Mechanistic Insights Into Cholinergic, Neurotrophic, and Glutamatergic Pathways in Fluoride Neurotoxicity.

1. Introduction

Fluoride is a naturally occurring trace element that plays a significant nutritional and public health role due to its essential contribution to the mineralization and proper development of hard tissues, particularly bones and teeth, with an estimated daily requirement of approximately 2.6 mg/kg body weight [1, 2]. However, chronic human exposure of higher fluoride levels (> 1.5 mg/L) via drinking water may cause fluorosis and other health outcomes, including neurotoxicity [3, 4]. It has been widely reported that millions of people throughout different regions of the world (particularly in South Asia) are at risk for fluorosis due to fluoride-contaminated drinking water, and a serious public health concern [5, 6]. Recently, it has also been documented that fluoride may be able to cross the blood-brain barrier, build up in brain tissues, and impair neuronal functions, and this could have a negative impact on the mammalian nervous system [7]. Additionally, excessive fluoride consumption may disrupt the various metabolic pathways by increasing lipid peroxidation and inhibiting several cholinergic enzymes [4, 8].

Several molecular mechanisms have been proposed in fluoride-induced neurotoxicity, including oxidative stress, neurotransmitter dysregulation, apoptosis (extrinsic, intrinsic, and ER-mediated pathways), and interactions between fluoride and other essential elements [9, 10]. In line with these proposed mechanisms, we previously reported that chronic fluoride exposure suppressed the antioxidant defense system by reducing SOD1 and GCLC expression [10], promoted mitochondrial apoptosis through upregulation of BAX and downregulation of BCL-2, and disrupted ER stress signaling by decreasing XBP1 and GRP78 expression [10]. Building upon these findings, the present study focuses on the cholinergic, neurotrophic, and glutamatergic pathways to further elucidate the molecular basis of fluoride-induced neurotoxicity.

Fluoride-induced neurotoxicity has been reported to be linked with disruption of key neurochemical pathways involved in regulating neurotransmitters [4]. For example, the cholinergic system, especially acetylcholinesterase (AChE) and Butyrylcholinesterase (BChE), plays a vital role in learning and memory processes [4]. Fluoride-induced inhibition of these enzymes may result in impaired synaptic transmission and neuronal excitability [11, 12]. Previous studies revealed that fluoride can directly affect neurons, myelin, and neurotransmitters in rats, which raises the concern of fluoride as a neurotoxin [13]. Previous results showed that higher fluoride accumulation in the various brain regions affects the levels (in a dose-dependent manner) of norepinephrine, acetylcholine, dopamine, adrenaline, histamine, serotonin, and glutamate [14]. Another study has reported that high fluoride exposure attenuated the expression of nicotinic acetylcholine receptors in the rat brain and cultured neuronal cells [15]. Similarly, [16] documented severe fluorosis in fluoride-exposed rats, and their progeny showed considerably lower expressions of both the a7 and a4 nAChRs as compared to controls and point to fluoride-induced neurotoxicity.

Brain-derived neurotrophic factor (BDNF) plays a critical role in the regulation and maintenance of neurotransmission and neuronal signaling pathways [17]. BDNF is one of the most extensively studied neurotrophins and has been identified as a key regulator of behavioral processes, including learning and memory [18]. It plays a critical role in neuronal survival, differentiation, synaptic plasticity, and hippocampal development [19, 20]. In addition, BDNF is essential for the remodeling and maintenance of the adult nervous system [21]. Studies revealed that exposure to chronic stress naturally decreases BDNF expression in hippocampus [22], while it has also been reported that rats exposed to fluoride had higher levels of BDNF in their brains [23, 24].

Glutamate, another neurotransmitter that is, crucial for learning and memory processes through ionotropic and metabotropic glutamate receptors 5 (mGluR5) and most of the excitatory synapses in the CNS contain glutamate [25]. It is essential for learning, memory, and cognitive abilities because of its function in synaptic plasticity and information processing [26]. However, it is critical to maintain a delicate glutamate balance since high amounts can cause excitotoxicity, a pathophysiological condition where sustained stimulation of glutamate receptors causes detrimental effects on neuronal cells [27]. Recent research has examined and described the function of mGluRs in the hippocampus’s synaptic transmission and plasticity, which are thought to be the foundation of memory and learning [27].

