Vagus Nerve Stimulation Enhances Memory and Synaptic Plasticity via the Hippocampal NE/β-AR Signaling Pathway in Pilocarpine Model Rats

Vagus Nerve Stimulation Enhances Memory and Synaptic Plasticity via the Hippocampal NE/β-AR Signaling Pathway in Pilocarpine Model Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Vagus Nerve Stimulation Enhances Memory and Synaptic Plasticity via the Hippocampal NE/β-AR Signaling Pathway in Pilocarpine Model Rats Renli Qi, Jun Wang, Honglong Pei, Mou Zhang, Jianing Shen, Yanmei Chen, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5869428/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Vagus nerve stimulation (VNS) is recognized for its therapeutic potential in various neurological disorders, with clinical and preclinical evidence suggesting its capacity to enhance memory function beyond symptom alleviation. However, the mechanisms underlying VNS-mediated memory enhancement are not well understood. Given that the hippocampal noradrenergic (NE) system and β-adrenergic receptor (β-AR) pathway are linked to memory processes, this study investigated the effects of VNS on memory and hippocampal neuroplasticity in pilocarpine-induced memory-impaired rats, with a focus on the role of the NE/β-AR signaling pathway. Methods VNS was administered to pilocarpine-treated rats for two weeks via electrodes placed on the left cervical vagus nerve, with parameters set at a current intensity of 1 mA, frequency of 30 Hz, and pulse width of 250 µs for 2 hours daily. Poststimulation, the rats underwent contextual fear conditioning (CFC) and hippocampal long-term potentiation (LTP) testing. The protein expression levels of NE, β2-AR, and key downstream signaling molecules (protein kinase A, CaMKII) were quantified. To ascertain β-AR receptor involvement, a β-AR antagonist was administered in the hippocampus prior to VNS. Results VNS increased hippocampal NE neurotransmitter release, and the expression of β-AR and its downstream pathway proteins PKA and CaMKII enhanced impaired hippocampal LTP and contextual fear conditioning memory in PILO rats. This VNS-induced increase was reversed by β-AR antagonist administration. Conclusion The enhancement of hippocampal neuroplasticity and memory by VNS is associated with the hippocampal NE/β-AR signaling pathway, indicating a potential therapeutic mechanism for VNS in memory-related disorders. VNS memory hippocampus LTP NE β-AR Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The vagus nerve, classified as the tenth cranial nerve, is the longest and most extensively distributed cranial nerve and comprises sensory, motor, and parasympathetic fibers [ 1 ]. It plays a significant role in various physiological processes. Vagus nerve stimulation (VNS) is a neuromodulation technique that employs an implantable pulse generator to deliver intermittent electrical stimuli to the vagus nerve, thereby transmitting signals to the brain and inducing broad changes in the nervous system. VNS has received US Food and Drug Administration approval for the adjunctive treatment of drug-resistant epilepsy [ 2 ], refractory major depressive disorder [ 3 ], and poststroke recovery [ 4 ]. Clinically, VNS has been shown to alleviate disease symptoms and has a significant positive impact on memory over the long term [ 5 , 6 ]. Despite these findings, the mechanisms through which VNS enhances memory are not fully understood. The hippocampus is pivotal in memory formation, and our previous research revealed severe hippocampal neuron loss in pilocarpine model rats [ 7 ], a finding supported by other studies that utilized this model to investigate memory impairments [ 8 , 9 ]. Contextual fear conditioning (CFC) is a well-established behavioral paradigm in rodents, where subjects learn to associate a neutral context with an aversive stimulus, thus exhibiting fear responses to contexts indicating danger[ 10 ]. Synaptic plasticity, which involves the activity-dependent modification of synaptic strength or efficacy within the nervous system, underlies learning and memory processes[ 11 ]. Long-term potentiation (LTP), characterized by enhanced postsynaptic responses following stimulation, is a form of hippocampal synaptic plasticity associated with synaptic strengthening and improved learning and memory[ 11 , 12 ]. Therefore, in this study, we investigated whether VNS could improve CFC performance and hippocampal LTP levels in pilocarpine-induced memory-impaired rats. From a functional neuroanatomical perspective, VNS transmits stimulation signals to the nucleus tractus solitarius (NTS) via vagus nerve afferents and then projects to the locus coeruleus (LC)[ 13 ], the principal norepinephrine (NE)-producing nucleus in the brain. The LC projects widely to other brain regions, including the cerebral cortex, dorsal raphe nucleus, hippocampus, amygdala, and cerebellum, where it modulates functions such as learning, memory, attention, and cognition[ 14 , 15 ]. While studies have indicated that VNS can promote NE release in the hippocampus [ 16 ], it remains unclear whether VNS enhances memory by increasing NE neurotransmission and which receptor signaling pathways are involved. Adrenergic receptors (ARs), a class of G protein-coupled receptors that mediate the effects of catecholamine neurotransmitters, include α-AR and β-AR receptors, with β-AR receptors being highly expressed in the rat hippocampus[ 17 ]. β-AR and its downstream pathways have been shown to play critical roles in memory and LTP formation[ 18 ]. NE, which acts on β-AR, stimulates an increase in cyclic adenosine monophosphate (cAMP) synthesis; cAMP activates protein kinase A (PKA) and indirectly activates extracellular signal-regulated kinase (ERK)[ 18 ]. PKA and ERK are important for long-term memory formation and synaptic plasticity across species [ 19 , 20 ]. One of the direct downstream signaling targets of β-AR is inhibitor-1 (Inh-1), which, when phosphorylated by PKA, inhibits the expression of protein phosphatase 1 (PP1). PP1 is responsible for dephosphorylating proteins critical for LTP induction, including CaMKII, NMDARs, and AMPARs. Thus, β-AR activation leads to the inhibition of PP1, enhancing the expression of synaptic plasticity-related proteins[ 22 ]. In this study, we aimed to determine whether VNS could improve memory and hippocampal synaptic plasticity in pilocarpine-induced memory-impaired rats and whether this effect is mediated by the hippocampal NE/β-AR signaling pathway. We specifically examined improvements in CFC performance and hippocampal LTP levels, as well as changes in the protein expression levels of NE, β-AR, and downstream signaling pathways (protein kinase A, CaMKII), following two weeks of VNS treatment. Additionally, we administered the β-AR antagonist propranolol (PR) to the hippocampus to determine the role of the β-AR receptor in this process. Materials and methods Sixty healthy adult male Sprague–Dawley rats, 6 weeks old and weighing 250–300 g, were obtained from the Experimental Animal Center of Kunming Medical University, which holds Production License SCXK (Dian) K2020–0004. All the rats were housed in a controlled environment of 20–25°C, with a humidity of 55 ± 2%, and in quiet states maintained under a 12 h/12 h light/dark cycle with ad libitum access to food and water. The rats were handled daily by experimenters for 1 week to reduce the stress response. A schematic of the experimental procedures is provided in Fig. 1 . Pilocarpine Rat Model The epileptic rat model was established via the lithium‒pilocarpine method as described in our previous research[ 7 ]. Lithium chloride (127 mg/kg, Sigma, USA) was initially administered intraperitoneally to the rats. A single dose of pilocarpine (350 mg/kg; Meilun Biotechnology Co., Jiangsu, China) was subsequently intraperitoneally injected to induce neural death 15–19 hours after lithium chloride administration. To counteract peripheral cholinergic effects, methylscopolamine (1 mg/kg; Yuanye Biotechnology Co., Shanghai, China) was intraperitoneally injected 30 minutes prior to pilocarpine application. Status epilepticus was terminated after 90 minutes using chloral hydrate (30 mg/kg, intraperitoneal; Hengrui Medical Co., Jiangsu, China). The control rats received injections of methylscopolamine and saline. Surgery for Electrode and Infusion Cannula Implantation The implantation of VNS electrodes was conducted one week after the establishment of the PILO model. The rats were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneal injection; Hengrui Medical Co., Jiangsu, China), and the depth of anesthesia was monitored via the toe-pinch reflex test. Following successful anesthesia, VNS electrodes were wrapped around the left vagus nerve in the neck and secured with nonabsorbable sutures (refer to our previous literature[ 7 ] for specific methods). Next, fixed fusion cannulas were implanted bilaterally in the hippocampus. According to the rat brain atlas by Paxinos and Watson, five holes (1.2 mm diameter) were drilled in the skull, with three for fixation and two for embedding drug infusion cannulas (RWD Co., Ltd., Shenzhen, China; outer diameter, 0.5 mm; inner diameter, 0.40 mm) into the CA1 region of the hippocampus (3.3 mm posterior to the bregma and 2 mm lateral to the midline). A solid stainless-steel wire was inserted into the drug infusion cannula for sealing. The final step involved securing the VNS electrode and drug infusion cannulas with dental acrylic. Penicillin was administered intramuscularly for three days postoperatively to prevent infection. VNS Protocol VNS was initiated three weeks after the establishment of the PILO model and continued for two weeks, with a total daily stimulation time of 120 minutes. VNS was divided into four 30-minute blocks with a 2-hour interval between each block. VNS was performed via a programmable pulse stimulator (Model Master8, A.M.P.I., Israel), with parameters set for bipolar balanced square waves (pulse width of 250 µs; frequency of 30 Hz; current intensity of 1 mA). Stimulation output was verified via a digital oscilloscope and by observing the cough reflex in the rats. The current of the electric pulse was set to 1 mA after a 5-minute adaptation period at 0.7 mA, and stimulation proceeded for two weeks with the rats in a free-moving state. Drug Infusion Prior to each VNS session, the rats in the PILO + PR + VNS group were administered the β-AR antagonist propranolol (PR, 5 µg/µL), following the methodology of previous research [ 21 ]. The rats were induced with 2% isoflurane anesthesia (Hebei Nine Sent Pharmaceutical Co., Ltd., Hebei, China) before drug infusion. A stainless-steel drug administration tube (outer diameter: 0.3 mm, with the tip extending 1.0 mm beyond the cannula, resulting in a drug injection site 2.7 mm below the skull surface) was carefully inserted at a rate of 0.5 µL/min, with 1 µL of propranolol being injected into each hippocampal region. The administration tube was left in place within the cannula for an additional minute postinjection to ensure complete drug delivery. The control groups received an equivalent volume of saline. Contextual fear conditioning When fearful, rodents exhibit a freezing response as a defensive posture. In our experiments, test subjects received a tone cue (conditional stimulus) followed by an electric shock (unconditioned stimulus), a process termed conditioning. Following this training, the animals were tested with a tone cue or context to assess memory retention, which is known as testing. Rodents