Enhancement of Memory and Synaptic Plasticity by Celastrus paniculatus Seed Extract: Upregulation of pSer831‐GluA1 Trafficking and Arc/PSD‐95 Expression in the Hippocampus of Male Rats

TL;DR

This study shows that Celastrus paniculatus seed extract improves memory and synaptic plasticity in rats by enhancing key proteins involved in brain signaling.

Contribution

The novel contribution is demonstrating that CP extract upregulates pSer831-GluA1 trafficking and Arc/PSD-95 expression in the hippocampus, linking it to improved memory.

Findings

CP-treated rats showed improved spatial memory performance comparable to donepezil-treated rats.

CP reversed scopolamine-induced memory deficits by enhancing platform crossings and reducing escape latency.

CP upregulated hippocampal pSer831-GluA1, Arc, and PSD-95, indicating enhanced synaptic plasticity.

Abstract

Celastrus paniculatus (CP) is a traditional medicinal plant widely used in Ayurveda and Southeast Asian medicine for enhancing memory and treating cognitive dysfunction. Although CP has been reported to exhibit antioxidant, anti‐inflammatory, and neuroprotective effects, its direct impact on activity‐dependent synaptic plasticity remains insufficiently characterized. This study is aimed at investigating the effects of CP seed extract on memory performance and synaptic plasticity in a rat model, with a particular focus on AMPA receptor modulation and associated synaptic proteins. Five‐week‐old male Sprague–Dawley rats were randomly assigned to five groups: control, CP (80 mg/kg), donepezil (1.5 mg/kg), scopolamine (1 mg/kg), and scopolamine followed by CP. Treatments were administered daily for 14 days. Spatial memory performance was assessed using the Morris water maze. Following behavioral testing, hippocampal tissue was collected for immunohistochemical analysis of Arc protein and Western blotting of pSer831‐GluA1, Arc, and PSD‐95. CP‐treated rats exhibited significantly reduced escape latency and increased time in the target quadrant, with outcomes comparable to those of donepezil‐treated rats. In scopolamine‐pretreated rats, CP administration reversed memory deficits by enhancing platform crossings and reducing escape latency. Molecular analysis revealed that CP significantly upregulated hippocampal expression of pSer831‐GluA1, Arc, and PSD‐95, indicating enhanced AMPA receptor trafficking and synaptic integrity. CP seed extract enhances spatial memory and synaptic plasticity by modulating critical molecular components of the glutamatergic synapse. These findings suggest that CP may support memory performance in both baseline conditions and in animals with scopolamine‐induced deficits.

Figures

Schematic representation of the experimental design. Five‐week‐old male Sprague–Dawley rats were divided into five groups: control (5% DMSO, 14 days), CP‐treated (80 mg/kg, 14 days), donepezil‐treated (1.5 mg/kg, 14 days), scopolamine‐treated (1.0 mg/kg, 7 days), and scopolamine+CP (scopolamine for 7 days followed by CP treatment for 14 days). After treatment, rats underwent behavioral assessment using the Morris water maze (MWM) test. Subsequently, brains were collected for molecular and histological analysis using Western blotting and immunohistochemistry to evaluate memory‐related protein expression. Intraperitoneal (ip) injections were administered as part of the experimental procedure.

Effects of donepezil, CP, and scopolamine on escape latency in the MWM. The graph shows the escape latency (s) over 7 days of training in the MWM. Escape latency refers to the time (in seconds) it takes for the animals to locate the hidden platform. The groups included the control (open circles), donepezil‐treated (gray circles), CP‐treated (gray diamonds), scopolamine‐treated (black squares), and scopolamine+CP (black diamonds) groups. Throughout the training days, a reduction in escape latency was observed in all groups, with the donepezil‐ and CP‐treated groups demonstrating a significantly shorter latency than the control and scopolamine‐treated groups. Statistical significance relative to the control group is indicated by  ∗ p < 0.05,  ∗∗ p < 0.01, and  ∗∗∗ p < 0.001, and significance compared with the scopolamine‐treated group is denoted by # p < 0.05 and ## p < 0.01. Data are presented as means ± SEM of the mean. n = 6.

Effects of CP treatments on PSD‐95 expression in the hippocampus. Representative Western blot images showing PSD‐95 and actin as a loading control across treatment groups: control, donepezil, CP, scopolamine, and scopolamine+CP. The graph quantifies the relative expression of PSD‐95 normalized to actin. Scopolamine significantly reduced PSD‐95 expression compared with that observed in the control group, whereas donepezil significantly increased PSD‐95 expression levels. CP treatment alone maintained PSD‐95 expression levels similar to those observed in the control group. Pretreatment with CP before scopolamine administration significantly restored PSD‐95 expression compared with scopolamine alone. Data are presented as means ± SEM. Statistical significance:  ∗∗∗∗ p < 0.0001 versus control and #### p < 0.0001 versus scopolamine (n = 6).

