Male C57BL/6 J mice were acquired from Southern Medical University’s Animal Center in Guangzhou, China. ChAT-Cre mice and Ai14 reporter mice were gifts from Tianming Gao at Southern Medical University (Guangzhou, China). ChAT-Cre mice were crossed with the Ai14 reporter mice to label the ChAT neurons with tdTomato (ChAT-Cre:Ai14 mice). All mice were housed in a controlled environment with a 12-h light/dark cycle (lights on at 08:00 AM) and were provided with ad libitum access to food and water under standard conditions (21–26 °C). The behavioral experiment adhered to the guidelines set by the Chinese Council on Animal Care, ensuring compliance with professional and ethical standards. All behavioral testing was undertaken during the light phase of the light/dark cycle. The mice were prepared for testing by being handled gently for at least 5 days to reduce the stress that comes with manipulation. A total of 105 male mice were used in the experiment. Control group (i.p. saline, #RE1330, G-CLONE, Beijing, China); 5-FU group (i.p. 5-FU, #F6627, Sigma-Aldrich, St. Louis, MO, USA); 5-FU+hM3Dq group (i.p. 5-FU with hM3Dq expressed in cholinergic neurons of MS); 5-FU+donepezil group (i.p. 5-FU followed by i.p. donepezil (#D6821, Sigma-Aldrich) before behavioral experiments). The ethical approval code and number for this experiment is 2023-F-09-01. The entirety of our endeavors was devoted to mitigating animal distress.

Mice in the 5-FU group received i.p. injections of 50 mg/kg b.w. 5-FU [25, 26]. An injection of the designer medication clozapine-N-oxide (CNO, 5 mg/kg, i.p.; #C0832, Sigma-Aldrich) was given 30 min before behavioral testing, in order to simplify chemogenetic manipulations [27]. To elevate ACh levels, donepezil was injected i.p. at a 1.0 mg/kg dose [28]. Control mice were injected with saline.

The microdialysis system was purchased from the Carnegie Medicine Associates (CMA, Stockholm, Sweden). A microdialysis probe (CMA/20, CMA) was inserted using a guide cannula into the dorsal hippocampal region (coordinates: AP, –2.0 mm; ML, –1.3 mm; DV, –1.6 mm; AP: anterior-posterior, ML: medial-lateral, DV: dorsal-ventral) to assess the amount of extracellular ACh in the hippocampus. Microdialysis began with the probe connected to a microinjection pump system (CMA/100, CMA). An artificial cerebrospinal fluid (CSF) solution ((125 mM NaCl (#S9888); 2.5 mM KCl (#P5405); 1.26 mM CaCl₂ (#C5670); 1.18 mM MgCl₂ (#M8266)); All chemicals used were purchased from Sigma-Aldrich) that included neostigmine at a concentration of 10 µM, was circulated continuously in the probe, maintaining a flow rate of 1 µL/min to inhibit the degradation of ACh. Before the experiments, the dialysate concentrations were adjusted by dialysis of a specifically formulated artificial CSF solution containing 5 nM ACh (#A9101, Sigma-Aldrich). In the microdialysis procedure for both control and 5-FU mice, the initial 60-min perfusion exudate was discarded to ensure system equilibrium, with subsequent samples collected after a minimum of 1 h. Prior to being analyzed using HPLC-MS/MS (1260 Infinity III, Agilent Technologies, Santa Clara, CA, USA), the samples had been frozen at –80 °C.

The mice were anesthetized with pentobarbital sodium (75 mg/kg, i.p., #57-33-0, Toronto Research Chemicals, Toronto, Ontario, Canada) and subsequently decapitated to collect hippocampal tissue. After weighing and homogenizing in PBS (#P924709-5EA, Macklin, Shanghai, China), the samples were centrifuged at 25,000 g for 20 min at 4 °C. AChE concentrations in the supernatants were assessed using an assay kit (#ab138871, Abcam, Cambridge, MA, USA). In a volume of 100 µL/well, 50 µL of ACh stock was added to the standard, blank, and test samples, followed by incubation for 30 min at room temperature away from light. Absorbances were read at OD = 410 $\pm$ 5 nm with a microplate reader (Varioskan LUX, Thermo Fisher, Waltham, MA, USA).