Previous studies have demonstrated that chronic fluoride exposure can impair cognitive function [28] and neurogenesis [29] via different mechanisms, including oxidative stress, neuronal apoptosis, and dysregulation of signaling pathways involved in neuronal survival and plasticity [30, 31]. Few recent studies have also suggested that fluoride-induced neurogenesis impairment is associated with alterations in the Notch1 signaling pathway, contributing to cognitive dysfunction [29]. Given that recent studies reported the detrimental effect of fluoride exposure on the brain [32], however, the detailed insights into molecular mechanisms underlying its impact on neurotoxicity are still lacking. Previous studies have focused on general neurobehavioral effects, but integrative research is needed to understand the link of fluoride exposure and its effects on cholinergic signaling, neurotrophic support (BDNF) and glutamatergic modulation (mGluR5). Therefore, present study addresses this critical gap by examining disrupted neurotransmitters associated neurological functions and related molecular pathways as well as histopathological changes in the hippocampus and cerebral cortex of males and females rat exposed to fluoride.

2. Materials and Methods

2.2. Animal Model and Experimental Design

The ethical approval was sought from the National Institute of Health (NIH), Pakistan (No.F-1/EAC/2024) and strictly followed. The NIH guidelines for the Care and Use of Laboratory Animals [33] were followed. Forty [34] adult male and female Sprague-Dawley rats, weighing between 150 – 200 g and aged 4-5 weeks, were acclimatized for 1 week [35], under standard laboratory conditions (25°C ± 4°C room temperature, 40%-60% humidity, 12 h light/dark cycle) [36] with free access to standard rodent chow and water [37]. Rats were randomly allocated into four groups (n = 10/group; 5 males and 5 females): control (< 0.5 ppm fluoride); treatment: low (50 ppm), medium (150 ppm), and high (300 ppm) NaF (Sigma, S7920), corresponding to approximately 2.6, 7.8, and 15.6 mM (7.5, 22.5, and 45 mg/kg/day), respectively [38]. Fluoride concentrations were selected based on previous studies of chronic fluoride toxicity [39] and administered in drinking water for 90 days.

2.2.1. Brain Tissues Collection, Histopathology, Total mRNA Extraction and cDNA Synthesis

2.2.1.1. Brain Tissue Collection

The brain tissues were collected as described by Dahiru et al. [10]. Samples designated for gene expression analysis were immersed in RNAlater solution and stored at -40°C until RNA extraction, following previously described procedures [40]. Additional tissue specimens were fixed in 10% neutral buffered formalin for subsequent histopathological examination.

2.2.1.2. Histopathological Analysis

Histopathological evaluation of brain tissues using hematoxylin and eosin (H&E) was previously performed to assess general anatomical and histoarchitectural alterations following chronic fluoride exposure [10]. In the present study, however, our primary objective was to conduct a detailed assessment of neuronal damage within specific hippocampal subregions, namely cornu ammonis 1 (CA1), cornu ammonis 3 (CA3), and dentate gyrus (DG). The selection of these hippocampal regions was based on the initial H&E-stained sections, which revealed pronounced histopathological alterations in hippocampus and cerebral cortex. The CA1 region is particularly susceptible to neurotoxic and oxidative stress. The CA3 region plays an essential role in hippocampal network function and memory processing, and the DG is important for neurogenesis and information encoding. Therefore, CA1, CA3, and DG were specifically selected for in-depth histopathological evaluation to characterize the extent and pattern of fluoride-induced neuronal injury. This targeted analysis distinguishes the current study from our previous work by providing a region-specific assessment of hippocampal neurodegeneration rather than a general evaluation of brain histoarchitecture. The prepared slides were coded numerically [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40], and the group identities were disclosed to the examiners only after completion of the histopathological assessment and scoring.

