The herbicide glyphosate inhibits hippocampal long-term potentiation and learning through activation of pro-inflammatory signaling

Background Glyphosate, a herbicide marketed under the trade name Roundup, is now widely used, in part because genetically modified organism plants that are resistant to this agent have been developed. Environmental or dietary exposure to glyphosate is omnipresent and there are concerns this exposure could impair cognitive function in addition to carcinogenicity. Methods Using hippocampal slices from juvenile male rats, we investigated whether glyphosate alters synaptic transmission and induction of long-term potentiation (LTP), a cellular model of learning and memory. Our hypothesis is that glyphosate alters neuronal function and impairs LTP induction via activation of pro-inflammatory processes, because increases in pro-inflammatory cytokines and neuroinflammation have been reported following glyphosate exposure. LTP was induced by delivery of 100 Hz x 1 sec high frequency stimulation (HFS) of the Schaffer collateral pathway and excitatory synaptic potentials (EPSPs) were monitored 60 min after HFS. Resulsts: We first tested effects of Roundup on basal synaptic function and LTP induction. Roundup depressed EPSPs in a dose-dependent manner. Basal synaptic transmission was completely suppressed by 2000 ppm. At concentrations ≤ 20 ppm Roundup did not affect basal transmission, but 4 ppm Roundup administered 30 min before HFS inhibited LTP induction. We also observed that acute administration of 10–100 μM glyphosate inhibits LTP induction. Minocycline, an inhibitor of microglial activation, and TAK-242, an inhibitor of toll-like receptor 4 (TLR4), both overcame the inhibitory effects of 100M glyphosate. Similarly, lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) overcame the inhibitory effects. In addition, ISRIB (integrated stress response inhibitor) and quercetin, an inhibitor of endoplasmic reticulum stress, allowed LTP induction in the presence of glyphosate. We also observed that in vivo glyphosate injection (16.9 mg/kg i.p.) impaired one-trial inhibitory avoidance learning. This learning deficit was overcome by TAK-242. Conclusion While Roundup inhibits LTP induction, these observations indicate that glyphosate alone, the major ingredient of Roundup, can impair cognitive function through pro-inflammatory signaling in microglia. Manipulation of pro-inflammatory signaling could be a useful strategy to prevent cognitive impairment after exposure to a glyphosate-based herbicide (GBH).

Glyphosate, which was rst developed in 1950, was originally used as a descaling agent to clean pipes in the 1960's.Because glyphosate inhibits the plant enzyme 5-enolpyruvylshikimate-3-phosphate synthase in the aromatic amino acid biosynthetic pathway, it was patented as a herbicide in 1970 and brought to market under the trade name Roundup.The market for glyphosate expanded exponentially as genetically modi ed plants were developed in the 1990's.Accordingly, human exposure to glyphosate has become routine across populations.In the US, glyphosate is commonly detected in stream water samples (Medalie et al., 2020).In France, it was detected in over 99% of human urine samples (Grau et al., 2022), while in central India, glyphosate was detected in 93% of the urine samples with a mean (SD) concentration of 3.4(1.2)µg/l(Parvez et al., 2018).
Given this vast exposure, a critical question is whether glyphosate is toxic.In addition to its potential carcinogenicity including non-Hodgkin's lymphoma and hepatic cancer, some studies have linked glyphosate with autism spectrum disorder (ASD) (Samsel & Seneff, 2013).In mice, maternal glyphosate exposure results in abnormal behaviors and growth retardation in offspring (Coullery et al., 2020;Pu et al., 2020), implying that exposure to glyphosate alters neuronal function directly or indirectly.Additionally, acute exposure of rats to glyphosate decreases monoamine levels in brain (Hernández-Plata et al., 2015; Martínez et al., 2018), supporting a possible link to Parkinson's disease (PD) (Wang et al., 2011).In mice, oral exposure to glyphosate is reported to cause depression-like behaviors (Aitbali et al., 2018;Cattani et al., 2017).Moreover, glyphosate exposure may diminish memory formation in mice (Ait Bali et al., 2017).
Although it is plausible that these neuronal sequelae are at least partially induced indirectly by intestinal microbial degradation (Rueda-Ruzafa et al., 2019), it is also possible that the herbicide directly impairs neuronal function because glyphosate passes the blood brain barrier (BBB) (Martinez & Al-Ahmad, 2019) and in ltrates the brain to induce neuroin ammation (Astiz et al., 2012;Sato et al., 2011;Winstone et al., 2022).
Using ex vivo rat hippocampal slices, we investigated whether glyphosate administered directly onto brain tissue alters synaptic transmission and long-term potentiation (LTP), a form of synaptic plasticity thought to contribute to learning and memory.We also examined whether activation of neural proin ammatory processes contributes to effects of glyphosate on hippocampal function.