Full text

1. Introduction

Cognitive health is increasingly recognized as a critical component of overall well‐being, particularly in aging populations at risk of neurodegenerative diseases. Nutritional and plant‐based interventions have emerged as promising strategies for preserving brain function and preventing cognitive decline. The hippocampus, a brain region essential for learning and memory, relies on synaptic plasticity—especially long‐term potentiation (LTP)—to mediate adaptive neural responses. LTP is a fundamental cellular process in which glutamate release from presynaptic terminals promotes the recruitment of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors (AMPARs) to postsynaptic membranes, thereby enhancing synaptic strength and reinforcing memory encoding [1–4]. Understanding the molecular mechanisms underlying synaptic plasticity provides insight into how dietary bioactive compounds may support cognitive function and brain health.

While NMDA receptors (NMDARs) contribute to synaptic plasticity, their insertion into synapses occurs more slowly than AMPAR cycling [5]. NMDAR trafficking is thought to regulate metaplasticity, modulating synaptic adaptability for future plastic changes [6, 7]. Given their direct role in modulating synaptic strength during LTP, this study focuses on AMPARs, which act as primary effectors in synaptic efficacy through dynamic trafficking. As AMPAR insertion and stabilization are key regulators of synaptic plasticity, this study is aimed at elucidating their specific contributions in the hippocampus, particularly in LTP induction and maintenance.

Posttranslational modifications are key regulators of synaptic transmission and plasticity, and AMPAR subunit phosphorylation (GluA1‐4) controls receptor stability, trafficking, interactions, and synaptic expression [2]. Among these, the GluA1 subunit plays a key role in synaptic plasticity; GluA1 knockout mice exhibit impaired LTP in the CA1 region of the adult hippocampus [8–10]. Interestingly, GluA2/GluA3 double knockout mice can still retain some functions, suggesting that GluA1 has unique regulatory mechanisms that allow it to support specific synaptic functions independently of other AMPAR subunits [3]. GluA1 has several identified phosphorylation sites on the intracellular carboxy terminus: serine (S) 818, S831, and threonine (T) 840 [11]. Many of these sites have been shown to contribute to synaptic AMPAR regulation and synaptic plasticity. The first to receive attention was S831, which exhibits increased phosphorylation following LTP [12, 13]. A previous study reported that S831 regulation on the GluA1 subunit may play a critical role in bidirectional synaptic plasticity in the Schaffer collateral inputs to CA1. In particular, gene knockin mice lacking S831 phosphorylation sites, in which S residues were replaced by another amino acid, exhibited a faster decaying LTP and alterations in synaptic plasticity [13–15].

Synaptic plasticity relies on the dynamic reorganization of AMPARs and their interaction with the PSD protein scaffold [16]. PSD‐95, a major scaffolding protein in the postsynaptic membrane and a member of the membrane‐associated guanylate kinase family [17], plays a key role. Increased synaptic PSD‐95 expression enhances memory formation [18], whereas its knockdown impairs synaptic plasticity and cognitive abilities [19]. Similarly, Arc is vital for neuronal plasticity at active synapses [20], interacting with PSD‐95 to regulate AMPAR homeostatic scaling [21]. The loss of Arc, either by knockout or knockdown, leads to deficits in synaptic plasticity and hippocampus‐dependent learning [22]. Therefore, the regulation of memory formation through the mechanisms of AMPARs, PSD‐95, and Arc is widely accepted [23].

Celastrus paniculatus (CP) Willd., a species from the Celastraceae family [24], is predominantly found across the tropical, subtropical, and temperate regions of Asia [25, 26]. It has been traditionally used in Ayurvedic medicine for enhancing intellect (Medhya Rasayana), treating forgetfulness, and cognitive dysfunction [25, 27]. In a previous study, we reported the protective effects of CP against cell death induced by MPP^+^, a neurotoxin used to model Parkinson′s disease. This protection occurs through modulation of the GSK‐3β pathway, a key regulator implicated in several neurodegenerative mechanisms [28]. These findings suggest that CP possesses neuroprotective properties in cellular models, particularly in terms of antiapoptotic effects and support of neuronal survival. In contrast, several studies have shown that CP seed extract enhances cognitive function by increasing acetylcholine levels in the brain, along with its antioxidant properties [29]. Recent results have shown that CP oil improves behavioral deficits by reducing latency in the Morris water maze (MWM), decreasing novel object exploration time, enhancing the discrimination index in the novel object recognition test, and normalizing responses in the conditioned avoidance test [27]. Furthermore, CP oil has been shown to enhance the levels of key neurotransmitters and antioxidants while reducing markers of oxidative stress, inflammation, and neurodegeneration. It improves cognitive function in rats with scopolamine‐induced amnesia by enhancing cholinergic function [30]. Several studies have identified major bioactive constituents in CP seed extracts, such as fatty acid esters, pilocarpine, and steroidal compounds, which are believed to contribute to its neuroprotective and memory‐enhancing effects [27]. These compounds, identified through GC‐MS analysis, are associated with antioxidant activity, acetylcholinesterase (AChE) inhibition, and behavioral improvements in animal models. However, its potential as a diet‐based cognitive enhancer that supports synaptic function and prevents memory impairment has not been fully explored. Although CP is rich in neuroactive and antioxidant compounds, its effects on synaptic plasticity—particularly GluA1 phosphorylation and its relationship with Arc and PSD‐95—remain unclear.