2.2.1.3. Total mRNA Extraction

Brain tissue (100 mg) was used for total mRNA extraction using TRIzol reagents according to manufacturer instructions. The concentration of extracted mRNA was measured using Nanodrop spectrophotometer apparatus and the purity was verified by assessing the A260/A280. Likewise, gel electrophoresis was performed to check the quality of extracted RNA [36].

2.2.1.4. cDNA Synthesis

Complementary DNA was synthesized from total RNA (1 µg) in a 20 µL reaction volume using RevertAidFirst-Strand cDNA synthesis kit in a two-steps method (Supporting Information S1: Table 1^–2). The synthesized cDNA was stored at -20°c until further use for RT-qPCR.

2.2.2. Primers Design and Genes Expression Using RT-qPCR

2.2.2.1. Primers Design

AChE, BChE, BDNF, mGluR5, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH- as a reference gene) genes were designed using PrimerQuest Tool provided by IDT (Integrated DNA Technology, IA, USA) (https://www.idtdna.com/PrimerQuest/Home/Index) and validated using NCBI primer BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The primer sequences and amplicon sizes are provided in supplementary Table 3. The primers were designed using axon-intron spanning on the basis of the complete cDNA sequences deposited in GenBank of NCBI (accession numbers: NM_172009.3 for AChE, NM_022942.1 for BChE, NM_001270630.1 for BDNF, NM_017012.2, for mGluR5, NM_001004210.2 and NM_017008.4 for GAPDH).

2.2.2.2. Genes Expression

RT-qPCR was performed where each 10 µL reaction included: 1 µL cDNA (20x dilution), 1 µL each for sense and antisense primers (x10 concentration), 2 µL of nuclease-free water, and 5 µL SYBR Green Master Mix. GAPDH served as internal control. The RT-qPCR reactions were carried out in duplicate, and threshold cycles (C _t) values were averaged. The relative fold change expression of target genes was calculated using the comparative Ct (2^???CT) method. The cycling conditions for RT-qPCR were as followed: an initial denaturation step at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 45 s. Amplification was completed with a final extension step at 72°C for 10 min. The same cycling conditions were applied to all target genes and the housekeeping gene. To verify the specificity of amplification and confirm the expected amplicon sizes, selected RT-qPCR products were resolved on 2% agarose gels stained with ethidium bromide and visualized using a UV gel documentation system.

2.2.3. Determination of AChE and BChE Enzymatic Activities

2.2.3.1. Preparation of PBS Solution

The solutions were prepared according to [34] protocol. Briefly: to prepare PBS solution. 1: 2.72 g of potassium phosphate monobasic was added to 200 mL of distilled water and mixed. 2: 17.8 g of sodium phosphate dibasic was added into 1000 mL of distilled water and mixed. 200 mL from 1000 mL solution of sodium phosphate dibasic solution was removed and 200 mL potassium phosphate monobasic prepared solution was added. The pH was adjusted to 7.4 and stored the solution in the refrigerator.

2.2.3.2. Preparation of Color Reagent (DTNB, 10 mmol/L)

The color reagent was prepared by dissolving 396.3 mg of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) in 100 mL of phosphate buffer (PP) using magnetic stirring. The prepared solution was aliquoted into 5 mL portions and stored at -20°C until use.

2.2.3.3. Preparation of Substrate Solutions (ASCh, 28.3 mmol/L; BSCh, 63.2 mmol/L)

ASCh and BSCh substrate solutions were prepared by dissolving 82.24 mg of ASCh or 200.47 mg of BSCh, respectively, in 10 mL of distilled water. The prepared substrate solutions were aliquoted into 1 mL portions and stored at -20°C until use.

2.2.3.4. Preparation of Ethopropazine (6 mmol/L)

Ethopropazine solution was prepared by dissolving 20.94 mg of ethopropazine in 10 mL of 12 mmol/L HCl with gentle stirring. The solution was aliquoted into 500 µL portions and stored at -20°C until use.