Animals
Sprague-Dawley albino rats were offspring of pregnant female rats obtained from Charles River Laboratories (Indianapolis IN) and were housed in approved facilities at Washington University.Animal use followed National Institute of Health (NIH) guidelines and was approved by the Washington University Institutional Animal Care and Use Committee (IACUC).The reporting in this manuscript follows recommendations in the ARRIVE guidelines.
For electrophysiological studies, slices were transferred to a submersion-recording chamber at 30 o C with ACSF and perfused continuously at 2 ml/min.Extracellular recordings were obtained from the apical dendritic layer (stratum radiatum) of area CA1 for monitoring excitatory postsynaptic potentials (EPSPs) with electrodes lled with 2 M NaCl (5-10 MΩ resistance).
Because LTP is a synaptic phenomenon, we focused on recordings of EPSP slope.EPSPs were evoked using 0.1 ms constant current pulses through a bipolar stimulating electrode in the Schaffer collateral (SC) pathway.Responses were monitored by applying single stimuli every 60 sec at half-maximal intensity based on a control input-output (IO) curve.After obtaining stable baseline recordings for at least 10 min, LTP was induced by a single 100 Hz x 1 s high frequency stimulation (HFS) using the same intensity stimulus.Following HFS, responses were monitored by single stimuli once per minute during the period of post-tetanic potentiation (PTP) and then every ve minutes for the remainder of an experiment.For display purposes, graphs show data every 5 minutes except during initial post-tetanic potentiation.
In vivo injection of glyphosate and behavioral studies Rats were tested for memory acquisition in a one-trial inhibitory avoidance learning task (Izumi, Mennerick, et al., 2021;Izumi & Zorumski, 2020;Whitlock et al., 2006).This task re ects explicitdeclarative fear memories and has been associated with hippocampal LTP; the task is relatively simple to administer with high reliability and clear behavioral endpoints (Izumi, Cashikar, et al., 2021;Izumi et al., 2022;Parent et al., 1994;Tokuda et al., 2010).The testing apparatus consists of two chambers, only one of which is lit.Both compartments have a oor of stainless steel rods (4 mm diameter, spaced 10 mm apart) through which an electrical shock could be delivered in the dark chamber (12 x 20 x 16 cm).The adjoining lit compartment (30 x 20 x 16 cm) was illuminated with four 13 W lights.Light intensity in the lit chamber was 1000 lux while that in the dark chamber was < 10 lux.On the rst day of testing, rats were brought to the lab for vehicle injection, placed in the lit chamber, and allowed to habituate to the apparatus by freely moving between chambers for 10 min without any foot shocks being administered.On the next day, rats were administered glyphosate (16.9 mg/kg ip) or vehicle (saline) 1 hour prior to training.TAK-242 (3 mg/kg i.p.) was injected 24 hours and 2 hours before glyphosate administration.At the time of training, animals were initially placed in the lit compartment and allowed to explore the apparatus freely for up to 300 s (5 min).When rats completely entered the dark chamber, they were immediately given a foot shock.After each 300 s session, rats were removed from the apparatus and returned to their home cages.On the next day of testing, rats were placed in the lit chamber without any drug treatment and the latency to enter the dark compartment was recorded over a 300s trial.Chemicals TAK-242 (CAS 243984-11-4 Cat 6587) was purchased from R&D Systems (Minneapolis MN).Lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) (Catalog # tlrl-rslps) and MCC950 (CAS 210826-40-7, Catalog # inh-mcc) were purchased from InvivoGen (San Diego CA).Trans-ISRIB (CAS 1597403-47-8, Cat 5284) was from Tocris (Ellisville MO).Other chemicals, including glyphosate (CAS 1071-83-6), minocycline (CAS 13614-98-7, Cat# M2280000) and IL1-Ra (Cat# SRP 3084), quercetin (CAS 849061-97-8, PHR1488) and salts were obtained from Millipore Sigma Chemical Company.Roundup, a herbicide containing glyphosate, was purchased from a local store.Drugs were prepared as stock solutions in either ACSF or DMSO and diluted to nal concentration at the time of experiment.The concentrations of TAK-242, LPS-RS and minocycline are based on our previous studies using those inhibitors against lipopolysaccharide (LPS) and acrylamide (Izumi, Cashikar, et al., 2021;Izumi et al., 2022).The concentrations of MCC950 were also based on our previous paper (Izumi et al., 2022).The dose of TAK-242 in the behavioral study followed a proceeding report by Ono et al. (Ono et al., 2020).