typically display a conditioned fear response, evident as freezing behavior, in response to both the associated context and the same tone cue presented in a different context. The duration of freezing in these contexts reflects an animal’s memory capacity. Contextual fear conditioning was conducted using a fear conditioning chamber (DL Naturegene Life Science, Inc., USA). The experimental protocol spanned three days and was performed at the same time each day, as depicted in Fig. 2 a. Day 1: Habituation. The rats were removed from their home cages and placed in the chamber for free exploration, adapting to two distinct environments (horizontal and vertical striped scenes) for 10 minutes each. Day 2: Training. The rats were placed in the chamber against one of the backgrounds for a total of 820 seconds and received five electric shocks (1-second duration, 0.78 mA intensity) at intervals of 140 seconds per session (19 seconds of sound followed by 1 second of electric stimulus). Day 3: Contextual memory test. Using the same background as the training day, the freezing behavior of the rats was observed and recorded for 10 minutes in the absence of electric shock stimuli. LTP Artificial cerebrospinal fluid was prepared and continuously oxygenated prior to the procedure. The rats were decapitated to collect brain tissue, which was subsequently sliced into 340 nm-thick hippocampal sections via a vibratome. Following a 1-hour incubation at a constant temperature, the slices were transferred to a recording chamber. The CA1 and CA3 regions of the hippocampus were identified via a multimicro manipulator system (MPC200, Sutter Corporation). Recording electrodes, pulled from borosilicate glass capillaries via a glass electrode puller (Sutter Corporation) and filled with 3 M NaCl electrolyte solution, were carefully lowered into the CA1 region, while stimulating electrodes, fashioned from twisted tungsten wire, were inserted into the CA3 region. A stimulator was employed to deliver continuous single biphasic pulse stimulation, and a multichannel amplifier (20 kHz, 700B, Axon Corporation) was utilized for recording purposes. The electrode positions were fine-tuned until field excitatory postsynaptic potentials (fEPSPs) were reliably evoked. The stimulation current was gradually increased to a level that elicited an fEPSP amplitude representing 50% of the maximum response (approximately 0.2–0.6 mA), which was then used as the baseline for synaptic transmission. Baseline responses were recorded at 30-second intervals for 15 minutes, after which high-frequency stimulation (HFS: 100 Hz, 10 trains of 200 ms duration each, repeated 3 times with a 10-second interval) was applied to induce LTP. After induction, the fEPSPs were monitored and recorded every 30 seconds for an additional 50 minutes. The fEPSP amplitudes following HFS were normalized to the pre-HFS baseline average. Electrophysiological data acquisition was facilitated by MultiClamp 700B Commander software and Clampex 10.7, with subsequent analysis and plotting conducted via GraphPad Prism. Molecular biology experiments The rats were anesthetized with an overdose of pentobarbital sodium (60 mg/kg, i.p.) and then subjected to cardiac perfusion. After the brain was removed, one side of the hippocampal tissue was rapidly frozen in liquid nitrogen and stored at -80°C for protein immunoblotting and NE content determination. The other side of the brain hemisphere was fixed in 4% paraformaldehyde for immunofluorescence staining. a. Western blot (WB) a. Western blot (WB) For the WB experiments, hippocampal tissues were homogenized in RIPA lysis buffer containing a protease inhibitor cocktail to extract proteins. The supernatant was collected and centrifuged at 13,000 rpm at 4°C for 20 minutes. Protein concentrations were determined via the bicinchoninic acid (BCA) assay. Equal amounts of protein samples were separated on 5%, 12%, or 15% SDS‒PAGE gels and transferred onto PVDF membranes. The membranes were blocked in 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween (BSA-TBST) for 1 hour at room temperature and incubated with the following primary antibodies overnight at 4°C: β-AR (1:1000, ab182136, Abcam), PKA (1:1000, ADI-KAS-PK017-D, Enzo Life Sciences), and CaMKII (1:1000, sc-5306, Santa Cruz Biotechnology). After three washes with TBST buffer, the membranes were incubated with secondary antibody solutions (HRP goat anti-rabbit 1:1000, Proteintech; HRP goat anti-mouse 1:5000, Proteintech) for 2 hours at room temperature. The signals were detected via an Image Quant LAS4000 image analyzer (GE Healthcare, Buckinghamshire, UK), and the band intensities were quantified via Quantity One software v.4.6.2 (Bio-Rad, Hercules, CA, USA). b. NE Content Detection Norepinephrine content was measured via a Norepinephrine ELISA Kit (EK2432, SAB). Hippocampal tissues were minced, weighed, mixed with 1x PBS at a ratio of 10 ml/g, homogenized at 45 Hz for 120 seconds and centrifuged at 5000 rpm for 5 minutes at 4°C. Standards and samples (50 µl each) were added to the wells, followed by the addition of 50 µl of detection solution A and incubation at room temperature for 1 hour. After three washes with 300 µl of 1x PBS, 100 µl of detection B was added, and the mixture was incubated at room temperature for 45 minutes. In the dark, 90 µl of substrate solution was added to each well and incubated at room temperature for 10–20 minutes after three washes. Finally, 50 µl of stop solution was added, and the optical density of each well was measured at a wavelength of 450 nm via an enzyme reader. Statistical analysis All the statistical analyses were performed via SPSS software version 26.0 (IBM Corporation, Armonk, NY, USA). The data are presented as the means ± standard errors of the means (SEMs). The freezing time during the CFC test, fEPSP amplitudes, NE content and WB results were evaluated via one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons or Student’s t test where appropriate. The freezing time during the CFC training period was analyzed via two-way repeated-measures ANOVA to assess the main effects and interactions, followed by Tukey’s post hoc test for pairwise comparisons. A p value of less than 0.05 was considered to indicate statistical significance. Graphs and additional data visualization were generated via GraphPad Prism software version 8. Results 1. VNS improved impaired memory and hippocampal LTP in PILO model rats As shown in Figure 2a, the contextual fear conditioning (CFC) test includes three sessions: habituation, conditioning and testing. The freezing time of the experimental animals during the testing period was used to characterize the memory of the subjects. As shown in the line graph in Figure 2b, for the freezing time in the conditioning period, two-way ANOVA with repeated measures revealed significant effects of trials on the training curve (F(3.643, 54.64)=35.6, p<0.0001), whereas no significant group × trial interactions were detected (F(10, 75)=1.108, p=0.3677), which indicated that all three groups of rats learned the relationship between electrical shock and the training environment. As shown in the bar graph in Figure 2c, for the freezing time in the testing period, there was a significant difference among the three groups (ANOVA, p=0.0162); the freezing time of the PILO group was significantly lower than that of the control group (p=0.0136), whereas that of the PILO+VNS group was significantly greater than that of the PILO group (p=0.0395), suggesting that the memory of the PILO group effectively improved in the VNS group. LTP induction is shown in Figure 3a. The stimulation electrodes were inserted into the CA3 region, the recording electrodes were inserted into the CA1 region, the baseline fEPSP was recorded for 15 minutes, HFS was used to induce LTP, the fEPSP was recorded for 50 minutes after stimulation, and the ratio of the amplitude of the fEPSP to the baseline value within 20–50 minutes after HFS was used to measure LTP (Figure 3b). After HFS, there was a significant difference in the increase in fEPSP amplitude among the control (n=6) , PILO (n=6) and PILO+VNS (n=6) groups (ANOVA, p=0.029), and the increase in fEPSP amplitude in the PILO group was significantly lower than that in the control group (p=0.0499), whereas that in the PILO+VNS group was significantly greater than that in the PILO group (p=0.0089), indicating that VNS effectively improved the impaired hippocampal LTP of the PILO model rats (Figure 3c). 2. VNS enhanced the function of the NE/β-AR system and its downstream pathways in the hippocampus of PILO rats a. VNS promoted NE neurotransmitter release in the hippocampus of PILO rats The NE content in the hippocampal tissues was detected via a general noradrenaline ELISA kit. Significant differences were not detected among the control (n=6) , PILO (n=6) and PILO+VNS (n=6) groups (ANOVA, p>0.05), but the NE content in the hippocampus of the PILO+VNS group was greater than that in the PILO group (t=3.104, p=0.0267) according to Student's t test, suggesting that VNS promoted NE neurotransmitter release in the hippocampal region of PILO rats (Figure 4a). b. VNS activated β-AR and downstream signaling pathways in the hippocampus of PILO rats The protein expression levels of β-AR, PKA, and CaMKII were quantified via Western blot analysis. One-way ANOVA revealed significant differences in β-AR protein expression among the control (n=6), PILO (n=6), and PILO +VNS (n=6) groups (p<0.0001). Compared with the control group, the PILO group presented a significant decrease in expression (p=0.0001), whereas the PILO+VNS group presented a significant increase in expression compared with the PILO group (p<0.0001), suggesting that VNS increased the expression of β-AR in the hippocampal region of PILO rats (Figure 4b). There were also significant differences in the PKA protein expression levels among the three groups (p<0.0001, one-way ANOVA). Compared with that in the control group, the protein expression level of PKA in the PILO group was significantly lower (p=0.0317), whereas that in the PILO+VNS group was significantly greater (p<0.0001), indicating that VNS upregulated PKA expression in the hippocampal region of PILO rats (Figure 4c). As shown in Figure 4d, there was no significant difference in the protein expression of hippocampal CaMKII among the three groups, but VNS tended to increase the protein expression of CaMKII in the hippocampal area of PIL rats. Therefore, VNS increased hippocampal NE neurotransmitter release and the expression of β-AR and its downstream pathway proteins PKA and CaMKII and enhanced impaired hippocampal LTP and contextual fear conditioning memory in PILO rats; this effect of VNS could be blocked by a β-AR antagonist. 