2.2.3.5. Spectrophotometric Determination of AChE and BChE Activities

AChE activity in whole blood was determined spectrophotometrically according to the method described by [34]. Briefly, 30 uL of EDTA-anticoagulated whole blood was transferred into a sterile 15 mL Falcon tube and mixed with 3 mL of phosphate-buffered saline (PBS) containing Triton X 100 to prepare the hemolysate, which was subsequently stored at 4°C until analysis. For the assay, 2 mL of PBS was mixed with 100 uL of 10 mmol/L 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and 10 uL of ethopropazine in a 15 mL Falcon tube. Subsequently, 1000 uL of the prepared hemolysate was added and incubated at 37°C for 20 min. The reaction mixture was transferred to a cuvette, and the enzymatic reaction was initiated by adding 50 uL of ASCh substrate. Absorbance was measured at 347 nm using a spectrophotometer, and readings were recorded at 1 min intervals for 5 min. AChE activity was calculated according to the method of [34].

BChE activity in serum was determined using a similar spectrophotometric procedure. Briefly, 10 uL of serum was added to a reaction mixture containing 3 mL of PBS and 100 uL of 10 mmol/L DTNB. The reaction was initiated by the addition of 50 uL of BSCh substrate, followed by incubation at 37°C for 20 min. Absorbance was measured at 347 nm using a spectrophotometer, and readings were recorded at 1 min intervals for 5 min. BChE activity was calculated according to the method described by [34], Figure 1.

Open in a separate windowFigure 1Illustrated diagram showing procedure for AChE and BChE enzymatic activities analysis.

3. Results

3.1. Histopathological Analysis of Brain Tissues of Rats Exposed to Fluoride

Chronic NaF exposure induced dose-dependent histopathological alterations in the hippocampus of both male and female rats (Figures 2 and 3). In male rats (Figure 2a), moderate and high dose NaF exposure resulted in marked neuronal degeneration, particularly within the hippocampal CA1, CA3, and DG regions. Histopathological changes included neuronal shrinkage, pyknosis, cytoplasmic eosinophilia with chromatolysis, vacuolization, spongiosis, and apoptotic features such as karyolysis and karyorrhexis. Chi-square analysis of hippocampal neuronal injury (Figure 2b) demonstrated an increasing frequency of neuronal damage across the regions with increasing fluoride exposure, a statistically significant association between fluoride exposure and neuronal injury was detected in the DG (X ² = 11.209, df = 3, p < 0.05), CA1 (X ² = 10.276, df = 3, p < 0.05) and CA3 (X ² = 9.873, df = 3, p < 0.05) regions.

Open in a separate windowFigure 2Chronic fluoride exposure induces hippocampal neuronal damage in male rats. (A) Representative H&E stained photomicrographs of hippocampal sections from male rats exposed to control, low-dose (50 ppm), medium-dose (150 ppm), and high-dose (300 ppm) NaF. Histopathological evaluation was performed in the hippocampal CA1, CA3, and DG regions. Images were acquired using a light microscope at the same magnification and anatomical orientation. (n = 5). (B) Quantitative analysis of neuronal damage in the CA1, CA3, and DG regions of the hippocampus in male rats following chronic NaF exposure. Data are presented as mean ± SD (n = 5 per group). Statistical analysis was performed using one-way ANOVA followed by the appropriate post hoc test. *p < 0.05 versus the control group.

Open in a separate windowFigure 3Chronic fluoride exposure induces hippocampal neuronal damage in male rats. (A) Representative H&E stained photomicrographs of hippocampal sections from male rats exposed to control, low-dose (50 ppm), medium-dose (150 ppm), and high-dose (300 ppm) NaF. Histopathological evaluation was performed in the hippocampal CA1, CA3, and DG regions. Images were acquired using a light microscope at the same magnification and anatomical orientation. (n = 5). (B) Quantitative analysis of neuronal damage in the CA1, CA3, and DG regions of the hippocampus in male rats following chronic NaF exposure. Data are presented as mean ± SD (n = 5 per group). Statistical analysis was performed using Chi-square followed by the appropriate post hoc test. *p < 0.05 versus the control group.