Biocytin was used in this study to investigate changes in the morphology of neurons. To prevent ATP/GTP from degrading, the solution and aliquots listed below were prepared and kept in a cold environment. The following ingredients (in mM) were added to a 25 mL solution: 10 NaCl; 0.3 Na-GTP (#51120, Sigma-Aldrich); 10 EGTA (#E3889, Sigma-Aldrich); 1 MgCl₂; 110 K-gluconic acid (#G4500, Sigma-Aldrich); 2 Mg²⁺-ATP (#A9187, Sigma-Aldrich); 40 HEPES (#H4034, Sigma-Aldrich); and 0.1% biocytin (#B4261, Sigma-Aldrich). The pH was adjusted to 7.3 using a solution of NaOH (5 M, #655104, Sigma-Aldrich) to ensure osmolarity ranges of 280 to 300 mOsm. Biocytin was included in the internal solution to visualize recorded cells, and slices were drop fixed in 4% paraformaldehyde (#158127, Sigma-Aldrich) for 12 h. Then, the slices were permeabilized, and stained for Streptavidin-AF488 (#S11223, Thermo Fisher).

To analyze the three-dimensional morphology of dendrites, we used Imaris software (Imaris 8.1, Oxford Instruments, Oxford, United Kingdom) to calculate the overall branching pattern and examine the characteristics of spines. In short, we imported a z-stack acquisition into Imaris and performed manual tracing after calibration. Subsequently, we determined the total length of dendrites. Sholl analysis was conducted with an interval set at 10 µm.

The brains were collected and refrigerated in a modified artificial cerebrospinal fluid (ACSF) maintained at a low temperature with ice after the mice were anesthetized with pentobarbital sodium. The ACSF solution consisted of glycerol (250 mM, #G6279); KCl (2 mM); MgSO₄ (10 mM, #M2643); CaCl₂ (0.2 mM); NaH₂PO₄ (1.3 mM, #S5011); NaHCO₃ (26 mM, #S5761); and glucose (10 mM, #G7021), All chemicals used were purchased from Sigma-Aldrich. The brain slices were prepared by cutting 300 µm horizontal sections using a vibrating microtome (VT1200S, Leica, Wetzlar, Germany). Before being recorded, these slices were allowed to sit at room temperature for 1 h. All solutions used during this process contained a mixture of 95% O₂ and 5% CO₂ to ensure proper saturation levels.

Slices were placed in the recording chamber in an ACSF solution maintained between 32 and 34 °C. We used ChAT-Cre:Ai14 mice to specifically label ChAT neurons with tdTomato. Neurons showing fluorescence were examined by a microscope (Eclipse FN1, Nikon, Tokyo, Japan) with an infrared-sensitive charge-coupled device camera. Spontaneous firing patterns were recorded in a cell-attached mode using an analog-to-digital converter (Digi data 1440A, Axon Instruments, Union City, CA, USA), and an amplifier (Multi Clamp 700B, Axon Instruments, San Jose, CA, USA).

Neuronal excitability was assessed by examining the rate of firing in response to depolarizing pulses. At the same time, CNO was present to confirm the regulation of these neurons using DREADD. An ACSF solution (18 mM NaCl; 0.6 mM EGTA; 133 mM potassium gluconate (#G4500, Sigma-Aldrich); 10 mM HEPES; 2 mM Mg²⁺-ATP; and 0.3 mM sodium glutamate (#G5889, Sigma-Aldrich)) at pH 7.2 and 280 mOsm was added to the pipettes. Steady current was applied with increments of +20 pA, from –60 pA to +220 pA.

Microelectrodes (with a resistance of 3–5 M$\mathrm{\Omega }$) were filled with a solution that contained 130 mM potassium gluconate; 20 mM KCl; 10 mM HEPES buffer; 2 mM MgCl₂$\cdot$6H₂O (#M2393, Sigma-Aldrich); 4 mM Mg²⁺-ATP; 0.3 mM Na-GTP; and 10 mM EGTA, to record spontaneous firing. A KOH (#484016, Sigma-Aldrich) solution with a 10 M concentration was used to bring the pH level down to 7.25. Spontaneous firing activity of each neuron was recorded for at least 2 min. Data were analyzed with Clampfit software (version 10.2, Axon Instruments, San Jose, CA, USA).