3. Blocking hippocampal β-AR function inhibited the enhancing effect of VNS on memory and LTP As indicated in Figure 5a, prior to each VNS treatment, the noncompetitive β-AR antagonist PR was microinjected bilaterally into the CA1 region of the hippocampus in PILO rats to block the action of β-AR. In the CFC test, the learning curves of the rats in the PILO saline + VNS group (n=6) and the PILO PR+VNS group (n=6) were not significantly different (Figure 5b). Compared with the PILO saline + VNS group, the PILO PR+VNS group presented a significant decrease in contextual fear memory (t=4.799, p=0.0007), suggesting that the β-AR antagonist blocked the ability of VNS to improve memory in the PILO rats (Figure 5c). In the LTP induction experiment, the level of LTP in the PILO PR+VNS group was significantly lower than that in the PILO saline +VNS group (t=20.38, p<0.0001), indicating that the β-AR antagonist blocked the effect of VNS on enhancing hippocampal neuroplasticity in the PILO rats (Figure 5d, e). Compared with that in the PILO saline +VNS group, the NE content in the hippocampal tissue of the rats in the PILO PR + VNS group was significantly lower (t=2.966, p=0.0141), suggesting that β-AR antagonists blocked the ability of VNS to promote NE neurotransmitter release in the hippocampus of the PILO rats (Figure 5f). Compared with those in the PILO saline + VNS group, the β-AR, PKA and CaMKII protein levels in the hippocampal tissue of the rats in the PILO+PR+VNS group were significantly lower (t=2.737, 2.412 and 2.329, respectively; p=0.021, 0.0366 and 0.0421, respectively). These results suggest that β-AR antagonists block the enhancement of hippocampal β-AR receptors and downstream signaling pathways by VNS in PILO rats (Figure 5g-i). Discussion VNS has been approved for the treatment of drug-resistant epilepsy, severe depression, and poststroke rehabilitation. During its clinical application in patients, VNS not only improved epilepsy or depression symptoms but also had a positive effect on memory[ 12 , 22 ]. In experimental animals, researchers have also reported that VNS can enhance the memory of normal animals[ 23 , 24 ]. Furthermore, studies on noninvasive vagus nerve electrical stimulation have shown that it can improve attention, working memory, memory consolidation, decision-making, and other cognitive functions[ 25 – 27 ]. Therefore, some researchers have proposed that noninvasive VNS could be used for the early treatment and prevention of Alzheimer's disease [ 28 ]. Although both invasive and noninvasive VNS have been shown to effectively enhance memory in clinical and experimental animal studies, the specific mechanisms involved remain unclear. Selecting an appropriate memory impairment animal model is crucial for elucidating how VNS improves memory. We used the pilocarpine rat model, which has been shown to exhibit severe apoptosis of hippocampal neurons, providing a pathological basis for memory impairment[ 7 ]; many drugs have been studied in this model for their effects on memory [ 8 , 9 ]. To reveal the long-term effects of VNS on memory, we delivered continuous stimulation for 14 days after the pilocarpine rat model was established, stimulated for 2 hours each day, and then assessed the rats’ memory, neurotransmitter changes, and receptor molecular pathways. The hippocampus plays a crucial role in establishing contextual fear memory [ 29 , 30 ]. Using the CFC test, we assessed memory in three groups of rats and found that all groups learned the associations between electrical shock and the training environment. During the testing period, pilocarpine-treated rats exhibited significant memory impairment, which was significantly improved after two weeks of continuous VNS treatment, indicating that VNS can improve impaired memory in pilocarpine-treated rats. Moreover, this effect could be blocked by β-AR antagonists, suggesting a key role for β-AR in VNS-mediated memory improvement. Additionally, we detected a significant decrease in LTP in the CA1 region of the hippocampus in pilocarpine-treated rats compared with control rats. This finding is consistent with previous reports, which have documented impaired LTP during both the latent[ 31 ] and chronic phases[ 32 ] of pilocarpine-treated animals. One study that monitored hippocampal synaptic plasticity changes over one week post- pilocarpine treatment found no significant alterations in LTP on the 1st day, but a marked reduction on the 3rd day, with further impairment observed by day 7 post-treatment[ 33 ]. The mechanisms underlying this LTP impairment likely involve disrupted neuron-glial interactions. Specifically, astrogliosis in the hippocampus leads to morphological changes in astrocytes, reducing the number of astrocytic leaflets near synapses[ 34 ]. This disrupts the local supply of D-serine to NMDARs, enhancing receptor desensitization[ 35 ]. D-serine application restored LTP and enhanced NMDAR-mediated currents in pilocarpine treated rats[ 33 ]. Additionally, impaired glutamate clearance mechanisms may also contribute to LTP deficits. Under normal conditions, high-affinity glial and neuronal excitatory amino acid transporters (EAATs) effectively clear glutamate released during HFS[ 36 ]. However, in epileptic tissue, glutamate clearance is further compromised due to alterations in EAAT functional activity and decreased astrocytic surface area at glutamatergic synapses[ 37 ]. This impaired clearance may lead to the accumulation of glutamate in the synaptic cleft, thereby exacerbating NMDAR desensitization and LTP impairment. VNS treatment effectively enhanced LTP in the CA1 region of pilocarpine-treated rats, confirming that VNS can improve hippocampal neuroplasticity and memory function. This result is consistent with previous studies on normal rats under freely moving conditions[ 23 , 38 , 39 ]. Electrophysiological and neurochemical data suggest that the enhanced effect of VNS on LTP may involve CNS noradrenergic systems. The initial increase in NE induced by VNS likely triggers changes in the localization and expression of pre- and post-synaptic receptors and proteins that are critical for maintaining LTP[ 40 ]. Activation of β-AR by NE facilitates the induction of LTP across all major regions of the hippocampal formation[ 41 ]. This effect is mediated through the activation of voltage-sensitive calcium channels, including NMDA receptor channels[ 42 ]. Furthermore, we found that the enhancing effect of VNS on LTP could be blocked by NE β-AR antagonists. Previous studies have reported that the enhancing effect of VNS on LTP in the CA3 region of the hippocampus in normal rats is dependent on the LC and hippocampal β-AR[ 39 ] [ 43 ], which is consistent with our findings in the CA1 region of pilocarpine-treated rats. However, an unexpected result in our study was that the amplitudes of fEPSPs in β-AR antagonist-treated rats tended to decrease below baseline following HFS. Previous research in normal rats has shown that acute VNS can enhance synaptic transmission in the PP-CA3 pathway[ 39 ]. This enhancement can be blocked by pre-treatment with β-AR antagonists, which can even reduce synaptic transmission below baseline levels. Notably, β-AR antagonists alone do not affect PP-CA3 synaptic transmission, suggesting that when β-ARs are saturated by antagonists, VNS may activate mechanisms that reduce synaptic strength.There is a complex relationship between β-ARs and synaptic plasticity. NE or β-AR agonists (such as isoproterenol, Iso) can enhance LTP induced by HFS[ 44 ]. Moreover, β-adrenergic stimulation with Iso enables LTP induction even after low-frequency stimuli (1–5 Hz for 15 min) that would otherwise induce LTD[ 45 ]. We speculate that in the hippocampus of pilocarpine-treated rats, impaired glutamate clearance at the synaptic cleft may lead to desensitization of NMDARs. Additionally, β-AR antagonists may further reduce the expression of NMDARs and AMPARs on the postsynaptic membrane. Under these conditions, HFS may induce glutamate excitotoxicity, causing damage to synapses and reducing synaptic transmission efficiency, or may trigger weak postsynaptic calcium influx, initiating LTD mechanisms. From a functional neuroanatomical perspective, VNS transmits stimulation information to the nucleus tractus solitarius (NTS) through the afferent fibers of the vagus nerve, which project to the locus coeruleus (LC), the primary brain nucleus producing NE, projecting diffusely to other brain areas, such as the cerebral cortex, dorsal raphe nucleus, hippocampus, amygdala, and cerebellum[ 14 , 46 ]. Robrecht Raedt et al. reported an increase in hippocampal NE content following 240 minutes of acute intermittent VNS (frequency = 30 Hz; intensity = 1 mA; pulse width = 250 µs; duty cycle = 7 s ON-18 s OFF)[ 16 ]. Our findings indicate that two weeks of continuous VNS can effectively increase hippocampal NE levels in pilocarpine-treated rats. Unlike the study by Robrecht Raedt, which compared the hippocampal NE content before and after VNS in pilocarpine-treated rats without contrasting it with that of controls, our experiment revealed no significant difference in the hippocampal NE content between pilocarpine-treated and control rats[ 16 ]. We hypothesize that the LC in pilocarpine-treated rats remains undamaged, maintaining consistent NE neurotransmitter release to the hippocampus, yet VNS can activate NE neurons in the LC, thereby increasing the hippocampal NE content. As shown in Fig. 6 shows, the mechanism by which increased hippocampal NE content ameliorates contextual fear memory and LTP involves VNS-induced upregulation of hippocampal β-AR and its downstream PKA and CaMKII proteins, which are integral to hippocampal LTP enhancement. Blocking β-AR reversed the VNS-induced improvements in contextual fear memory and hippocampal LTP in pilocarpine-treated rats, suggesting that VNS enhances memory and LTP in pilocarpine-treated rats through the hippocampal NE/β-AR signaling pathway. Our findings suggest that VNS rescues pilocarpine induced PKA suppression via the NE/β-AR pathway. Elevated PKA activity may enhance AMPA receptor function through GluA1-Ser845 phosphorylation[ 47 ]. Moreover, this effect could be blocked by β-AR antagonists, suggesting a key role for β-AR/PKA pathway in VNS-mediated memory improvement. While PP1 and downstream targets (e.g., CaMKII, NMDAR/AMPAR subunits) were not directly examined here, future studies should evaluate these pathways to fully elucidate the molecular basis of VNS induced synaptic recovery. Interestingly, despite the lack of a significant difference in the hippocampal NE content between pilocarpine-treated and control rats, β-AR expression was diminished in pilocarpine-treated rats. Therefore, why does hippocampal β-AR expression decrease in PILO rats? Our previous research indicated that VNS can protect CA1 region neurons from pilocarpine-induced damage. Further investigations are needed to determine whether VNS can mitigate inflammatory responses or apoptosis in the hippocampus through the NE/β-AR signaling pathway, thereby improving memory in pilocarpine-treated rats. Conclusion VNS ameliorated impaired memory and hippocampal LTP in pilocarpine-treated rats, potentially through enhancing the activity of the hippocampal NE/β-AR signaling pathway. Abbreviations VNS, vagus nerve stimulation; NE, norepinephrine; β-AR, β-adrenergic receptor; LTP, long-term potentiation; fEPSP, field excitatory postsynaptic potential; CFC, contextual fear conditioning; LC, locus coeruleus; PILO, pilocarpine. Declarations Funding This work was supported by several grants: the National Natural Science Foundation of China [Grant Numbers 82360270 and 32260197]; the Key Project of the Natural Science Foundation of Yunnan Province [Grant Number 202401AS070040]; the Yunnan Provincial Education Department Scientific Research Fund [Grant Number 2023Y0636]; the Yunnan Provincial Department of Science and Technology-Kunming Medical University Joint Program on Basic Research [Grant Number 202401AY070001-236]; the First-Class Discipline Team of Kunming Medical University [Grant Number 2024XKTDPY02]; and the Yunnan Revitalization Talent Support Program for Hualin Yu and Jinghui Li. Additional support was provided by the NHC Key Lab of Drug Addiction Medicine at Kunming Medical University through its Open Projects Fund [Project Number KN202424]. Ethical Approval This study was conducted in accordance with the ethical standards of the institution or practice at which the studies were performed. All procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Kunming Medical University (IACUC, SYXK(Dian) K2020-0006). Consent to Participate As this study involved animals, consent to participate is not applicable. Consent to Publish The results of this study have been published with the consent of all authors and the responsible authorities at the institution where the work was conducted. References Berthoud, H.R. and W.L. Neuhuber, Functional and chemical anatomy of the afferent vagal system. Auton Neurosci, 2000. 