Similarly, in female rats (Figure 3a), NaF exposure induced histopathological alterations in the hippocampus, characterized by neuronal degeneration, pyknosis, vacuolization, and apoptotic changes. Chi-square analysis of hippocampal neuronal injury (Figure 3b) demonstrated an increasing frequency of neuronal damage across the regions with increasing fluoride exposure, a statistically significant association between fluoride exposure and neuronal injury was detected in the CA1 (X ² = 9.254, df = 3, p < 0.05), CA3 (X ² = 8.653, df = 3, p < 0.05) and DG (X² = 8.209, df = 3, p < 0.05) regions. Comparative analysis between male and female rats demonstrated a greater susceptibility of male rats to fluoride-induced hippocampal injury.

3.2. Fluoride Exposure Affects Cholinesterases, Neurotrophic and Glutamatergic Gene Expression

Chronic fluoride exposure resulted in significant downregulation of AChE expression as compared to control (p < 0.05). However, no significant differences in sex (p > 0.05) and dose*sex interaction (p > 0.05). Tukey post hoc revealed statistically significant difference between control and low exposed (p < 0.05), medium exposed (p < 0.05) and high exposed (p < 0.05) groups (Figure 4A). However, BChE mRNA expression showed no statistically significance between fluoride exposed groups and control (p > 0.05), with no significant differences in sex (p > 0.05) and dose*sex interaction (p > 0.05). (Figure 4B). The gel electrophoresis revealed the presence of distinct band of BChE at 168 bp (Supporting Information S1: Figure 1).

Open in a separate windowFigure 4Mean ± SD of the relative fold expression of AChE (A), BChE (B), BDNF (C), and mGluR5 (D) in the brain tissues of male and female rats exposed to control (0.5 ppm), 50, 150, and 300 ppm of sodium fluoride for 90 days. Asterisks (*) indicate level of significance as p < 0.05, while ns (non-significant) at p > 0.05.

Exposure to fluoride also significantly decreased BDNF expression in fluoride exposed groups (p < 0.05), with no significant differences in sex (p > 0.05) and dose*sex interaction (p > 0.05). Tukey post hoc least significant difference revealed statistically significant difference between control versus high and medium exposed groups (p  0.05). However, there is no significant difference between the treatment groups (p > 0.05) (Figure 4C). The gel results revealed amplification at 130 bp (Supporting Information S1: Figure 1).

mGluR5 expression was remarkably downregulated in fluoride exposed groups (p < 0.05), with no significant difference in sex (p > 0.05) and dose*sex interaction (p > 0.05). Tukey post hoc least significant difference revealed statistically significant difference between control and medium (p < 0.05); high (p < 0.05) exposed groups, however, no significant difference between control and low exposed group (p >0 .05) (Figure 4D). The gel results revealed distinct amplificon at 151 bp (Supporting Information S1: Figure 1). Schematic illustration is presented to show the neurotransmitter-associated pathway linked to chronic fluoride neurotoxicity (Figure 5).

Open in a separate windowFigure 5Proposed cholinergic, neurotrophic, and glutamatergic mechanisms involved in fluoride-induced neurotoxicity. Fluoride crosses the blood-brain barrier and accumulates in neural tissues, where it disrupts neurotransmission, receptor expression, calcium homeostasis, and intracellular signaling pathways at the presynaptic terminal, synaptic cleft, and postsynaptic membrane. AChE/BChE: In the cholinergic system, acetylcholine (ACh) is an important neurotransmitter involved in neuronal communication and synaptic regulation. Fluoride inhibits the activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), the enzymes responsible for ACh hydrolysis in the synaptic cleft. The resulting accumulation of ACh leads to prolonged activation of postsynaptic cholinergic receptors, receptor desensitization, and disruption of neuronal signaling pathways. BDNF: Plays a critical role in neuronal survival, differentiation, synaptic maintenance, and neuronal plasticity. Chronic fluoride exposure reduces BDNF expression in the cerebral cortex and hippocampus, contributing to impaired neurogenesis, reduced synaptic repair, and disruption of neuronal homeostasis. mGluR5: Glutamate is the principal excitatory neurotransmitter in the central nervous system and exerts its effects through metabotropic glutamate receptor 5 (mGluR5), a G protein-coupled receptor involved in calcium signaling, intracellular signaling cascades, and synaptic homeostasis. Fluoride disrupts glutamatergic neurotransmission by altering glutamate metabolism and reducing mGluR5 activation, resulting in impaired intracellular calcium signaling, altered synaptic remodeling, and disturbances in neuronal network homeostasis. Collectively, these molecular alterations contribute to fluoride-induced neurotoxicity through dysregulation of cholinergic, neurotrophic, and glutamatergic signaling pathways.

