 cm; Clever System Inc., Virginia, USA) with aluminum sidewalls and a Plexiglas rear wall. The floor of each chamber comprised 18 stainless steel rods (5-mm diameter) spaced 1.6 cm apart (center to center). The rods were wired to an electric shock producer for delivery of the footshock. Ventilation fans positioned into the sound-attenuating chests were used to supply background noise (60 dB), and illumination were provided with yellow lights within the chambers. The chambers were cleaned with 70% Ethyl alcohol. Stainless steel pans placed underneath the grid floors were sprayed with a thin film of 70% Ethyl alcohol before the animals were placed inside the boxes. Animals were transported to the experimental rooms in their homecages, which were covered with a black trash back and arranged on the top of the plastic cart. Context B was composed of white Plexiglas cages (42.5 cm × 26.2 cm × 5.8 cm) with corn-cob granule in another independent experimental room. Animals were transported to the Context B in their homecages on the top of the cart and covered with a white trash bag.
Statistical analysis: Freezing behavior in the test session were analyzed using a factorial repeated measures analysis of variance (ANOVA) with group as the between-subjects factor and test time point as the within-subjects factor. Group differences were further analyzed using simple main effect tests (one-way ANOVA) followed by LSD post hoc tests. Difference between groups at the specific test time bin was investigated using one-way ANOVA, followed by post hoc LSD tests. Statistical significance was accepted at p<0.05, twotailed.
Effects of scopolamine on the context-shock associative learning: The objective of experiment 1 is to determine whether scopolamine has effects on context-shock associative learning. Previous research revealed that intra-dorsal hippocampal infusion of scopolamine immediately after context-shock training impaired conditioned freezing on the test. The results indicated that cholinergic systems are involved in the consolidation of the context-shock associative memory. However, the results did not determine the role of cholinergic systems on the acquisition of the context-shock associative memory. In the present study, animals were given scopolamine (1.0 mg/kg) or vehicle (0.9% saline) 15 min before or after contextual conditioning training, which would revealed the fact whether scopolamine has effects on the acquisition or consolidation of context-shock associative memory.
Experimental design: Thirty-two rats were randomly assigned to vehicle, pre-scopolamine, post-scopolamine or pseudoexposure groups. There were two training phases in the twoprocess contextual fear conditioning paradigm in two days as previously described. The experimental process is shown in Figure 1. Briefly, in the pre-exposure step on the first day, rats were placed in Context A for 15 min pre-exposure except for the pseudo-exposure group animals which were exposed to Context B for the same time. After exposure, the animals were returned to their vivarium.
In the context-shock session on the second day, fifteen minutes prior to training, pre-scopolamine animals received scopolamine (1.0 mg/kg) in their homecages (n=8) and vehicle (n=8), post-scopolamine (n=8), pseudo-exposure (n=8) animals received saline (1.0 ml/kg). All animals were then transported to Context A and received an unsignal footshock (2 s, 0.9 mA) 15 s after being placed into conditioning chambers. Rats were removed from the Context A immediately after the shock and post-scopolamine animals were immediately administrated with scopolamine (1.0 mg/kg), while other animals were injected with saline (1.0 ml/kg). The animals were then transported back to the vivarium. Based on the previous study, 15 s of re-exposure was enough to retrieve the pre-exiting contextual memory of pre-exposure but not enough for animals to encode a new contextual representation. Thus only the preexiting context representation can be associated to shock in this session.
Twenty-four hours later, all animals were placed in the Context A for a 6-min retention test. Behavior was recorded with video tracking equipment and analyzed with computer software (FreezingScan, Clever System Inc., Virginia, USA). Freezing behavior, an index of conditional fear in rat was defined as the absence of any visible movement for longer than 1 s except for respiration. Freezing was scored in the test session and blocked into 6 bins of each minute. Freezing was normalized to 60 s and averaged within each bin.