85 (1-3): p. 1-17. Wheless, J.W., A.J. Gienapp, and P. Ryvlin, Vagus nerve stimulation (VNS) therapy update. 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Sanders, T.H., et al., Cognition-Enhancing Vagus Nerve Stimulation Alters the Epigenetic Landscape. J Neurosci, 2019. 39 (18): p. 3454-3469. Jacobs, H.I., et al., Transcutaneous vagus nerve stimulation boosts associative memory in older individuals. Neurobiol Aging, 2015. 36 (5): p. 1860-7. Klaming, R., et al., Effects of Noninvasive Cervical Vagal Nerve Stimulation on Cognitive Performance But Not Brain Activation in Healthy Adults. Neuromodulation, 2022. 25 (3): p. 424-432. Martin, C.O., et al., The effects of vagus nerve stimulation on decision-making. Cortex, 2004. 40 (4-5): p. 605-12. Vargas-Caballero, M., et al., Vagus Nerve Stimulation as a Potential Therapy in Early Alzheimer's Disease: A Review. Front Hum Neurosci, 2022. 16 : p. 866434. Kim, W.B. and J.H. Cho, Encoding of contextual fear memory in hippocampal-amygdala circuit. Nat Commun, 2020. 11 (1): p. 1382. Melo, M.B., V.M. Favaro, and M.G.M. Oliveira, The dorsal subiculum is required for contextual fear conditioning consolidation in rats. Behav Brain Res, 2020. 390 : p. 112661. Kryukov, K.A., et al., Status epilepticus alters hippocampal long-term synaptic potentiation in a rat lithium-pilocarpine model. Neuroreport, 2016. 27 (16): p. 1191-5. Zhang, Y., et al., Time-dependent changes in learning ability and induction of long-term potentiation in the lithium-pilocarpine-induced epileptic mouse model. Epilepsy Behav, 2010. 17 (4): p. 448-54. Postnikova, T.Y., et al., Impairments of Long-Term Synaptic Plasticity in the Hippocampus of Young Rats during the Latent Phase of the Lithium-Pilocarpine Model of Temporal Lobe Epilepsy. Int J Mol Sci, 2021. 22 (24). Plata, A., et al., Astrocytic Atrophy Following Status Epilepticus Parallels Reduced Ca(2+) Activity and Impaired Synaptic Plasticity in the Rat Hippocampus. Front Mol Neurosci, 2018. 11 : p. 215. Henneberger, C., et al., Long-term potentiation depends on release of D-serine from astrocytes. Nature, 2010. 463 (7278): p. 232-6. Diamond, J.S. and C.E. Jahr, Synaptically released glutamate does not overwhelm transporters on hippocampal astrocytes during high-frequency stimulation. J Neurophysiol, 2000. 83 (5): p. 2835-43. Clarkson, C., et al., Ultrastructural and functional changes at the tripartite synapse during epileptogenesis in a model of temporal lobe epilepsy. Exp Neurol, 2020. 326 : p. 113196. Zuo, Y., D.C. Smith, and R.A. Jensen, Vagus nerve stimulation potentiates hippocampal LTP in freely-moving rats. Physiol Behav, 2007. 90 (4): p. 583-9. Shen, H., et al., Vagus nerve stimulation enhances perforant path-CA3 synaptic transmission via the activation of beta-adrenergic receptors and the locus coeruleus. Int J Neuropsychopharmacol, 2012. 15 (4): p. 523-30. Maity, S., et al., Norepinephrine triggers metaplasticity of LTP by increasing translation of specific mRNAs. Learn Mem, 2015. 22 (10): p. 499-508. Stanton, P.K. and J.M. Sarvey, Norepinephrine regulates long-term potentiation of both the population spike and dendritic EPSP in hippocampal dentate gyrus. Brain Res Bull, 1987. 18 (1): p. 115-9. Gray, R. and D. Johnston, Noradrenaline and beta-adrenoceptor agonists increase activity of voltage-dependent calcium channels in hippocampal neurons. Nature, 1987. 327 (6123): p. 620-2. Lv, J., et al., Activation of β-adrenoceptor facilitates active avoidance learning through enhancement of glutamate levels in the hippocampal dentate gyrus. Neuroreport, 2017. 28 (15): p. 973-979. Jedrzejewska-Szmek, J., et al., beta-adrenergic signaling broadly contributes to LTP induction. PLoS Comput Biol, 2017. 13 (7): p. e1005657. Larsen, M.E., et al., Stimulating beta-adrenergic receptors promotes synaptic potentiation by switching CaMKII movement from LTD to LTP mode. J Biol Chem, 2023. 299 (6): p. 104706. Berger, A., et al., How Is the Norepinephrine System Involved in the Antiepileptic Effects of Vagus Nerve Stimulation? Front Neurosci, 2021: p. 15：790943. Banke, T.G., et al., Control of GluR1 AMPA receptor function by cAMP-dependent protein kinase. J Neurosci, 2000. 20 (1): p. 89-102. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5869428","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":440518946,"identity":"33d8646c-0c90-4a57-a7b7-a83c23453cd9","order_by":0,"name":"Renli Qi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYDACZhBRICHHzwPhMzYQp8VAwliyh2gtYGDAkGhwhlgtfMd5Hz4uMLBIMD5zOnUzD4ON7IYDzM8e4NMieZjd2HiGgUSe2dnebbd5GNKMNxxgMzfA66TDbGzSPAYSxWbneUFaDiduOMDDJkFAC/tvoJbEzf1gLf+J0sLGDNKygRfssAOEtUgeZmMGOcxY4szZbTfnGCQbzzzMZoZXC9/5Y4yfeSrq5Ph7crfdeFNhJ9t3vPkZXi0MB1DdyQCNXOK1jIJRMApGwSjAAgDupUM7XhAtwgAAAABJRU5ErkJggg==","orcid":"","institution":"First Affiliated Hospital of Kunming Medical University","correspondingAuthor":true,"prefix":"","firstName":"Renli","middleName":"","lastName":"Qi","suffix":""},{"id":440518947,"identity":"4d1b743a-478d-4521-b3cd-2f3a1e2a08a5","order_by":1,"name":"Jun Wang","email":"","orcid":"","institution":"First Affiliated Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Wang","suffix":""},{"id":440518948,"identity":"42b5a09e-d929-4bdf-807e-26a2a2c22a86","order_by":2,"name":"Honglong Pei","email":"","orcid":"","institution":"First Affiliated Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Honglong","middleName":"","lastName":"Pei","suffix":""},{"id":440518949,"identity":"4ecc0b2a-a9b0-490f-a075-4291c3ae9b15","order_by":3,"name":"Mou Zhang","email":"","orcid":"","institution":"First Affiliated Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mou","middleName":"","lastName":"Zhang","suffix":""},{"id":440518950,"identity":"658dc6de-b563-476e-b7df-7f5530a2ccd5","order_by":4,"name":"Jianing Shen","email":"","orcid":"","institution":"Kunming University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jianing","middleName":"","lastName":"Shen","suffix":""},{"id":440518951,"identity":"ece09141-9af8-49aa-89f7-92f3e6143438","order_by":5,"name":"Yanmei Chen","email":"","orcid":"","institution":"Kunming University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yanmei","middleName":"","lastName":"Chen","suffix":""},{"id":440518952,"identity":"f95f1172-8649-4928-9e50-85fd16a916a5","order_by":6,"name":"Hualin Yu","email":"","orcid":"","institution":"First Affiliated Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hualin","middleName":"","lastName":"Yu","suffix":""},{"id":440518953,"identity":"9decef20-1547-4365-9f4a-62e15580df57","order_by":7,"name":"Jinghui Li","email":"","orcid":"","institution":"First Affiliated Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jinghui","middleName":"","lastName":"Li","suffix":""},{"id":440518954,"identity":"7d53dcfd-62ab-41a6-931b-810837973d70","order_by":8,"name":"Jinkuan Wei","email":"","orcid":"","institution":"Kunming University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jinkuan","middleName":"","lastName":"Wei","suffix":""}],"badges":[],"createdAt":"2025-01-21 02:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5869428/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5869428/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80294383,"identity":"184a6d59-b6ab-4d70-ac98-6f7828cbdc2c","added_by":"auto","created_at":"2025-04-10 08:24:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":102369,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of the experimental design. The pilocarpine/Control rat model was established on day -28; VNS electrodes and infusion cannulas were implanted on day -14; VNS /Sham VNS was administered on day 0 and continued for 14 days, with behavioral testing for CFC or LTP conducted from day 15 to day 17. In the\u003cem\u003e PILO saline + VNS\u003c/em\u003e or \u003cem\u003ePILO PR + VNS\u003c/em\u003e groups, saline or propranolol (PR) was injected into the hippocampus before each VNS session. Abbreviations: VNS, vagus nerve stimulation; PILO, pilocarpine; PR, propranolol; CFC, contextual fear conditioning; LTP, long-term potentiation.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5869428/v1/67b4dbb3ab1304057e3d7b14.png"},{"id":80294384,"identity":"45270163-f8fb-4f46-9706-5d644a83ab1e","added_by":"auto","created_at":"2025-04-10 08:24:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":141931,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of VNS on CFC memory. \u0026nbsp;(a) Schematic diagram of the CFC test. (b) The training curves in the conditioning period indicate thatall three groups of rats learned the relationship between electrical shock and the training environment. (c) The freezing time of the \u003cem\u003ePILO\u003c/em\u003e group was significantly lower \u003cem\u003ethan that of the control\u003c/em\u003e group, whereas that ofthe \u003cem\u003ePILO+VNS\u003c/em\u003e group was significantly greater than that ofthe\u003cem\u003e PILO\u003c/em\u003e group. The data are presented as the means ± SEMs, with n=6 for each group. *p \u0026lt; 0.05, **p \u0026lt; 0.01 by ANOVA.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5869428/v1/587578f7a980ff7545dc6b3c.png"},{"id":80294389,"identity":"c2e6c4b7-3934-4059-949d-6808419be6d6","added_by":"auto","created_at":"2025-04-10 08:24:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":177908,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of VNS on hippocampal LTP. (a) Schematic diagram of the hippocampal LTP induction experiment. (b) Time course of changes in hippocampal fEPSP amplitudes in the three groups. (c) The increase in fEPSP amplitude (LTP) in the \u003cem\u003ePILO \u003c/em\u003egroup was significantly lower than that in the \u003cem\u003econtrol\u003c/em\u003e group, whereas the \u003cem\u003ePILO+VNS\u003c/em\u003e group presented a significantly greater increase than the \u003cem\u003ePILO\u003c/em\u003egroup did. The dataare presented as the means ± SEMs, with n=6 for each group. *p \u0026lt; 0.05, **p \u0026lt; 0.01 by ANOVA.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5869428/v1/9796513322981b6c5519863b.png"},{"id":80294388,"identity":"bdd6277e-c35e-4cb7-aacb-da8afdf9cbbc","added_by":"auto","created_at":"2025-04-10 08:24:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":174624,"visible":true,"origin":"","legend":"\u003cp\u003eVNS affects the hippocampal NE/β-AR system and downstream pathways\u003c/p\u003e\n\u003cp\u003e(a) ELISA measurements revealedincreased NE levels in the \u003cem\u003ePILO+VNS \u003c/em\u003egroup compared with the\u003cem\u003e PILO group\u003c/em\u003e, with no difference between the \u003cem\u003eControl \u003c/em\u003eand\u003cem\u003ePILO\u003c/em\u003e groups. (b) β-AR protein expression decreased in the \u003cem\u003ePILO \u003c/em\u003egroup but increased in the \u003cem\u003ePILO+VNS \u003c/em\u003egroup relative to the \u003cem\u003ePILO group\u003c/em\u003e. (c) Similar patterns were observed for PKA protein expression. (d) CaMKII protein expression did not differ across groups, although VNS tended to increase CaMKII expression in \u003cem\u003ePILO\u003c/em\u003e rats. The data are presented as the means ± SEMs (n=6 per group). *p \u0026lt; 0.05, **p \u0026lt; 0.01; ANOVA for β-AR, PKA, and CaMKII; Student’s t test for NE.