Moreover, correlation analysis revealed associations between fluoride exposure levels and the expression of cholinergic, neurotrophic, and glutamatergic markers (Figure 6). A strong positive correlation between BChE and BDNF (r = 0.518; p < 0.05), mGluR5 (r = 0.782; p < 0.05). Likewise positive correlations were observed between BDNF and mGluR5 (r = 0.429; p < 0.05), indicating that increasing fluoride exposure was associated with progressive downregulation of these neuroprotective and neurotransmission-related genes. Nevertheless, a weak positive correlation was observed between AChE vs BChE (r = 0.372; p > 0.05), BDNF (r = 0.173; p > 0.05), and mGluR5 (r = 0.305; p > 0.05).

Open in a separate windowFigure 6Scatter plots showing the associations among the relative expression levels of AChE, BChE, BDNF, and mGluR5 in control and fluoride exposed rats. Spearman’s rank correlation analysis was performed to evaluate the strength and direction of the relationships between the investigated genes. Each point represents an individual animal, and the red line indicates the fitted regression trend: AChE vs BChE (r = 0.372; p > 0.05), BDNF (r = 0.173; p > 0.05), and GluR5 (r = 0.305; p > 0.05). BChE versus BDNF (r = 0.518; p < 0.05) and mGluR5 (r = 0.782; p < 0.05), and BDNF versus mGluR5 (r = 0.429; p < 0.05).

3.3. Chronic Fluoride Exposure Altered AChE and BChE Enzymatic Activities

Chronic fluoride exposure resulted in a significant decrease (p < 0.05) in blood AChE activity in fluoride-exposed groups compared with the control. AChE activity declined in a dose-dependent manner (low to high doses) from 1.025 mU/mL in controls to 0.987, 0.678, and 0.604 mU/mL in males and from 1.095 mU/mL to 0.957, 0.824, and 0.694 mU/mL in females, respectively. However, no significant effects of sex (p > 0.05) and/or fluoride dose*sex interaction (p > 0.05) were observed (Figure 7A). Tukey’s post hoc analysis further revealed significant differences between the control group and the low- (p < 0.05), medium (p < 0.05), and high (p < 0.05) dose fluoride exposure groups.

Open in a separate windowFigure 7Mean ± SD of the enzymatic activities of (A) AChE and (B) BChE in control and fluoride-exposed groups. Asterisks (***) indicate statistically significant differences, while ns (non-significant) at p > 0.05. (C) Principal component analysis (PCA) of AChE and BChE gene expression and enzymatic activity, illustrating the clustering patterns and separation of experimental groups according to fluoride exposure levels. (D) Cluster mean analysis showing dose-dependent differences in AChE and BChE enzymatic activities and gene expression profiles among the experimental groups.

Similarly, serum BChE activity was also significantly decreased (p < 0.05) in fluoride-exposed groups compared to control. BChE activity decreased in a dose-dependent manner (low to high doses) from 0.568 mU/mL to 0.476, 0.407, and 0.357 mU/mL in males and from 0.575 mU/mL to 0.571, 0.326, and 0.311 mU/mL in females, respectively. A significant fluoride dose * sex interaction was also observed (p < 0.05) (Figure 7B). Tukey’s post hoc analysis revealed significant differences between the control group and the medium (p < 0.05) and high (p < 0.05) dose fluoride exposure groups.