Therefore, this study investigated the effects of CP on spatial memory, GluA1 phosphorylation, and the expression of Arc and PSD‐95 in the hippocampus of male rats and compared them with those of donepezil. This research is aimed at providing insight into how CP, as a plant‐derived bioactive compound, may influence memory‐related processes and hippocampal plasticity under the experimental conditions used in this study.

2. Materials and Methods

2.1. Preparation of the CP Seed Extract

CP seeds were generously provided by the Queen Sirikit Botanic Garden, a member of the Botanical Garden Organization under the Ministry of Natural Resources and Environment, Mae Rim, Chiang Mai, Thailand. The seeds were air‐dried and coarsely milled using a stainless steel grinder for 3 min, after which the resulting powder was passed through a 60‐mesh sieve to obtain an approximate particle size of ~250 μm. A total of 300 g of the sieved seed powder was extracted using the maceration method with 95% ethanol at room temperature for 72 h, followed by filtration to separate the filtrate from the residue. This extraction procedure was repeated three times under identical conditions. The combined filtrates were then evaporated under reduced pressure at 50°C to obtain the crude ethanol extract, which was stored at −20°C until further use.

A portion of the crude extract was dissolved in a methanol–water solution (1:4 v/v). The solution was transferred to a separating funnel, followed by the addition of n‐hexane (H) in a 1:1 volume ratio to the aqueous (H_2_O) phase. The mixture was shaken thoroughly until the two phases were well mixed. The solution was then left to stand until the water and H phases separated. The H phase was collected in a beaker. This process was repeated three times until the H phase was nearly clear. The water phase was subsequently subjected to fractionation by adding ethyl acetate in a 1:1 ratio using a separating funnel, and the same procedure was followed for the H fraction. Each fraction was then evaporated to dryness using a rotary evaporator to obtain the H, ethyl acetate, and H_2_O fractions. All extracts were stored under refrigeration until further use. The H_2_O fraction was used in all experiments.

2.2. Experimental Animals

A total of 30 five‐week‐old male Sprague–Dawley rats (Rattus norvegicus) were used and randomly assigned to five experimental groups (n = 6 per group): control, CP‐treated, donepezil‐treated, scopolamine‐treated, and scopolamine followed by CP treatment (scopolamine+CP). The animals were acclimatized for 7 days, during which they were provided with standard pellet chow and reverse osmosis‐treated water ad libitum. Daily records for general health, body weight, and food intake were maintained. All experimental procedures were performed according to the guidelines approved by the Ethics Committee on Animal Experimentation of Burapha University, Thailand (ID 010/2565).

During the 14‐day treatment period, the control group received 5% dimethyl sulfoxide, and the CP‐treated group received 80 mg/kg of CP seed extract. The selected dose of 80 mg/kg was based on previous studies that demonstrated cognitive‐enhancing and neuroprotective effects of CP extract at this dose [25, 27]. As this study focused on the mechanistic evaluation of synaptic markers, dose–response evaluation was beyond its scope. The donepezil‐treated group received 1.5 mg/kg of donepezil [31], the scopolamine‐treated group received 1 mg/kg of fraction scopolamine, and the scopolamine+CP group received 1 mg/kg of scopolamine followed by 80 mg/kg of CP seed extract. All treatments were administered via intraperitoneal (ip) injections. The scopolamine dosage used in this study was based on previous research [32], which demonstrated that administration of scopolamine at 1 mg/kg (ip) for 7 days effectively induced memory deficits in mice, supporting its use as an appropriate dose for cognitive impairment modeling, as illustrated in the schematic diagram (Figure 1).