Under pentobarbital anesthesia (75 mg/kg, i.p.), a stereotactic brain injection was administered to mice using a stereotaxic instrument (RWD, Shenzhen, China). We used adeno-associated virus (AAV), AAV-Ef1$\alpha$-Dio-hM3Dq-mCherry (titer: 2.27 $\times$ 10¹³ v.g./mL, #PT-0042, BrainVTA, Wuhan, China) or the control virus, AAV-Ef1$\alpha$-Dio-mCherry (titer: 2.03 $\times$ 10¹³ v.g./mL), for chemogenetic regulation of neurons. The virus was administered at 50 nl/min using calibrated glass microelectrodes attached to an infusion pump (Nanoliter 2010 Injector, WPI, Sarasota, FL, USA). The MS of each Chat-Cre mouse was injected with 300 nL of AAV virus at a 10° outward angle in the coronal plane (+1.6 mm AP; $\pm$0.65 mm ML; –4.0 mm DV). After infusion, the pipette was kept in the injection site for about 10 min in order to stop virus flow. The mouse was maintained on a heating pad until it regained consciousness. Experiments were conducted 3 weeks after stereotactic injection.

All statistical analyses were conducted using the GraphPad Prism (GraphPad Prism 8, Dotmatics, Boston, MA, USA). We used Student’s t-test for simple statistical comparisons. Post hoc analyses and 1- and 2-way ANOVAs were used to analyze the data from the experimental groups with multiple comparisons. According to the following standards, the figures show significant statistical results: *p 0.05, **p 0.01, ***p 0.001. Data are displayed as mean $\pm$ SEM.

To examine cognitive function in mice after treatment, we treated mice with 5-FU (50 mg/kg) once daily for 10 consecutive days [25, 26]. After the administration of the chemotherapy drug, behavioral tests such as the MWM, OFT, and NOR tests were conducted (Fig. 1A). 5-FU treatment had no impact on mouse locomotor activity (Fig. 1B). There was no significant difference in the duration that the two groups spent in the center during the OFT, indicating that the administration of 5-FU did not affect the anxiety levels of the mice (Fig. 1C).

Fig. 1.

The administration of 5-FU impaired cognitive performance of mice. (A) Schematic diagrams of the 5-FU treatment and behavioral tests. Created with Adobe illustrator (CC 2014, Adobe Inc., San Jose, CA, USA). (B) Effects of 5-FU on mouse locomotion (n = 10/group). (C) Effect of 5-FU on anxiety levels. (D) Design of experiments for the Novel Object Recognition test. (E–G) The duration of exploration during the learning phase (E), the duration of exploration during 24-h retrieval assessments (F), and discrimination index of novel object recognition (G) were quantified for mice across different experimental groups. (H) The escape time required for relocating the platform during testing (n = 10/group). (I) Exemplary patterns of swimming trajectories were observed during the probe trial. (J) Times spent in Q1 during the test. (K) The total number of platform crossings throughout the test. (L) Speed of movement in the MWM. (M) Latency during the cued test. Data are displayed as mean $\pm$ SEM. OFT, open field test; NOR, novel object recognition; MWM, morris water maze; 5-Fu, 5-fluorouracil. ** p 0.01; *** p 0.001.

To assess cognitive performance related to 5-FU treatment, we performed behavioral assessments, including NOR and MWM tests. During the NOR test, the mice that received 5-FU exhibited a reduction in their 24-h item recall (Fig. 1D–G; Fig. 1F, p 0.01, Fig. 1G, p 0.01). Furthermore, during the MWM test training session, the mice receiving the 5-FU treatment demonstrated a discernible increase in the time needed to locate the hidden platform. This indicated that 5-FU therapy had a negative influence on mouse spatial learning (Fig. 1H, p 0.001). Moreover, the 5-FU mice exhibited a reduction in the time allocated to the desired quadrant and a diminished frequency of entering the target area, although their speed remained unchanged (Fig. 1H–M; Fig. 1J, p 0.01, Fig. 1K, p 0.01). These findings suggested that treatment with 5-FU had a negative impact on the cognitive abilities of the mice.