Effects of scopolamine on the formation of a conjunctive contextual representation: The objective of the experiment 2 is to determine whether scopolamine affects contextual learning, namely, the session to encode the configural context representation. The previous research reported that pre-training administration of scopolamine at 0.5 mg/kg disrupted the acquisition of contextual fear. However, there is no evidence to show the dissociable roles of scopolamine in the acquisition and the consolidation of the conjunctive contextual representation. In the experiment 2, animals were given scopolamine (1.0 mg/kg) or vehicle (0.9% saline) 15 min before or after pre-exposure session in a two-process paradigm, which would reveal the fact whether scopolamine has effects on the formation of a conjunctive contextual representation.
Experimental design: Thirty-two rats were randomly assigned to vehicle, pre-scopolamine, post-scopolamine groups or pseudo-exposure groups. The two-process training paradigm was used as previously described in Experiment 1 with some modification (Figure 2). Briefly, in the pre-exposure session on the first day, rats were placed in Context A for pre-exposure of 15 min except for animals in the pseudo-exposure group which were exposed to Context B for the same time. Fifteen minutes prior to pre-exposure, pre-scopolamine animals (n=8) received scopolamine (1.0 mg/kg) in their homecages and vehicle (n=8), post-scopolamine (n=8), pseudo-exposure (n=8) animals were administrated with saline (1.0 ml/kg). After exposure, postscopolamine animals received scopolamine (1.0 mg/kg), while other animals received saline (1.0 ml/kg). And then, the animals were returned to their vivarium. Twenty-four hours after training, all animals were then transported to Context A and received an unsignal footshock (2 s, 0.9 mA) 15 s after be placed into conditioning chambers. Rats were removed from the Context A immediately after shock, and then returned to the vivarium. In the test session on the secondary day, all rats were transported back to the Context A for a 6-min retention test as in experiment 1.
Effects of scopolamine on the context-shock associative learning: Figure 3 shows the results of scopolamine on the context-shock associative learning in the retention test. Contextual freezing was significantly impaired in animals treated with scopolamine (1.0 mg/kg) prior to contextual fear conditioning. A 4 (Group) × 6 (minute) repeated measures ANOVA on retention test freezing revealed significant main effects of group [F(3,28)= 4.770, p=0.008], and test time [F(5,140)=7.684, p=0.000] and group × minute interaction [F(15, 140)=2.252, p=0.007] were significant. Post hoc tests indicated that a pre-conditioning administration of 1.0 mg/g scopolamine severely impaired the acquisition of contextshock associative memory (p=0.016, n=8), but postadministration injection had no effect on the consolidation of context-shock associative memory (p=0.740, n=8). Additionally, the animals in pseudo-exposure group froze significantly less than the control group (p=0.022, n=8), which can be attributed to the “immediate shock freezing deficit” whereby negligible levels of fear freezing are observed when shock is delivered immediately after placement in the conditioning chamber. To identify the time point(s) at which pre-conditioning administrated rats differed from the vehicletreated rats, one-way ANOVAs followed by post hoc LSD tests were used. Results showed that animals pre-conditioning injected with scopolamine froze less than vehicle-treated ones during minutes 1-4 after placement in the Context A (all ps<0.04) (Figure 3).
Freezing behavior (percentage time, mean ± SEM) was scored during the 6-min testing session. Rats received 1.0 mg/kg scopolamine 15 min prior to context-shock conditioning showed significant less freezing than saline control rats (p=0.016, n=8). Ones received scopolamine 15 min post to conditioning did not differ significantly (p=0.740, n=8). The animals in pseudo-exposure group froze significantly less than the saline control group (p=0.022, n=8). *p<0.05, **p<0.01, the difference between groups at the specific test time bin analyzed using one-way ANOVA.
Effects of scopolamine on the formation of conjunctive contextual representation: Figure 4 shows the results of scopolamine on the formation of the conjunctive context representation in the retention test. Contextual freezing was significantly impaired in animals treated with scopolamine prior or post to the pre-exposure (Figure 2B). A 4 (Group) × 6 (minute) repeated measures ANOVA on retention test freezing showed significant main effects of group [F(3,28)=7.234, p=0.001], and test time [F(5,140)=4.888, p=0.000] were significant, but no group × minute interaction [F(15,140)=1.048, p=0.410]. Post hoc tests indicated that a preand post-pre-exposure administration of 1.0 mg/g scopolamine severely impaired the formation of the configural contextual representation (all ps=0.001, n=8). Additionally, the animals in the pseudo-exposure group froze significantly less than the saline control group (p=0.000, n=8). To identify the time point(s) at which pre- or post-pre-exposure administrated rats differed from the vehicle-treated rats, one-way ANOVAs followed by post hoc LSD tests were used. Results showed that animals pre-exposure injected with scopolamine froze less than vehicle-treated during minutes 1-6 after placement in the Context A (all ps<0.035), and post-pre-exposure injected animals also froze less than vehicle-treated ones during all time (all ps<0.027) (Figure 4).