Schematic representation of the experimental design. Five‐week‐old male Sprague–Dawley rats were divided into five groups: control (5% DMSO, 14 days), CP‐treated (80 mg/kg, 14 days), donepezil‐treated (1.5 mg/kg, 14 days), scopolamine‐treated (1.0 mg/kg, 7 days), and scopolamine+CP (scopolamine for 7 days followed by CP treatment for 14 days). After treatment, rats underwent behavioral assessment using the Morris water maze (MWM) test. Subsequently, brains were collected for molecular and histological analysis using Western blotting and immunohistochemistry to evaluate memory‐related protein expression. Intraperitoneal (ip) injections were administered as part of the experimental procedure.

At 24 h prior to the experimental endpoint, the rats were housed in a metabolic cage to collect food and water intake. Further, information on urinary and fecal output was collected. The rats were sacrificed by an overdose of 100 mg/kg thiopental (Anesthal, Jagsonpal Pharmaceuticals Ltd., India). The hippocampus was harvested via rapid dissection and immediately frozen at −80°C. No blood biochemical tests for liver or kidney function were performed; toxicity assessment was based solely on general observations.

2.3. MWM Test

Spatial memory was assessed 2 weeks before euthanasia using the MWM, a standard paradigm for hippocampus‐dependent spatial navigation. The pool (153 cm diameter, 60 cm height) was filled to a depth of 30 cm and divided into four quadrants, with distinct visual cues positioned around the perimeter. A hidden platform (10 cm diameter) was placed in a single quadrant and submerged 1.5–2 cm below the water surface. Water temperature was maintained at 25^°^C ± 1^°^C, and water opacity was standardized by adding a fixed amount of nontoxic white paint. Animal behavior was recorded by an overhead camera and analyzed using identical tracking parameters.

Training was performed over seven consecutive days with four trials per day. Rats were released from semirandomized start points. If the platform was located within 60 s, the animal was allowed to remain on it for 10 s; otherwise, it was guided to the platform. Escape latency was recorded for each trial. After each session, animals were towel‐dried and placed in a heated cage.

A probe trial was conducted 2 weeks after treatment, during which the platform was removed, and each rat was allowed to swim for 60 s. Two standard memory parameters were analyzed: (i) number of platform crossings, defined as the number of times the trajectory intersected the predefined circular region of interest (ROI) corresponding to the former platform location, and (ii) time spent in the target quadrant. All analyses were performed using fixed ROIs and identical tracking settings across animals.

2.4. Quantification of GluA1, Arc, and PSD‐95 Protein Expression Levels by Western Blotting

Western blotting was conducted as previously described [28]. Hippocampal tissues were collected immediately after the probe test to assess posttraining protein expression associated with memory retrieval. The tissues were lysed using radioimmunoprecipitation assay buffer, and membrane proteins were extracted following the manufacturer′s instructions using the ProteoExtract Native Membrane Protein Extraction Kit (Calbiochem, Sigma‐Aldrich, Merck; Cat. No. 44810). Equal amounts of protein were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions and then transferred onto nitrocellulose membranes. The membranes were incubated with a 1:1000 dilution of mouse anti‐pSerGluA1 and GluA1 antibodies (Sigma‐Aldrich, St. Louis, Missouri, United States), mouse anti‐Arc antibody (Santa Cruz Biotechnology, Santa Cruz, California), rabbit anti‐PSD‐95 antibody (1:1000, Abcam, Cambridge, United Kingdom), or β‐actin antibody (Sigma‐Aldrich). Subsequently, the membranes were incubated with a 1:5000 dilution of the horseradish peroxidase (HRP)–conjugated secondary antibody (Santa Cruz Biotechnology, Inc.). The signal was detected using an ECL Western blotting substrate (Pierce, Rockford, Illinois, United States) and visualized using a Gel Doc imaging system.

2.5. Arc Protein Expression Using the Immunohistochemistry Technique

For immunostaining analysis, 4‐μm‐thick sections were prepared from the formalin‐fixed, paraffin‐embedded hippocampal tissue of the treated animals. The sections were subjected to a series of alcohol and phosphate‐buffered saline (PBS) rinses. Antigen retrieval was performed by heating the sections in a 0.01 M citric acid buffer (pH 6.0) for 20 min using a microwave. The sections were then washed three times with PBS and incubated in blocking buffer (DAKO) for 30 min at room temperature. The sections were then incubated with an anti‐Arc antibody (1:300 dilution) at 4°C overnight. The sections were then incubated with a biotinylated goat antimouse IgG secondary antibody (1:500 dilution) for 60 min at room temperature. After incubation with HRP, the sections were immersed in a peroxidase reaction solution containing diaminobenzidine for color development. The negative control sections were processed in the same manner, except that the primary antibody was omitted. For histological and immunohistochemical analyses, images were acquired using an Olympus BX51 microscope equipped with an Olympus DP22 camera. To ensure consistency, images were captured using 4× and 20× objectives, autoexposure time, and Brightfield illumination. All image acquisition parameters were maintained across samples to ensure comparability and standardization throughout the study.