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5869428/v1/c0c58b68283d38ad49c58031.png"},{"id":80295118,"identity":"b1a4f0bd-de58-4b99-8fe4-32d72e613648","added_by":"auto","created_at":"2025-04-10 08:32:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":150543,"visible":true,"origin":"","legend":"\u003cp\u003eHippocampal β-AR antagonists reduce the memory- and LTP-enhancing effects of VNS\u003c/p\u003e\n\u003cp\u003e(a) Schematic of the administration site. (b) No difference in learning curves between the PILO saline +VNS and PILO PR+VNS groups during testing. (c) Compared with the PILO saline + VNS group, the PILO PR+VNS group presented reduced contextual fear memory. (d) Time course of hippocampal fEPSP amplitude changes. (e) Lower LTP levels in the PILO PR+VNS group than in the PILO saline +VNS group. (f, h, i) Decreased β-AR, PKA, and CaMKII protein levels in the PILO+PR+VNS group. The data are presented as the means ± SEMs (n=6 per group). *p \u0026lt; 0.05, **p \u0026lt; 0.01; Student’s t test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5869428/v1/7a2bdeae43421ea1c23075c8.png"},{"id":80294399,"identity":"fc9bc6dd-61d1-4907-b59e-6a702fff26a9","added_by":"auto","created_at":"2025-04-10 08:24:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":149647,"visible":true,"origin":"","legend":"\u003cp\u003eVNS enhances memory and LTP by increasing the activity of the hippocampal NE/β-AR signaling pathway. The VNS transmits stimulation information to the nucleus solitary tract (NST) via the vagus nerve afferent fibers and then ascends to the locus coeruleus (LC). The LC, the primary nucleus in the brain for NE production, projects to the hippocampus. VNS promotes the release of NE neurotransmitters in the hippocampus and increases the activity of the hippocampal NE/β-AR signaling pathway, thereby increasing CFC memory and hippocampal LTP.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5869428/v1/f62c17ac8591994b8cd669cb.png"},{"id":86024215,"identity":"00c382b8-569e-43f0-a99b-86b5f2983445","added_by":"auto","created_at":"2025-07-04 12:39:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1605945,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5869428/v1/e1b9d849-7fff-4b56-a27b-bae1d4cdc337.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Vagus Nerve Stimulation Enhances Memory and Synaptic Plasticity via the Hippocampal NE/β-AR Signaling Pathway in Pilocarpine Model Rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe vagus nerve, classified as the tenth cranial nerve, is the longest and most extensively distributed cranial nerve and comprises sensory, motor, and parasympathetic fibers [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It plays a significant role in various physiological processes. Vagus nerve stimulation (VNS) is a neuromodulation technique that employs an implantable pulse generator to deliver intermittent electrical stimuli to the vagus nerve, thereby transmitting signals to the brain and inducing broad changes in the nervous system. VNS has received US Food and Drug Administration approval for the adjunctive treatment of drug-resistant epilepsy [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], refractory major depressive disorder [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and poststroke recovery [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Clinically, VNS has been shown to alleviate disease symptoms and has a significant positive impact on memory over the long term [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Despite these findings, the mechanisms through which VNS enhances memory are not fully understood.\u003c/p\u003e \u003cp\u003eThe hippocampus is pivotal in memory formation, and our previous research revealed severe hippocampal neuron loss in pilocarpine model rats [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], a finding supported by other studies that utilized this model to investigate memory impairments [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Contextual fear conditioning (CFC) is a well-established behavioral paradigm in rodents, where subjects learn to associate a neutral context with an aversive stimulus, thus exhibiting fear responses to contexts indicating danger[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Synaptic plasticity, which involves the activity-dependent modification of synaptic strength or efficacy within the nervous system, underlies learning and memory processes[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Long-term potentiation (LTP), characterized by enhanced postsynaptic responses following stimulation, is a form of hippocampal synaptic plasticity associated with synaptic strengthening and improved learning and memory[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, in this study, we investigated whether VNS could improve CFC performance and hippocampal LTP levels in pilocarpine-induced memory-impaired rats.\u003c/p\u003e \u003cp\u003eFrom a functional neuroanatomical perspective, VNS transmits stimulation signals to the nucleus tractus solitarius (NTS) via vagus nerve afferents and then projects to the locus coeruleus (LC)[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], the principal norepinephrine (NE)-producing nucleus in the brain. The LC projects widely to other brain regions, including the cerebral cortex, dorsal raphe nucleus, hippocampus, amygdala, and cerebellum, where it modulates functions such as learning, memory, attention, and cognition[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. While studies have indicated that VNS can promote NE release in the hippocampus [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], it remains unclear whether VNS enhances memory by increasing NE neurotransmission and which receptor signaling pathways are involved.\u003c/p\u003e \u003cp\u003eAdrenergic receptors (ARs), a class of G protein-coupled receptors that mediate the effects of catecholamine neurotransmitters, include α-AR and β-AR receptors, with β-AR receptors being highly expressed in the rat hippocampus[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. β-AR and its downstream pathways have been shown to play critical roles in memory and LTP formation[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. NE, which acts on β-AR, stimulates an increase in cyclic adenosine monophosphate (cAMP) synthesis; cAMP activates protein kinase A (PKA) and indirectly activates extracellular signal-regulated kinase (ERK)[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. PKA and ERK are important for long-term memory formation and synaptic plasticity across species [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. One of the direct downstream signaling targets of β-AR is inhibitor-1 (Inh-1), which, when phosphorylated by PKA, inhibits the expression of protein phosphatase 1 (PP1). PP1 is responsible for dephosphorylating proteins critical for LTP induction, including CaMKII, NMDARs, and AMPARs. Thus, β-AR activation leads to the inhibition of PP1, enhancing the expression of synaptic plasticity-related proteins[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In this study, we aimed to determine whether VNS could improve memory and hippocampal synaptic plasticity in pilocarpine-induced memory-impaired rats and whether this effect is mediated by the hippocampal NE/β-AR signaling pathway. We specifically examined improvements in CFC performance and hippocampal LTP levels, as well as changes in the protein expression levels of NE, β-AR, and downstream signaling pathways (protein kinase A, CaMKII), following two weeks of VNS treatment. Additionally, we administered the β-AR antagonist propranolol (PR) to the hippocampus to determine the role of the β-AR receptor in this process.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eSixty healthy adult male Sprague\u0026ndash;Dawley rats, 6 weeks old and weighing 250\u0026ndash;300 g, were obtained from the Experimental Animal Center of Kunming Medical University, which holds Production License SCXK (Dian) K2020\u0026ndash;0004. All the rats were housed in a controlled environment of 20\u0026ndash;25\u0026deg;C, with a humidity of 55\u0026thinsp;\u0026plusmn;\u0026thinsp;2%, and in quiet states maintained under a 12 h/12 h light/dark cycle with \u003cem\u003ead libitum\u003c/em\u003e access to food and water. The rats were handled daily by experimenters for 1 week to reduce the stress response. A schematic of the experimental procedures is provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePilocarpine Rat Model\u003c/h2\u003e \u003cp\u003eThe epileptic rat model was established via the lithium‒pilocarpine method as described in our previous research[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Lithium chloride (127 mg/kg, Sigma, USA) was initially administered intraperitoneally to the rats. A single dose of pilocarpine (350 mg/kg; Meilun Biotechnology Co., Jiangsu, China) was subsequently intraperitoneally injected to induce neural death 15\u0026ndash;19 hours after lithium chloride administration. To counteract peripheral cholinergic effects, methylscopolamine (1 mg/kg; Yuanye Biotechnology Co., Shanghai, China) was intraperitoneally injected 30 minutes prior to pilocarpine application. Status epilepticus was terminated after 90 minutes using chloral hydrate (30 mg/kg, intraperitoneal; Hengrui Medical Co., Jiangsu, China). The control rats received injections of methylscopolamine and saline.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSurgery for Electrode and Infusion Cannula Implantation\u003c/h3\u003e\n\u003cp\u003eThe implantation of VNS electrodes was conducted one week after the establishment of the PILO model. The rats were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneal injection; Hengrui Medical Co., Jiangsu, China), and the depth of anesthesia was monitored via the toe-pinch reflex test. Following successful anesthesia, VNS electrodes were wrapped around the left vagus nerve in the neck and secured with nonabsorbable sutures (refer to our previous literature[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] for specific methods). Next, fixed fusion cannulas were implanted bilaterally in the hippocampus. According to the rat brain atlas by Paxinos and Watson, five holes (1.2 mm diameter) were drilled in the skull, with three for fixation and two for embedding drug infusion cannulas (RWD Co., Ltd., Shenzhen, China; outer diameter, 0.5 mm; inner diameter, 0.40 mm) into the CA1 region of the hippocampus (3.3 mm posterior to the bregma and 2 mm lateral to the midline). A solid stainless-steel wire was inserted into the drug infusion cannula for sealing. The final step involved securing the VNS electrode and drug infusion cannulas with dental acrylic. Penicillin was administered intramuscularly for three days postoperatively to prevent infection.