Decreased ACh levels in the hippocampus have been linked to cognitive impairments [21, 30, 31, 32]. The present study sought to test whether the 5-FU treatment reduced the ACh levels within the hippocampus. A microdialysis component with HPLC-MS/MS was used to quantify extracellular ACh levels in the hippocampal tissue during the 5-FU treatment (Fig. 2A). The data showed that mice treated with 5-FU for 6 days had notably lower extracellular ACh levels within the hippocampus than did the control group (Fig. 2B; Day 6 p 0.05, Day 8 p 0.01, Day 10 p 0.01,). The hippocampal ACh is released mainly by cholinergic projections from the MS, and broken down by AChE [22]. The findings of our study demonstrated that although the administration of 5-FU reduced hippocampal ACh, it did not affect hippocampal AChE activity (Fig. 2C). Therefore, the above findings showed that 5-FU treatment impaired septal-hippocampal ACh transmission.

Fig. 2.

Hippocampus cholinergic transmission is attenuated in mice treated with 5-FU. (A) Left: ACh levels in the hippocampus were determined by microdialysis and analyzed via HPLC-MS/MS. Right: the position of the cannulae and probe tracks. Scale bar = 200 µm. (B) ACh levels in the hippocampus during 5-FU or saline treatment (n = 5–6/group). (C) The AChE activity in the hippocampus after 10 days of drug delivery between the control and the 5-FU groups (n = 6/group). AChE, acetylcholinesterase. * p 0.05; ** p 0.01.

To determine whether 5-FU influenced neuronal functions, we evaluated the dendritic structure of ChAT neurons in the MS. Markedly lower numbers of dendritic branches were found in mice treated with 5-FU than in the control mice (Fig. 3A,B, p 0.001). The dendritic spine distributions in MS ChAT neurons were also examined. The mice treated with 5-FU exhibited lower spine density than did the controls (Fig. 3C,D, p 0.01). These findings showed that 5-FU treatment reduced the dendritic complexity of MS ChAT neurons in the experimental mice. The integrity of neural morphology is essential for neural activity. Consistently, our electrophysiological recordings revealed that the spontaneous activity of ChAT neurons was impaired after 5-FU treatment (Fig. 3E–G, p 0.01).

Fig. 3.

5-FU treatment impaired the neuronal structure and activity of cholinergic (ChAT) neurons in the medial septum (MS). (A) An illustration of the MS ChAT neurons’ typical morphology across many groupings. (B) Sholl analysis results (n = 9–10 neurons from three mice in each group). Scale bar = 50 µm. (C) An illustrative depiction of the neural dendrite segment from MS ChAT that was used to examine dendritic spines. Scale bar = 5 µm. (D) Determining the MS ChAT neurons’ spine number (n = 9–10 neurons from 3 mice/group). (E) Left: MS ChAT neuron preparation and electrophysiological recording. Right: example of patch-clamp recording of cholinergic fluorescent neuron under whole-cell configuration. (F) Representative spontaneous firing traces of the MS ChAT neurons from control and 5-FU mice. (G) The spontaneous firing rate of the MS ChAT neurons was quantified (n = 20 neurons from 4 mice/group). ** p 0.01; *** p 0.001. Two-way ANOVA with Sidak’s t-test (B) and unpaired t-test (D,G).