2.6. Statistical Analysis

The data are presented as means ± SEM. Statistical significance was determined using one‐way analysis of variance, followed by Tukey′s post hoc test. p values < 0.05 were used to denote statistical significance.

3. Results

3.1. General Observations and Toxicity

No signs of general toxicity were observed throughout the study. Body weight, food intake, and water consumption did not differ significantly between groups (Figures S1 and S2).

3.2. Effect of CP on Spatial Memory

The results show spatial learning and memory in male rats from the MWM test, comparing the control group with the donepezil‐treated, CP‐treated, scopolamine‐treated, and scopolamine+CP groups. The results show the escape latency over 7 days of training (Figure 2). Both the donepezil‐ and CP‐treated groups exhibited a significant reduction in escape latency compared with the control group (p = 0.0321 and p = 0.0316, respectively), indicating enhanced learning and memory retention. In contrast, the scopolamine‐treated group showed a markedly slower reduction in escape latency compared with the control group (p < 0.001). However, the scopolamine+CP group exhibited a restoration of escape latency, with a significant improvement compared with the scopolamine‐treated group (p = 0.0401), suggesting that CP mitigates the memory‐impairing effects of scopolamine. No signs of general toxicity were observed throughout the study. Body weight, food intake, and water consumption did not differ significantly between groups (Figures S1 and S2).

Effects of donepezil, CP, and scopolamine on escape latency in the MWM. The graph shows the escape latency (s) over 7 days of training in the MWM. Escape latency refers to the time (in seconds) it takes for the animals to locate the hidden platform. The groups included the control (open circles), donepezil‐treated (gray circles), CP‐treated (gray diamonds), scopolamine‐treated (black squares), and scopolamine+CP (black diamonds) groups. Throughout the training days, a reduction in escape latency was observed in all groups, with the donepezil‐ and CP‐treated groups demonstrating a significantly shorter latency than the control and scopolamine‐treated groups. Statistical significance relative to the control group is indicated by  ∗ p < 0.05,  ∗∗ p < 0.01, and  ∗∗∗ p < 0.001, and significance compared with the scopolamine‐treated group is denoted by # p < 0.05 and ## p < 0.01. Data are presented as means ± SEM of the mean. n = 6.

In the probe trials (Figure 3), the CP‐treated group crossed the previous platform location more frequently than the control group (p = 0.0040), indicating better retention of spatial memory. In contrast, the scopolamine‐treated group showed significantly fewer platform crossings (p < 0.0001). Notably, scopolamine pretreatment followed by CP administration significantly increased platform crossings compared with scopolamine alone (p < 0.0001), suggesting that CP alleviates scopolamine‐induced memory impairment. The CP‐treated group also spent an amount of time in the target quadrant comparable to that of the control group. Conversely, the scopolamine‐treated group spent significantly less time in the target quadrant (p < 0.0001), reflecting impaired spatial memory. Importantly, CP pretreatment significantly increased target‐quadrant retention time relative to scopolamine alone (p < 0.0001), further supporting CP′s protective effect. Regarding escape latency during the probe test, the CP‐treated group exhibited a significantly shorter latency compared with the control group (p = 0.0002), indicating enhanced memory retention. In contrast, the scopolamine‐treated group demonstrated a marked increase in escape latency (p = 0.0031), consistent with impaired memory performance. However, scopolamine pretreatment followed by CP treatment significantly reduced escape latency compared with scopolamine alone, further highlighting the neuroprotective effects of CP.

Figure 3. Effects of donepezil, CP, and scopolamine on memory performance in the MWM probe trial. (a) Number of platform crossings: Scopolamine significantly reduced platform crossings. CP and donepezil improved platform crossings. Scopolamine+CP also improved platform crossings. (b) Target quadrant retention time: Scopolamine reduced the retention time, whereas CP and donepezil increased it. Pretreatment with CP before scopolamine treatment significantly increased the retention time. (c) Escape latency: Scopolamine increased the escape latency. CP and donepezil decreased the escape latency, with further improvement in the scopolamine+CP group. Data are presented as means ± SEM. Statistical significance: ^##^ p < 0.01 and ^####^ p < 0.0001 versus control (n = 6).(a)(b)(c)

3.3. Effects of CP on GluA1 Expression in the Hippocampus

The results demonstrate the expression of pSer831‐GluA1 (surface) and total GluA1, normalized to β‐actin as a loading control, under different treatment conditions (Figure 4). CP and donepezil significantly increased the expression of surface pSer831‐GluA1 compared with the control group (p = 0.0143 and p = 0.0303, respectively). Scopolamine treatment markedly reduced surface pSer831‐GluA1 expression (p = 0.0017). Notably, scopolamine pretreatment followed by CP administration significantly increased surface pSer831‐GluA1 levels compared with scopolamine alone (p < 0.0001), while total GluA1 levels remained unchanged across groups. These findings suggest that CP helps restore the balance of pSer831‐GluA1 expression, potentially acting to reverse scopolamine‐induced impairments in synaptic plasticity.