\u003c/p\u003e\n\u003ch3\u003eVNS Protocol\u003c/h3\u003e\n\u003cp\u003eVNS was initiated three weeks after the establishment of the PILO model and continued for two weeks, with a total daily stimulation time of 120 minutes. VNS was divided into four 30-minute blocks with a 2-hour interval between each block. VNS was performed via a programmable pulse stimulator (Model Master8, A.M.P.I., Israel), with parameters set for bipolar balanced square waves (pulse width of 250 \u0026micro;s; frequency of 30 Hz; current intensity of 1 mA). Stimulation output was verified via a digital oscilloscope and by observing the cough reflex in the rats. The current of the electric pulse was set to 1 mA after a 5-minute adaptation period at 0.7 mA, and stimulation proceeded for two weeks with the rats in a free-moving state.\u003c/p\u003e\n\u003ch3\u003eDrug Infusion\u003c/h3\u003e\n\u003cp\u003ePrior to each VNS session, the rats in the \u003cem\u003ePILO\u0026thinsp;+\u0026thinsp;PR\u0026thinsp;+\u0026thinsp;VNS\u003c/em\u003e group were administered the β-AR antagonist propranolol (PR, 5 \u0026micro;g/\u0026micro;L), following the methodology of previous research [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The rats were induced with 2% isoflurane anesthesia (Hebei Nine Sent Pharmaceutical Co., Ltd., Hebei, China) before drug infusion. A stainless-steel drug administration tube (outer diameter: 0.3 mm, with the tip extending 1.0 mm beyond the cannula, resulting in a drug injection site 2.7 mm below the skull surface) was carefully inserted at a rate of 0.5 \u0026micro;L/min, with 1 \u0026micro;L of propranolol being injected into each hippocampal region. The administration tube was left in place within the cannula for an additional minute postinjection to ensure complete drug delivery. The control groups received an equivalent volume of saline.\u003c/p\u003e\n\u003ch3\u003eContextual fear conditioning\u003c/h3\u003e\n\u003cp\u003eWhen fearful, rodents exhibit a freezing response as a defensive posture. In our experiments, test subjects received a tone cue (conditional stimulus) followed by an electric shock (unconditioned stimulus), a process termed conditioning. Following this training, the animals were tested with a tone cue or context to assess memory retention, which is known as testing. Rodents typically display a conditioned fear response, evident as freezing behavior, in response to both the associated context and the same tone cue presented in a different context. The duration of freezing in these contexts reflects an animal\u0026rsquo;s memory capacity.\u003c/p\u003e \u003cp\u003eContextual fear conditioning was conducted using a fear conditioning chamber (DL Naturegene Life Science, Inc., USA). The experimental protocol spanned three days and was performed at the same time each day, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. Day 1: Habituation. The rats were removed from their home cages and placed in the chamber for free exploration, adapting to two distinct environments (horizontal and vertical striped scenes) for 10 minutes each. Day 2: Training. The rats were placed in the chamber against one of the backgrounds for a total of 820 seconds and received five electric shocks (1-second duration, 0.78 mA intensity) at intervals of 140 seconds per session (19 seconds of sound followed by 1 second of electric stimulus). Day 3: Contextual memory test. Using the same background as the training day, the freezing behavior of the rats was observed and recorded for 10 minutes in the absence of electric shock stimuli.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLTP\u003c/h2\u003e \u003cp\u003eArtificial cerebrospinal fluid was prepared and continuously oxygenated prior to the procedure. The rats were decapitated to collect brain tissue, which was subsequently sliced into 340 nm-thick hippocampal sections via a vibratome. Following a 1-hour incubation at a constant temperature, the slices were transferred to a recording chamber. The CA1 and CA3 regions of the hippocampus were identified via a multimicro manipulator system (MPC200, Sutter Corporation). Recording electrodes, pulled from borosilicate glass capillaries via a glass electrode puller (Sutter Corporation) and filled with 3 M NaCl electrolyte solution, were carefully lowered into the CA1 region, while stimulating electrodes, fashioned from twisted tungsten wire, were inserted into the CA3 region. A stimulator was employed to deliver continuous single biphasic pulse stimulation, and a multichannel amplifier (20 kHz, 700B, Axon Corporation) was utilized for recording purposes. The electrode positions were fine-tuned until field excitatory postsynaptic potentials (fEPSPs) were reliably evoked. The stimulation current was gradually increased to a level that elicited an fEPSP amplitude representing 50% of the maximum response (approximately 0.2\u0026ndash;0.6 mA), which was then used as the baseline for synaptic transmission. Baseline responses were recorded at 30-second intervals for 15 minutes, after which high-frequency stimulation (HFS: 100 Hz, 10 trains of 200 ms duration each, repeated 3 times with a 10-second interval) was applied to induce LTP. After induction, the fEPSPs were monitored and recorded every 30 seconds for an additional 50 minutes. The fEPSP amplitudes following HFS were normalized to the pre-HFS baseline average. Electrophysiological data acquisition was facilitated by MultiClamp 700B Commander software and Clampex 10.7, with subsequent analysis and plotting conducted via GraphPad Prism.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular biology experiments\u003c/h3\u003e\n\u003cp\u003eThe rats were anesthetized with an overdose of pentobarbital sodium (60 mg/kg, i.p.) and then subjected to cardiac perfusion. After the brain was removed, one side of the hippocampal tissue was rapidly frozen in liquid nitrogen and stored at -80\u0026deg;C for protein immunoblotting and NE content determination. The other side of the brain hemisphere was fixed in 4% paraformaldehyde for immunofluorescence staining.\u003c/p\u003e\n\u003ch3\u003ea. Western blot (WB)\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003ea. Western blot (WB)\u003c/div\u003e \u003cp\u003eFor the WB experiments, hippocampal tissues were homogenized in RIPA lysis buffer containing a protease inhibitor cocktail to extract proteins. The supernatant was collected and centrifuged at 13,000 rpm at 4\u0026deg;C for 20 minutes. Protein concentrations were determined via the bicinchoninic acid (BCA) assay. Equal amounts of protein samples were separated on 5%, 12%, or 15% SDS‒PAGE gels and transferred onto PVDF membranes. The membranes were blocked in 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween (BSA-TBST) for 1 hour at room temperature and incubated with the following primary antibodies overnight at 4\u0026deg;C: β-AR (1:1000, ab182136, Abcam), PKA (1:1000, ADI-KAS-PK017-D, Enzo Life Sciences), and CaMKII (1:1000, sc-5306, Santa Cruz Biotechnology). After three washes with TBST buffer, the membranes were incubated with secondary antibody solutions (HRP goat anti-rabbit 1:1000, Proteintech; HRP goat anti-mouse 1:5000, Proteintech) for 2 hours at room temperature. The signals were detected via an Image Quant LAS4000 image analyzer (GE Healthcare, Buckinghamshire, UK), and the band intensities were quantified via Quantity One software v.4.6.2 (Bio-Rad, Hercules, CA, USA).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eb. NE Content Detection\u003c/h2\u003e \u003cp\u003eNorepinephrine content was measured via a Norepinephrine ELISA Kit (EK2432, SAB). Hippocampal tissues were minced, weighed, mixed with 1x PBS at a ratio of 10 ml/g, homogenized at 45 Hz for 120 seconds and centrifuged at 5000 rpm for 5 minutes at 4\u0026deg;C. Standards and samples (50 \u0026micro;l each) were added to the wells, followed by the addition of 50 \u0026micro;l of detection solution A and incubation at room temperature for 1 hour. After three washes with 300 \u0026micro;l of 1x PBS, 100 \u0026micro;l of detection B was added, and the mixture was incubated at room temperature for 45 minutes. In the dark, 90 \u0026micro;l of substrate solution was added to each well and incubated at room temperature for 10\u0026ndash;20 minutes after three washes. Finally, 50 \u0026micro;l of stop solution was added, and the optical density of each well was measured at a wavelength of 450 nm via an enzyme reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll the statistical analyses were performed via SPSS software version 26.0 (IBM Corporation, Armonk, NY, USA). The data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors of the means (SEMs). The freezing time during the CFC test, fEPSP amplitudes, NE content and WB results were evaluated via one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test for multiple comparisons or Student\u0026rsquo;s t test where appropriate. The freezing time during the CFC training period was analyzed via two-way repeated-measures ANOVA to assess the main effects and interactions, followed by Tukey\u0026rsquo;s post hoc test for pairwise comparisons. A p value of less than 0.05 was considered to indicate statistical significance. Graphs and additional data visualization were generated via GraphPad Prism software version 8.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e1.\u0026nbsp; \u0026nbsp;\u0026nbsp;VNS improved impaired memory and hippocampal LTP in PILO model rats\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 2a, the contextual fear conditioning (CFC) test includes three sessions: habituation, conditioning and testing. The freezing time of the experimental animals during the testing period was used to characterize the memory of the subjects. As shown in the line graph in Figure 2b, for the freezing time in the conditioning period, two-way ANOVA with repeated measures revealed significant effects of trials on the training curve (F(3.643, 54.64)=35.6, p\u0026lt;0.0001), whereas no significant group \u0026times; trial interactions were detected (F(10, 75)=1.108, p=0.3677), which indicated that all three groups of rats learned the relationship between electrical shock and the training environment. As shown in the bar graph in Figure 2c, \u0026nbsp;for the freezing time in the testing period, there was a significant difference among the three groups (ANOVA, p=0.0162); the freezing time of the \u003cem\u003ePILO\u0026nbsp;\u003c/em\u003egroup was significantly lower \u003cem\u003ethan that of the control\u003c/em\u003e group (p=0.0136), whereas that of the \u003cem\u003ePILO+VNS\u003c/em\u003e group was significantly greater than that of the \u003cem\u003ePILO\u003c/em\u003e group (p=0.0395), suggesting that the memory of the \u003cem\u003ePILO\u003c/em\u003e group effectively improved in the VNS group.\u003c/p\u003e\n\u003cp\u003eLTP induction is shown in Figure 3a. The stimulation electrodes were inserted into the CA3 region, the recording electrodes were inserted into the CA1 region, the baseline fEPSP was recorded for 15 minutes, HFS was used to induce LTP, the fEPSP was recorded for 50 minutes after stimulation, and the ratio of the amplitude of the fEPSP to the baseline value within 20\u0026ndash;50 minutes after HFS was used to measure LTP (Figure 3b). After HFS, there was a significant difference in the increase in fEPSP \u003cem\u003eamplitude among the control\u0026nbsp;\u003c/em\u003e(n=6)\u003cem\u003e, PILO\u0026nbsp;\u003c/em\u003e(n=6) and \u003cem\u003ePILO+VNS\u003c/em\u003e (n=6) groups (ANOVA, p=0.029), and the increase in fEPSP amplitude in the \u003cem\u003ePILO\u003c/em\u003e group was significantly lower than that in the \u003cem\u003econtrol\u003c/em\u003e group (p=0.0499), whereas that in the \u003cem\u003ePILO+VNS\u003c/em\u003e group was significantly greater than that in the\u003cem\u003e\u0026nbsp;PILO\u003c/em\u003e group (p=0.0089), indicating that VNS effectively improved the impaired hippocampal LTP of the PILO model rats (Figure 3c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.