Freezing behavior (percentage time, mean ± SEM) was scored during the 6-min testing session. Rats received 1.0 mg/kg scopolamine 15 min prior or immediate post to pre-exposure session showed significant less freezing than saline control rats (all ps=0.001, n = 8). *p<0.05, **p<0.01, the difference between groups at the specific test time bin analyzed using one-way ANOVA. The animals in pseudo-exposure group froze significantly less than the saline control group (p = 0.000, n=8).
This study aimed to investigate the role of muscarinic receptors underlying the formation of the conjunctive contextual representation and the context-shock association in a twoprocess contextual fear conditioning paradigm. Results showed that pre-conditioning administration of scopolamine disrupted the formation of context-shock associative memory, and administration of scopolamine prior or post to pre-exposure both impaired the configural contextual representation formation. However, scopolamine did not produce retrograde amnesia if administrated after conditioning.
In the first experiment, scopolamine impaired contextual fear conditioning only if it was administrated before the conditioning session. This result is in line with previous findings showing that pertaining, but not posts training; scopolamine administration selectively impaired typical contextual fear conditioning . These data suggest that scopolamine preferred to disrupt the acquisition, but not consolidation, of context-shock associative memory. It has been reported that the hippocampus is necessary for conditioning to the conjunctive contextual memory representation . And the effects of anticholinergic action mimic those observed as hippocampal inactivation . Thus, the effect of scopolamine may be involved in the integration of pre-existing neutral configural contextual representation into an emotional memory . In our study, post training administration of scopolamine had no retrograde effect on the context-shock associative memory. Anagnostaras et al. reported that administration of scopolamine after the conditioning session did not disrupt the formation of contextual fear memory in the typical contextual fear paradigm . These results indicate that muscarinic cholinergic neurotransmission is not involved in the consolidation of the context-shock associative memory.
In the second experiment, we found that scopolamine produced an acquisition (anterograde) and consolidation (retrograde) deficits in the formation of a conjunctive contextual representation. These results are consistent with a growing body of evidence suggesting the involvement of cholinergic neurotransmission in many forms of contextual learning . Matus-Amat et al. revealed that hippocampal lesions by muscimol before pre-exposure produced deficits in acquiring a representation of context . Thus, it is possible that systemic injections (intraperitoneal injection, i.p.) of scopolamine before pre-exposure prevented rats from learning about the context by the inactivation of the hippocampus. However, hippocampal lesions by muscimol after pre-exposure did not disrupt the formation of contextual representation . In addition, intradorsal hippocampus infusion of scopolamine after preexposure session did not impair the formation of the conjunctive contextual representation yet . As such, the impairment of the consolidation of a contextual representation by scopolamine (i.p.) may not be attributable to the disruption of the hippocampus functions. We suggest that although the amnesia produced by scopolamine is always attributed to hippocampal lesions, other brain regions, such as medial prefrontal cortex, may be involved in the effect of muscariniccholinergic antagonism on the consolidation of contextual representation.
Taken together, we used the two-process paradigm to separate contextual fear conditioning into contextual pre-exposure and context-shock association sessions. By this method we found that muscarinic cholinergic neurotransmission is involved in the acquisition, but not consolidation of context-shock associative memory, while the acquisition and the consolidation of contextual representation depend on the activation of the muscarinic receptors.
This work was supported by the National Natural Science Foundation of China (81302757), the National Natural Science Foundation of China (31371042), the National Natural Science Foundation of China (31170989) and the Postdoctoral Science Foundation of China (2012M521662).
Conflicts of Interest
The authors have no conflicts of interest.
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