Figure 4. Effects of CP treatment on pSer831‐GluA1 and GluA1 expression in the hippocampus. (a) Western blots showing surface pSer831‐GluA1, total GluA1, and β‐actin expression across the control, donepezil‐treated, CP‐treated, scopolamine‐treated, and scopolamine+CP groups. (b) Quantification of pSer831‐GluA1 (top) and total GluA1 (bottom). Scopolamine reduced pSer831‐GluA1 expression (^∗∗^ p < 0.01), whereas donepezil and CP increased levels (^∗^ p < 0.05 versus control). Scopolamine + CP significantly increased pSer831‐GluA1 compared with scopolamine alone (^####^ p < 0.0001) and also differed from control (^∗^ p <  0.05) [n = 6].(a)(b)

3.4. Effects of CP on Arc Protein Expression in the Hippocampus

The results of the Western blot analysis demonstrate the levels of Arc protein expression in the hippocampus following treatment with donepezil, CP, scopolamine, and scopolamine with CP pretreatment (Figure 5). The blot shows that Arc protein expression varied across the different treatment groups. Compared with the control group, the donepezil‐treated group exhibited a slight increase in Arc expression. The CP‐treated group showed a more pronounced increase in Arc levels (p < 0.0001). In contrast, scopolamine treatment markedly reduced Arc expression (p < 0.0001), indicating potential impairment of neuronal activity associated with learning and memory. Pretreatment with CP partially rescued the scopolamine‐induced reduction in Arc expression, suggesting a protective or restorative effect of CP against scopolamine‐induced memory impairment. Actin was used as the loading control to ensure equal protein loading across all lanes. The actin bands were consistent across all treatment groups, confirming that the observed changes in Arc expression were not caused by differences in protein loading. The bar graph quantifies the relative expression levels of Arc, normalized to the control. CP treatment resulted in the highest increase in Arc expression, which was significantly higher than that observed in the control and donepezil‐treated groups. Scopolamine treatment significantly decreased Arc expression, highlighting its negative effects on memory‐related protein expression. The combination of scopolamine and CP significantly increased Arc expression compared with scopolamine alone, highlighting CP′s protective effect against the adverse effects of scopolamine. In summary, the results suggest that CP and donepezil enhance Arc expression in the hippocampus, whereas scopolamine reduces Arc expression. The group pretreated with CP before scopolamine exposure exhibited a reversal of the scopolamine‐induced reduction in Arc expression, indicating that CP has a potential protective effect. The expression of Arc in the hippocampus was analyzed using immunohistochemistry, revealing distinct differences between the control and CP‐treated groups (Figure 6). In the control group (Figure 6a,b), Arc expression was relatively low, with fewer Arc‐positive neurons distributed sparsely in the dentate gyrus. This suggests normal baseline neuronal activity without significant stimulation or plasticity changes. In contrast, the CP‐treated group (Figure 6c,d) exhibited increased Arc expression in the hippocampus. The number of Arc‐positive neurons increased, particularly in the dentate gyrus, and the staining intensity was noticeably higher than that in the control group. This indicates enhanced neuronal activity and possibly increased synaptic plasticity in response to treatment. These results suggest that CP treatment enhances neuronal activity in the hippocampus, which plays a critical role in learning and memory. The increased Arc expression observed in the treated group highlights CP′s potential involvement in promoting synaptic plasticity, a key mechanism in cognitive function.

Figure 5. Effects of CP treatments on Arc protein expression in the hippocampus. (a) Representative Western blot images showing Arc protein and actin as the loading control for different treatment groups: control, donepezil, CP, scopolamine, and scopolamine+CP. (b) Quantification of Arc protein expression relative to actin. Scopolamine significantly decreased Arc expression. Donepezil and CP significantly increased Arc expression. Pretreatment with CP before scopolamine administration improved Arc expression compared with that observed after treatment with scopolamine alone. Data are presented as means ± SEM. Statistical significance:  ^∗∗∗∗^ p < 0.0001 versus control and ^##^ p < 0.01 versus scopolamine (n = 6).(a)(b)