\u0026nbsp; \u0026nbsp;\u0026nbsp;VNS enhanced the function of the NE/\u0026beta;-AR system and its downstream pathways in the hippocampus of PILO rats\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ea.\u0026nbsp; \u0026nbsp;\u0026nbsp;VNS promoted NE neurotransmitter release in the hippocampus of PILO rats\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe NE content in the hippocampal tissues was detected via a general noradrenaline ELISA kit. Significant differences were not detected among the\u0026nbsp;\u003cem\u003econtrol\u0026nbsp;\u003c/em\u003e(n=6)\u003cem\u003e, PILO\u0026nbsp;\u003c/em\u003e(n=6) and \u003cem\u003ePILO+VNS\u003c/em\u003e (n=6) groups (ANOVA, p\u0026gt;0.05), but the NE content in the hippocampus of the \u003cem\u003ePILO+VNS\u003c/em\u003e group was greater than that in the \u003cem\u003ePILO\u0026nbsp;\u003c/em\u003egroup (t=3.104, p=0.0267) according to Student\u0026apos;s t test, suggesting that VNS promoted NE neurotransmitter release in the hippocampal region of PILO rats (Figure 4a).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb. \u003cem\u003eVNS activated \u0026beta;-AR and downstream signaling pathways in the hippocampus of PILO rats\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protein expression levels of \u0026beta;-AR, PKA, and CaMKII were quantified via Western blot analysis. One-way ANOVA revealed significant differences in \u0026beta;-AR protein expression among the\u003cem\u003e\u0026nbsp;control\u0026nbsp;\u003c/em\u003e(n=6),\u003cem\u003e\u0026nbsp;PILO\u0026nbsp;\u003c/em\u003e(n=6), and \u003cem\u003ePILO +VNS\u003c/em\u003e (n=6) groups (p\u0026lt;0.0001). Compared \u003cem\u003ewith the control\u003c/em\u003e group, the \u003cem\u003ePILO\u003c/em\u003e group presented a significant decrease in expression (p=0.0001), whereas the \u003cem\u003ePILO+VNS\u003c/em\u003e group presented a significant increase in expression compared with the \u003cem\u003ePILO\u0026nbsp;\u003c/em\u003egroup (p\u0026lt;0.0001), suggesting that VNS increased the expression of \u0026beta;-AR in the hippocampal region of PILO rats (Figure 4b). There were also significant differences in the PKA protein expression levels among the three groups (p\u0026lt;0.0001, one-way ANOVA). Compared with that in the control group, the protein expression level of PKA in the \u003cem\u003ePILO\u003c/em\u003e group was significantly lower (p=0.0317), whereas that in the \u003cem\u003ePILO+VNS\u0026nbsp;\u003c/em\u003egroup was significantly greater (p\u0026lt;0.0001), indicating that VNS upregulated PKA expression in the hippocampal region of PILO rats (Figure 4c). As shown in Figure 4d, there was no significant difference in the protein expression of hippocampal CaMKII among the three groups, but VNS tended to increase the protein expression of CaMKII in the hippocampal area of PIL rats.\u003c/p\u003e\n\u003cp\u003eTherefore, VNS increased hippocampal NE neurotransmitter release and the expression of \u0026beta;-AR and its downstream pathway proteins PKA and CaMKII and enhanced impaired hippocampal LTP and contextual fear conditioning memory in PILO rats; this effect of VNS could be blocked by a \u0026beta;-AR antagonist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3. \u0026nbsp; \u0026nbsp;Blocking hippocampal \u0026beta;-AR function inhibited the enhancing effect of VNS on memory and LTP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs indicated in Figure 5a, prior to each VNS treatment, the noncompetitive \u0026beta;-AR antagonist PR was microinjected bilaterally into the CA1 region of the hippocampus in PILO rats to block the action of \u0026beta;-AR.\u003c/p\u003e\n\u003cp\u003eIn the CFC test, the learning curves of the rats in the \u003cem\u003ePILO saline + VNS\u003c/em\u003e group (n=6) and the \u003cem\u003ePILO PR+VNS\u0026nbsp;\u003c/em\u003egroup (n=6) were not significantly different (Figure 5b). Compared with the PILO saline + VNS group, the PILO PR+VNS group presented a significant decrease in contextual fear memory (t=4.799, p=0.0007), suggesting that the \u0026beta;-AR antagonist blocked the ability of VNS to improve memory in the PILO rats (Figure 5c).\u003c/p\u003e\n\u003cp\u003eIn the LTP induction experiment, the level of LTP in the \u003cem\u003ePILO PR+VNS\u0026nbsp;\u003c/em\u003egroup was significantly lower than that in the \u003cem\u003ePILO saline +VNS\u003c/em\u003e group (t=20.38, p\u0026lt;0.0001), indicating that the \u0026beta;-AR antagonist blocked the effect of VNS on enhancing hippocampal neuroplasticity in the PILO rats (Figure 5d, e).\u003c/p\u003e\n\u003cp\u003eCompared with that in the PILO saline +VNS group, the NE content in the hippocampal tissue of the rats in the \u003cem\u003ePILO PR + VNS\u003c/em\u003e group was significantly lower (t=2.966, p=0.0141), suggesting that \u0026beta;-AR antagonists blocked the ability of VNS to promote NE neurotransmitter release in the hippocampus of the PILO rats (Figure 5f).\u003c/p\u003e\n\u003cp\u003eCompared with those in the \u003cem\u003ePILO saline + VNS\u003c/em\u003e group, the \u0026beta;-AR, PKA and CaMKII protein levels in the hippocampal tissue of the rats in the\u003cem\u003e\u0026nbsp;PILO+PR+VNS\u003c/em\u003e group were significantly lower (t=2.737, 2.412 and 2.329, respectively; p=0.021, 0.0366 and 0.0421, respectively). These results suggest that \u0026beta;-AR antagonists block the enhancement of hippocampal \u0026beta;-AR receptors and downstream signaling pathways by VNS in PILO rats (Figure 5g-i).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eVNS has been approved for the treatment of drug-resistant epilepsy, severe depression, and poststroke rehabilitation. During its clinical application in patients, VNS not only improved epilepsy or depression symptoms but also had a positive effect on memory[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In experimental animals, researchers have also reported that VNS can enhance the memory of normal animals[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Furthermore, studies on noninvasive vagus nerve electrical stimulation have shown that it can improve attention, working memory, memory consolidation, decision-making, and other cognitive functions[\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, some researchers have proposed that noninvasive VNS could be used for the early treatment and prevention of Alzheimer's disease [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough both invasive and noninvasive VNS have been shown to effectively enhance memory in clinical and experimental animal studies, the specific mechanisms involved remain unclear. Selecting an appropriate memory impairment animal model is crucial for elucidating how VNS improves memory. We used the pilocarpine rat model, which has been shown to exhibit severe apoptosis of hippocampal neurons, providing a pathological basis for memory impairment[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]; many drugs have been studied in this model for their effects on memory [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. To reveal the long-term effects of VNS on memory, we delivered continuous stimulation for 14 days after the pilocarpine rat model was established, stimulated for 2 hours each day, and then assessed the rats\u0026rsquo; memory, neurotransmitter changes, and receptor molecular pathways.\u003c/p\u003e \u003cp\u003eThe hippocampus plays a crucial role in establishing contextual fear memory [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Using the CFC test, we assessed memory in three groups of rats and found that all groups learned the associations between electrical shock and the training environment. During the testing period, pilocarpine-treated rats exhibited significant memory impairment, which was significantly improved after two weeks of continuous VNS treatment, indicating that VNS can improve impaired memory in pilocarpine-treated rats. Moreover, this effect could be blocked by β-AR antagonists, suggesting a key role for β-AR in VNS-mediated memory improvement.\u003c/p\u003e \u003cp\u003eAdditionally, we detected a significant decrease in LTP in the CA1 region of the hippocampus in pilocarpine-treated rats compared with control rats. This finding is consistent with previous reports, which have documented impaired LTP during both the latent[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and chronic phases[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] of pilocarpine-treated animals. One study that monitored hippocampal synaptic plasticity changes over one week post- pilocarpine treatment found no significant alterations in LTP on the 1st day, but a marked reduction on the 3rd day, with further impairment observed by day 7 post-treatment[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The mechanisms underlying this LTP impairment likely involve disrupted neuron-glial interactions. Specifically, astrogliosis in the hippocampus leads to morphological changes in astrocytes, reducing the number of astrocytic leaflets near synapses[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This disrupts the local supply of D-serine to NMDARs, enhancing receptor desensitization[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. D-serine application restored LTP and enhanced NMDAR-mediated currents in pilocarpine treated rats[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Additionally, impaired glutamate clearance mechanisms may also contribute to LTP deficits. Under normal conditions, high-affinity glial and neuronal excitatory amino acid transporters (EAATs) effectively clear glutamate released during HFS[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, in epileptic tissue, glutamate clearance is further compromised due to alterations in EAAT functional activity and decreased astrocytic surface area at glutamatergic synapses[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. This impaired clearance may lead to the accumulation of glutamate in the synaptic cleft, thereby exacerbating NMDAR desensitization and LTP impairment.