We assessed the effect of activating MS ChAT neurons on reduction of cognitive function impairments that had been observed in 5-FU-treated mice. To simulate clinical treatment administration and ensure prolonged activation of MS ChAT neurons, we used chemogenetics techniques to stimulate ChAT neurons specifically. Using clozapine-N-oxide (CNO) as an activator, we were able to successfully activate MS ChAT neurons with the excitatory hM3Dq designer receptor by stereotaxically delivering a Cre-dependent virus (AAV-EF1a-Dio-hM3Dq-mCherry) into the MS region of ChAT-Cre mice (Fig. 4A,B, p 0.001). The findings indicated that activation of MS ChAT neurons did not affect the locomotion and anxiety levels (Fig. 4C,D). The NOR memory deficits shown in mice given 5-FU were successfully reduced by activating MS ChAT neurons. (Fig. 4E–G; Fig. 4F control A vs control B p 0.001, 5-FU+hM3Dq A vs 5-FU+hM3Dq B p 0.01, Fig. 4G control vs 5-FU p 0.001, 5-FU vs 5-FU+hM3Dq p 0.01). We also found that giving CNO to 5-FU-treated mice corrected their memory and spatial learning deficiencies in the MWM test (Fig. 4H–M; Fig. 4H p 0.001, Fig. 4J control vs 5-FU p 0.001, 5-FU vs 5-FU+hM3Dq p 0.05, Fig. 4K control vs 5-FU p 0.01, 5-FU vs 5-FU+hM3Dq p 0.01).

Fig. 4.

Chemogenetic stimulation of MS ChAT neurons improved the compromised cognitive performance of 5-FU-treated mice. (A) Left: representation of viral injection and optical fiber implantation site. Right: hM3Dq expression in ChAT neurons in the MS. Scale bar = 200 µm. (B) Left: before and during CNO perfusion (5 µM), typical traces show action potentials produced by a 100-pA current in MS ChAT neurons. Right: Quantification of 100-pA current-induced action potentials hM3Dq expression neurons (n = 6–7). (C,D) The locomotion (C) and central time (D) during OFT of mice (n = 9–10/group) in different groups. (E–G) Times spent exploring during the learning phase in the different groups (E), the duration of exploration over 24-hour retrieval tests (F), and the NOR discrimination index (G) are shown. (H) During acquisition training (n = 10/group), the escape time traveled to locate the platform. (I) Swimming patterns during the probe trial. (J) Time spent in Q1 during the probe trial. (K) Platforms crossings during the probe trial. (L) Swimming speeds. (M) The cued-test latencies. AAV, adeno-associated virus; CNO, clozapine-N-oxide. * p 0.05; ** p 0.01; *** p 0.001.

Acetylcholinesterase inhibitors (AChEIs) are extensively used in clinical practice to treat cognitive impairments associated with neurodegenerative diseases by elevating ACh levels [33, 34]. Donepezil, a clinically utilized AChEI, was injected at a dose of 1.0 mg/kg, i.p. [28] to investigate its potential in ameliorating cognitive impairments induced by 5-FU treatment (Fig. 5A). The donepezil treatment did not affect the mice’s anxiety levels or movement (Fig. 5B,C). However, in mice treated with 5-FU, we found that administration of donepezil ameliorated cognitive impairments (Fig. 5D–L; Fig. 5E control A vs control B p 0.01, 5-FU+Donepezil A vs 5-FU+Donepezil B p 0.05, Fig. 5F control vs 5-FU p 0.01, 5-FU vs 5-FU+Donepezil p 0.05, Fig. 5G p 0.001, Fig. 5I control vs 5-FU p 0.001, 5-FU vs 5-FU+Donepezil p 0.01, Fig. 5G control vs 5-FU p 0.001, 5-FU vs 5-FU+Donepezil p 0.001).

Fig. 5.

Donepezil ameliorated cognitive impairments in 5-FU-treated mice. (A) Schematic diagrams of the drug treatment and behavioral tests. (B,C) The locomotion (B) and central time (C) during OFT of mice in various experimental groups (n = 10/group). (D–F) Duration of exploration during the learning phase (D), duration of exploration in the 24-h retrieval tests (E), and NOR discrimination index (F) for mice in various experimental groups. (G) During acquisition training, the escape time spent searching for the platform. (H) During the probing trial, exemplary swimming trajectory patterns were noted. (I) The duration allocated to Q1 in the probe trial. (J) The number of platform crossings observed during the probe trial. (K) Swimming speeds in the MWM. (L) Latency in the cued test. Two-way ANOVA with Sidak’s t-test (B–E,G), One-way ANOVA with Tukey’s t-test (F,I–L). Data are shown as mean $\pm$ SEM. * p 0.05; ** p 0.01; *** p 0.001.