Furthermore, because gene expression and enzyme activity represent distinct biological measurements, PCA was performed using standardized variables to investigate the overall multivariate response to fluoride exposure rather than direct quantitative comparisons between mRNA abundance and enzymatic activity (Figure 7C). Cluster mean analysis further demonstrated dose-dependent effects of fluoride exposure on cholinergic gene expression and enzymatic activities (Figure 7D). To enable comparison between expression and enzymatic changes, the relative fold changes in AChE and BChE expression together with the corresponding enzyme activities for studied male and female rats are summarized in Supporting Information S1: Table 4 and 5, respectively.

4. Discussion

Fluoride exposure pathways included consumption of water, tea, food, supplements, dental products, industrial emissions, and pesticide residues [41, 42]. It has been documented that fluoride intake is beneficial in small amounts and required for preventing dental cavities, however, chronic exposure to high concentration (particularly via drinking water) is detrimental to people’s health and may cause fluorosis and broader systemic harm [43]. Despite recent research on fluoride toxicity, the underlying molecular mechanisms of fluoride-induced neurotoxicity is still not completely understood. The current study used rat model and investigated the molecular and histopathological aspects of fluoride-induced neurotoxicity, focusing on the disruption of cholinergic, neurotrophic and glutamatergic pathways. Our findings demonstrate that chronic fluoride exposure reduced the expression of AChE, BDNF, and mGluR5, as well as decreased in AChE and BChE enzymatic activities. These molecular alterations were further supported by histopathological findings, which revealed fluoride-induced structural changes including neuronal degeneration, and cellular disorganization in the hippocampus and cerebral cortex. Collectively, these findings suggest that chronic fluoride exposure alters multiple interconnected molecular pathways regulating neuronal survival, neurotransmission and synaptic plasticity, providing mechanistic evidence for its neurotoxic effects. Our findings are consistent with those of previous reports demonstrating that fluoride exposure may alter neurotransmitter-related pathways and induce neurotoxic changes in the central nervous [16, 44].

In this study, we observed a decrease in AChE expression in the hippocampus and cerebral cortex and reduced enzymatic activities in the blood of both male and female rats, which is in accordance with previous studies [44]. These alterations indicated a fluoride-induced suppression of cholinergic system components across central and peripheral tissues. Given the sensitivity of the hippocampus and cerebral cortex to fluoride toxicity [45], the observed decreases in AChE further support their vulnerability to fluoride exposure. AChE is the primary enzyme responsible for terminating cholinergic neurotransmission through hydrolyzing acetylcholine. Therefore, diminished AChE expressions and activity may impairs cholinergic signaling, disrupt synaptic communication, and promote neuronal dysfunction. AChE is a membrane-associated glycoprotein synthesized and folded in the endoplasmic reticulum (ER) [46]. Our previous study revealed that fluoride triggers ER stress via reactive oxygen species (ROS) generation [10], which may impair protein synthesis and folding, leading to decreased AChE expression and activity. These findings suggest that ER stress may serve as molecular link between fluoride-induced oxidative damage and impaired cholinergic neurotransmission.

Likewise, we found a dose-dependent reduction in BDNF expression in the hippocampus and cerebral cortex of fluoride exposed male and female rats. This finding is consistent with the study of [47] but contrasts with earlier reports of [23, 24], highlighting the complexity of fluoride induced neurotoxicity. BDNF is a key neurotrophins involved in neuronal survival, differentiation, and synaptic plasticity and is highly expressed in the hippocampus and cerebral cortex [19]. Therefore, fluoride-induced downregulation of BDNF suggests impaired neurotrophic support and disruption of neuronal homeostasis. This downregulation may be associated with fluoride-induced oxidative stress and ER stress, which have shown in our previous study [10]. Oxidative stress has also been shown to suppress CREB-dependent BDNF transcription, thereby impairing neuronal survival and synaptic plasticity. Consequently, reduced BDNF expression may contribute to neuronal dysfunction and neurotoxicity by compromising neuronal maintenance and adaptive synaptic integrity.