\u003c/p\u003e \u003cp\u003eVNS treatment effectively enhanced LTP in the CA1 region of pilocarpine-treated rats, confirming that VNS can improve hippocampal neuroplasticity and memory function. This result is consistent with previous studies on normal rats under freely moving conditions[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Electrophysiological and neurochemical data suggest that the enhanced effect of VNS on LTP may involve CNS noradrenergic systems. The initial increase in NE induced by VNS likely triggers changes in the localization and expression of pre- and post-synaptic receptors and proteins that are critical for maintaining LTP[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Activation of β-AR by NE facilitates the induction of LTP across all major regions of the hippocampal formation[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This effect is mediated through the activation of voltage-sensitive calcium channels, including NMDA receptor channels[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, we found that the enhancing effect of VNS on LTP could be blocked by NE β-AR antagonists. Previous studies have reported that the enhancing effect of VNS on LTP in the CA3 region of the hippocampus in normal rats is dependent on the LC and hippocampal β-AR[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], which is consistent with our findings in the CA1 region of pilocarpine-treated rats. However, an unexpected result in our study was that the amplitudes of fEPSPs in β-AR antagonist-treated rats tended to decrease below baseline following HFS. Previous research in normal rats has shown that acute VNS can enhance synaptic transmission in the PP-CA3 pathway[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This enhancement can be blocked by pre-treatment with β-AR antagonists, which can even reduce synaptic transmission below baseline levels. Notably, β-AR antagonists alone do not affect PP-CA3 synaptic transmission, suggesting that when β-ARs are saturated by antagonists, VNS may activate mechanisms that reduce synaptic strength.There is a complex relationship between β-ARs and synaptic plasticity. NE or β-AR agonists (such as isoproterenol, Iso) can enhance LTP induced by HFS[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Moreover, β-adrenergic stimulation with Iso enables LTP induction even after low-frequency stimuli (1\u0026ndash;5 Hz for 15 min) that would otherwise induce LTD[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. We speculate that in the hippocampus of pilocarpine-treated rats, impaired glutamate clearance at the synaptic cleft may lead to desensitization of NMDARs. Additionally, β-AR antagonists may further reduce the expression of NMDARs and AMPARs on the postsynaptic membrane. Under these conditions, HFS may induce glutamate excitotoxicity, causing damage to synapses and reducing synaptic transmission efficiency, or may trigger weak postsynaptic calcium influx, initiating LTD mechanisms.\u003c/p\u003e \u003cp\u003eFrom a functional neuroanatomical perspective, VNS transmits stimulation information to the nucleus tractus solitarius (NTS) through the afferent fibers of the vagus nerve, which project to the locus coeruleus (LC), the primary brain nucleus producing NE, projecting diffusely to other brain areas, such as the cerebral cortex, dorsal raphe nucleus, hippocampus, amygdala, and cerebellum[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Robrecht Raedt et al. reported an increase in hippocampal NE content following 240 minutes of acute intermittent VNS (frequency\u0026thinsp;=\u0026thinsp;30 Hz; intensity\u0026thinsp;=\u0026thinsp;1 mA; pulse width\u0026thinsp;=\u0026thinsp;250 \u0026micro;s; duty cycle\u0026thinsp;=\u0026thinsp;7 s ON-18 s OFF)[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Our findings indicate that two weeks of continuous VNS can effectively increase hippocampal NE levels in pilocarpine-treated rats. Unlike the study by Robrecht Raedt, which compared the hippocampal NE content before and after VNS in pilocarpine-treated rats without contrasting it with that of controls, our experiment revealed no significant difference in the hippocampal NE content between pilocarpine-treated and control rats[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. We hypothesize that the LC in pilocarpine-treated rats remains undamaged, maintaining consistent NE neurotransmitter release to the hippocampus, yet VNS can activate NE neurons in the LC, thereby increasing the hippocampal NE content.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows, the mechanism by which increased hippocampal NE content ameliorates contextual fear memory and LTP involves VNS-induced upregulation of hippocampal β-AR and its downstream PKA and CaMKII proteins, which are integral to hippocampal LTP enhancement. Blocking β-AR reversed the VNS-induced improvements in contextual fear memory and hippocampal LTP in pilocarpine-treated rats, suggesting that VNS enhances memory and LTP in pilocarpine-treated rats through the hippocampal NE/β-AR signaling pathway.\u003c/p\u003e \u003cp\u003eOur findings suggest that VNS rescues pilocarpine induced PKA suppression via the NE/β-AR pathway. Elevated PKA activity may enhance AMPA receptor function through GluA1-Ser845 phosphorylation[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Moreover, this effect could be blocked by β-AR antagonists, suggesting a key role for β-AR/PKA pathway in VNS-mediated memory improvement. While PP1 and downstream targets (e.g., CaMKII, NMDAR/AMPAR subunits) were not directly examined here, future studies should evaluate these pathways to fully elucidate the molecular basis of VNS induced synaptic recovery.\u003c/p\u003e \u003cp\u003eInterestingly, despite the lack of a significant difference in the hippocampal NE content between pilocarpine-treated and control rats, β-AR expression was diminished in pilocarpine-treated rats. Therefore, why does hippocampal β-AR expression decrease in PILO rats? Our previous research indicated that VNS can protect CA1 region neurons from pilocarpine-induced damage. Further investigations are needed to determine whether VNS can mitigate inflammatory responses or apoptosis in the hippocampus through the NE/β-AR signaling pathway, thereby improving memory in pilocarpine-treated rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eVNS ameliorated impaired memory and hippocampal LTP in pilocarpine-treated rats, potentially through enhancing the activity of the hippocampal NE/β-AR signaling pathway.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eVNS, vagus nerve stimulation; NE, norepinephrine; β-AR, β-adrenergic receptor; LTP, long-term potentiation; fEPSP, field excitatory postsynaptic potential; CFC, contextual fear conditioning; LC, locus coeruleus; PILO, pilocarpine.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by several grants: the National Natural Science Foundation of China [Grant Numbers 82360270 and 32260197]; the Key Project of the Natural Science Foundation of Yunnan Province [Grant Number 202401AS070040]; the Yunnan Provincial Education Department Scientific Research Fund [Grant Number 2023Y0636]; the Yunnan Provincial Department of Science and Technology-Kunming Medical University Joint Program on Basic Research [Grant Number 202401AY070001-236]; the First-Class Discipline Team of Kunming Medical University [Grant Number 2024XKTDPY02]; and the Yunnan Revitalization Talent Support Program for Hualin Yu and Jinghui Li. Additional support was provided by the NHC Key Lab of Drug Addiction Medicine at Kunming Medical University through its Open Projects Fund [Project Number KN202424].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the ethical standards of the institution or practice at which the studies were performed. All procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Kunming Medical University (IACUC, SYXK(Dian) K2020-0006).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs this study involved animals, consent to participate is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of this study have been published with the consent of all authors and the responsible authorities at the institution where the work was conducted.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBerthoud, H.R. and W.L. 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Johnston, \u003cem\u003eNoradrenaline and beta-adrenoceptor agonists increase activity of voltage-dependent calcium channels in hippocampal neurons.\u003c/em\u003e Nature, 1987. \u003cstrong\u003e327\u003c/strong\u003e(6123): p. 620-2.\u003c/li\u003e\n \u003cli\u003eLv, J., et al., \u003cem\u003eActivation of \u0026beta;-adrenoceptor facilitates active avoidance learning through enhancement of glutamate levels in the hippocampal dentate gyrus.\u003c/em\u003e Neuroreport, 2017. \u003cstrong\u003e28\u003c/strong\u003e(15): p. 973-979.\u003c/li\u003e\n \u003cli\u003eJedrzejewska-Szmek, J., et al., \u003cem\u003ebeta-adrenergic signaling broadly contributes to LTP induction.\u003c/em\u003e PLoS Comput Biol, 2017. \u003cstrong\u003e13\u003c/strong\u003e(7): p. e1005657.\u003c/li\u003e\n \u003cli\u003eLarsen, M.E., et al., \u003cem\u003eStimulating beta-adrenergic receptors promotes synaptic potentiation by switching CaMKII movement from LTD to LTP mode.\u003c/em\u003e J Biol Chem, 2023. \u003cstrong\u003e299\u003c/strong\u003e(6): p. 104706.\u003c/li\u003e\n \u003cli\u003eBerger, A., et al., \u003cem\u003eHow Is the Norepinephrine System Involved in the Antiepileptic Effects of Vagus Nerve Stimulation?\u003c/em\u003e Front Neurosci, 2021: p. 15：790943.\u003c/li\u003e\n \u003cli\u003eBanke, T.G., et al., \u003cem\u003eControl of GluR1 AMPA receptor function by cAMP-dependent protein kinase.\u003c/em\u003e J Neurosci, 2000. \u003cstrong\u003e20\u003c/strong\u003e(1): p. 89-102.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"VNS, memory, hippocampus, LTP, NE, β-AR","lastPublishedDoi":"10.21203/rs.3.rs-5869428/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5869428/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eVagus nerve stimulation (VNS) is recognized for its therapeutic potential in various neurological disorders, with clinical and preclinical evidence suggesting its capacity to enhance memory function beyond symptom alleviation. However, the mechanisms underlying VNS-mediated memory enhancement are not well understood. Given that the hippocampal noradrenergic (NE) system and β-adrenergic receptor (β-AR) pathway are linked to memory processes, this study investigated the effects of VNS on memory and hippocampal neuroplasticity in pilocarpine-induced memory-impaired rats, with a focus on the role of the NE/β-AR signaling pathway.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eVNS was administered to pilocarpine-treated rats for two weeks via electrodes placed on the left cervical vagus nerve, with parameters set at a current intensity of 1 mA, frequency of 30 Hz, and pulse width of 250 \u0026micro;s for 2 hours daily. Poststimulation, the rats underwent contextual fear conditioning (CFC) and hippocampal long-term potentiation (LTP) testing. The protein expression levels of NE, β2-AR, and key downstream signaling molecules (protein kinase A, CaMKII) were quantified. To ascertain β-AR receptor involvement, a β-AR antagonist was administered in the hippocampus prior to VNS.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eVNS increased hippocampal NE neurotransmitter release, and the expression of β-AR and its downstream pathway proteins PKA and CaMKII enhanced impaired hippocampal LTP and contextual fear conditioning memory in PILO rats. This VNS-induced increase was reversed by β-AR antagonist administration.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe enhancement of hippocampal neuroplasticity and memory by VNS is associated with the hippocampal NE/β-AR signaling pathway, indicating a potential therapeutic mechanism for VNS in memory-related disorders.\u003c/p\u003e","manuscriptTitle":"Vagus Nerve Stimulation Enhances Memory and Synaptic Plasticity via the Hippocampal NE/β-AR Signaling Pathway in Pilocarpine Model Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-10 08:24:05","doi":"10.21203/rs.3.rs-5869428/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a1573555-82fe-4977-b137-2bae257cf5aa","owner":[],"postedDate":"April 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-04T12:23:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-10 08:24:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5869428","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5869428","identity":"rs-5869428","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}