Figure 6. Histological and immunohistochemical analyses of Arc expression in the hippocampus. Immunohistochemical staining for Arc‐positive neurons in the hippocampus. (a, b) The control group shows sparse Arc‐positive neurons in the dentate gyrus ([a] low magnification and [b] high magnification). (c, d) The CP‐treated group exhibited increased Arc‐positive neurons in the dentate gyrus ([c] low magnification and [d] high magnification). Arrows indicate Arc‐positive neurons. Scale bar = 500 * μ*m and 50 μm. n = 6.(a)(b)(c)(d)

3.5. Effects of CP on PSD‐95 Protein Expression in the Hippocampus

The effects of the different treatments on hippocampal PSD‐95 expression are shown in Figure 7. Scopolamine significantly decreased PSD‐95 expression compared with the control group. Treatment with donepezil significantly increased PSD‐95 levels (p < 0.0001). CP treatment maintained PSD‐95 expression at levels comparable to those of the control group. Notably, pretreatment with CP before scopolamine administration significantly restored PSD‐95 expression (p < 0.0001), bringing it closer to normal levels. These findings suggest that CP mitigates the negative effect of scopolamine on PSD‐95 expression.

Effects of CP treatments on PSD‐95 expression in the hippocampus. Representative Western blot images showing PSD‐95 and actin as a loading control across treatment groups: control, donepezil, CP, scopolamine, and scopolamine+CP. The graph quantifies the relative expression of PSD‐95 normalized to actin. Scopolamine significantly reduced PSD‐95 expression compared with that observed in the control group, whereas donepezil significantly increased PSD‐95 expression levels. CP treatment alone maintained PSD‐95 expression levels similar to those observed in the control group. Pretreatment with CP before scopolamine administration significantly restored PSD‐95 expression compared with scopolamine alone. Data are presented as means ± SEM. Statistical significance:  ∗∗∗∗ p < 0.0001 versus control and #### p < 0.0001 versus scopolamine (n = 6).

4. Discussion

This study demonstrated the effects of CP on memory ability using the MWM test. Treatment with 80 mg/kg CP for 14 days effectively improved memory performance by reducing escape latency, increasing the number of platform crossings, and extending the time spent in the target quadrant. These results are consistent with findings more than 20 years ago, which investigated the effects of CP on young adult rats′ performance in a navigational memory task using the MWM [33]. In line with previous research, a dose of 200 mg/kg administered for 14 days enhanced cognitive function in the shuttle‐box and step‐through paradigms [24]. Similarly, Arya et al. reported comparable findings, in which 14 days of oral CP administration was evaluated using the elevated plus maze and passive avoidance apparatus in Swiss albino mice [27]. These findings further support the role of CP seed extract as a natural compound with cognitive‐enhancing potential, aligning with the increasing interest in diet‐based approaches for maintaining brain health. However, the exact mechanism through which CP enhances memory remains unclear. Previous studies have found that CP possesses properties as an inhibitor of AChE and antioxidant activity [29]. Previous studies have shown that CP extract significantly improves memory and reduces AChE activity, supporting the enhanced memory performance of rats. Furthermore, some studies have reported the antioxidant properties of CP seeds [34]. According to Kumar and Gupta, the seed extracts used in vivo in rats demonstrated strong cognitive‐enhancing properties, possibly due to their high antioxidant potential. The seed extracts exert their cognitive‐enhancing properties by decreasing the brain levels of malondialdehyde, with simultaneous significant increases in the levels of glutathione and catalase [29]. We previously showed that CP has a neuroprotective effect by consistently attenuating MPP^+^‐induced toxicity before and after treatment. Posttreatment with CP could inhibit MPP^+^‐induced cell death via the induction of the antiapoptotic protein Bcl‐2, and Bcl‐2 expression was increased with the expression of p‐GSK‐3β (Ser9) in SH‐SY5Y cells [28].

Although the aforementioned mechanisms contribute to CP‐enhanced memory formation, they remain indirect and do not directly involve the cellular changes in the hippocampus responsible for long‐term memory. AMPAR trafficking plays a central role in cellular and electrophysiological studies that have uncovered LTP, which are cellular models for testing the plasticity mechanisms underlying memory formation. In this study, we are the first to provide evidence of CP′s influences on membranous AMPAR, Arc, and PSD‐95 expression, which are crucial components of LTP and synaptic plasticity–driven memory formation. A key mechanism underlying AMPAR trafficking and synaptic plasticity in the hippocampus is the phosphorylation of GluA1 (Ser831 and Ser845) and GluA2 (Ser880), which regulates AMPAR membrane expression. A previous study reported that some types of memory tasks physiologically and selectively induce an LTP‐like molecular change, including phosphorylation of GluA1 (Ser831) without affecting the phosphorylation of GluA1 (Ser845), in the hippocampus [35]. Moreover, hyperammonemia reduces GluA1 membrane expression and decreases GluA1 phosphorylation at Ser831, confirming that phosphorylation promotes the recycling of GluA1 to the membrane [36]. Therefore, this study demonstrated that CP enhances spatial memory by promoting GluA1 phosphorylation at Ser831, suggesting that CP improves memory formation through GluA1 phosphorylation, increased membrane expression, enhanced LTP, and subsequent memory induction.