Similarly, our study revealed a decrease in the expression of mGluR5 in the hippocampus and cerebral cortex. mGluR5 is a G protein-coupled receptor predominantly expressed in postsynaptic neurons and plays a crucial role in glutamatergic neurotransmission and synaptic plasticity [25]. Therefore, the observed downregulation of mGluR5 suggests disruption of glutamatergic signaling, a key feature of fluoride-induced neurotoxicity. This reduction may be associated with fluoride-induced oxidative stress and ER stress [10], which can impair the synthesis and trafficking of membrane proteins, leading to translational inhibition and degradation of misfolded proteins, including membrane receptors like mGluR5. Given the role of mGluR5 in activating intracellular signaling pathways that regulate CREB activation and BDNF expression, its downregulation may further exacerbate neuronal dysfunction by disrupting glutamatergic neurotransmission while simultaneously diminishing neurotrophic support.

Rather than representing independent molecular alterations, the reductions in AChE, BDNF, and mGluR5 expression suggest that chronic fluoride exposure disrupts an interconnected network. Since cholinergic and glutamatergic signaling, together with BDNF-mediated neurotrophic support, are functionally integrated, suggesting potential contribution toward defective neuronal communication, impaired synaptic function, and enhanced susceptibility to neurodegeneration, which might explain the structural brain modifications induced by chronic fluoride exposure.

The observed reductions in AChE, BChE, BDNF, and mGluR5 expression suggest that chronic fluoride exposure disrupts molecular pathways involved in cholinergic neurotransmission and neuronal function. Previous studies have reported that similar alterations are associated with cognitive impairment following fluoride exposure [29]. However, as behavioral assessments were not performed in the present study, no direct conclusions regarding cognitive or memory function can be drawn. These molecular alterations were reflected in the histopathological lesions observed in our study, including neuronal shrinkage, vacuolization, spongiosis, gliosis, eosinophilic cytoplasm, karyolysis, and frank neuronal apoptosis in the hippocampus and cerebral cortex. Neuronal shrinkage, pyknosis, karyolysis, vacuolization, and spongiosis are characteristic features of irreversible neuronal degeneration and glial activation following chronic fluoride exposure. These histopathological changes corroborate the molecular disturbances observed in cholinergic, neurotrophic, and glutamatergic signaling pathways. Notably, male rats exhibited more severe lesions than female rats, suggesting greater susceptibility to fluoride-induced neurotoxicity, possibly due to the neuroprotective effects of estrogen in females [48].

The present study has several limitations viz; molecular analysis was limited to mRNA level without corresponding protein quantification/validation, and behavioral assessments were not performed to determine whether the observed molecular and histopathological changes were associated with cognitive impairment. Future studies incorporating protein analyses, behavioral evaluations, and mechanistic interventions will further clarify the causal relationships among fluoride exposure, molecular signaling pathways, and neurotoxicity.

5. Conclusion

Chronic exposure to sodium fluoride via drinking water for 90 days altered cholinergic, neurotrophic, and glutamatergic markers and also induced histopathological lesions in the hippocampus and cerebral cortex of male and female rats in a dose and sex-dependent manner. The concurrent impairment of these interconnected pathways provides an explanation at molecular basis for the observed neuronal damage. Overall, this study provides important insights into the mechanisms underlying chronic fluoride-induced brain injury and highlights the cholinergic, neurotrophic, and glutamatergic pathways as potential targets for therapeutic intervention. Further studies incorporating behavioral assessments and neuroprotective interventions are required to determine the functional consequences of these molecular and histopathological alterations on cognition and memory, as well as to evaluate the potential of therapeutic strategies to prevent or reverse fluoride-induced neuronal damage.

Author Contributions

Ashiru Dahiru: writing – original draft, methodology, investigation, formal analysis. Ismat Nawaz: funding acquisition, resources, supervision. Syeda Kiran Riaz: supervision. Nazneen Bangash: validation, supervision. Muhammad Abbas: formal analysis, resources. Summaya Sohail Chaudry: visualization. Muhammad Jadoon Khan: supervision, methodology. Qingyu Huang: writing – review and editing. Syed Ali Musstjab Akber Shah Eqani: supervision, resources, writing – review and editing.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability StatementThe data that support the findings of this study are available from the corresponding author upon reasonable request.