Although this study focused on pSer831‐GluA1, Arc, and PSD‐95 as key markers of synaptic plasticity and LTP, we note that other molecular markers, such as amyloid precursor protein (APP), cyclin Y, and calcium‐binding proteins, also play essential roles in synaptic remodeling, neurogenesis, and memory regulation. These proteins were not assessed in the current study, representing a limitation.

Memory and spatial learning impairments are distinctive aspects of neurodegeneration and aging. In our study, we demonstrated that scopolamine administration caused significant deficits in cognitive function, as evaluated using the MWM. These findings are consistent with previous findings [37]. The combination of CP and scopolamine reduced scopolamine‐induced cognitive impairment and enhanced memory function, with the CP extract showing the highest efficacy. Cognitive decline is associated with cholinergic neuronal loss and reduced ACh levels in the brain. Scopolamine can block cholinergic signaling, leading to impaired learning and memory [38]. CP attenuates scopolamine‐induced memory impairment, potentially through interactions with both cholinergic and glutamatergic signaling pathways involving AMPAR. However, this study did not measure AChE activity and expression.

Despite promising findings, several limitations should be noted. First, the CP extract was not chemically characterized, and the specific active constituents remain unidentified. Future work should isolate and characterize these compounds to clarify their contribution to memory enhancement. Second, only the MWM test was used for behavioral assessment; including additional paradigms, such as novel object recognition or Y‐maze tests, would provide a more comprehensive evaluation of learning and memory. Third, while the present study focused on glutamatergic mechanisms, CP may also influence the cholinergic system, warranting further investigation of potential pathway interactions.

Although detailed phytochemical profiling of the current extract was not performed, previous studies indicate that C. paniculatus seeds contain multiple bioactive constituents—including sesquiterpenes, polyunsaturated fatty acids, alkaloids, and dihydroagarofuran derivatives—with reported antioxidant, neuroprotective, and cognition‐enhancing properties. These compounds may contribute collectively to the observed effects; however, their specific roles remain to be elucidated. Comprehensive fractionation and LC–MS/HPLC‐based characterization, combined with biological validation, will be essential to identify the precise active components responsible for CP′s cognitive effects.

This study also quantified protein expression using whole hippocampal homogenates rather than region‐specific dissections (e.g., CA1, CA3, and dentate gyrus), which may mask subfield‐specific variations in plasticity markers. In addition, although basic observational measures suggested no overt toxicity, detailed liver and kidney function assessments were not performed. The study included only male rats, precluding evaluation of sex‐dependent differences in synaptic plasticity or cholinergic–glutamatergic interactions; thus, future studies should incorporate both sexes or monitor the estrous cycle in females. Finally, pharmacokinetic parameters and blood–brain barrier penetration of CP components were not evaluated, representing important future directions to clarify brain exposure and mechanistic relevance.

5. Conclusions

This study showed that CP seed extract enhances memory and synaptic plasticity, as evidenced by increased pSer831‐GluA1, Arc, and PSD‐95 expression in the hippocampus. CP treatment improved memory performance in the MWM, similar to donepezil, and mitigated scopolamine‐induced memory deficits. These findings support the potential role of CP as a plant‐derived cognitive enhancer that may contribute to the prevention of memory impairment. By modulating key synaptic plasticity markers, CP demonstrates promise as a bioactive dietary compound that supports hippocampal function and memory consolidation. However, future studies should incorporate chemical profiling and quality control to ensure reproducibility and facilitate clinical application.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding

The study was funded by (i) Burapha University (BUU); (ii) Thailand Science Research and Innovation (TSRI); (iii) the National Science Research and Innovation Fund (NSRF), 54/2567; and the Faculty of Allied Health Sciences, Burapha University, AHS03/2565.

Supporting Information

Additional supporting information can be found online in the Supporting Information section.

Supporting information

Supporting Information 1 Figure S1: General observations of body weight–related parameters across experimental groups. Bar graphs represent mean ± SEM values measured at two experimental time points (left and right panels, separated by the dashed line). No significant differences were observed among groups, indicating the absence of overt systemic toxicity during the experimental period.

Supporting Information 2 Figure S2. General physiological observations across experimental groups. Bar graphs represent mean ± SEM values for (a) body weight, (b) food intake, (c) water intake, and (d) urinary output measured during the experimental period. No statistically significant differences were observed among groups, suggesting that CP treatment did not induce adverse systemic effects.