Statistical analysis
Physiological data were collected and analyzed using PClamp software (Molecular Devices, San Jose CA).Data are expressed as mean ± SEM 60 min following HFS, and are normalized with respect to initial baseline recordings (taken as 100%).Statistical comparisons in physiological studies were based on IO curves at baseline and sixty minutes following HFS to determine the degree of change in EPSP slope at the 50% maximal point with p < 0.05 considered signi cant.Data in gures for physiological studies are from continuous monitoring of EPSPs at low frequency during the course of experiments and thus may differ from numerical results described in the text, which represent analyses based on comparison of input-output curves.Statistics were performed using commercial software (GraphPad Prism 9.2.0,GraphPad Software, La Jolla California).For comparisons of LTP results among 0 ppm, 0.4 ppm and 4 ppm Roundup, data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test.For comparisons of LTP results with 100 µM glyphosate, data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnet's multiple comparison test.For non-normally distributed data analysis of one-trial learning after in vivo injection of glyphosate, Kruskal-Wallis test followed by Dunn's multiple comparison test was used.

Glyphosate inhibits hippocampal LTP
In initial experiments, we exposed hippocampal slices to increasing doses of Roundup (a glyphosatebased herbicide, GBH) to determine whether it affects basal synaptic transmission in the CA1 region.When GBH was perfused in increasing concentrations every 30 min, EPSPs were suppressed by high concentrations of GBH and the suppression was irreversible after wash out of the herbicide (N=3, Fig. 1A).Although 20 ppm or less of GBH did not affect baseline EPSPs, administration of a 100 Hz x 1 sec HFS failed to induce LTP in slices pretreated with 4 ppm GBH for 2-4 hours (97.1 ± 2.2%, N=5, Fig. 1B).This is statistically smaller than matching control LTP in the absence of GBH (146.5 ± 11.6%, N=5, P=.0006).The degree of LTP induced in slices pretreated with 0.4 ppm GBH for 2-4 hours (124.2 ± 2.6%, N=5, Fig. 1B) is not statistically different from control LTP (P=0.851) but is larger than changes observed at 4 ppm (P=0.0351).

<Fig.1 near here>
Because glyphosate is the main ingredient in GBH, we next examined whether glyphosate itself alters basal transmission or LTP induction.In the absence of glyphosate, HFS consistently induced LTP in control slices (Control LTP: 135.0 ± 2.8% of baseline measured 60 min after HFS, N=5, Fig. 2A).

Glyphosate inhibits LTP & learning via pro-in ammatory signaling
Based on a recent study indicating that glyphosate evokes in ammatory responses (Winstone et al., 2022), we examined whether microglial activation is involved in the adverse effects on LTP.For these studies, we used minocycline, an agent that is known to inhibit microglial activation and to have antiin ammatory effects (Tikka & Koistinaho, 2001;Wu et al., 2015).We found that pre-treatment with minocycline overcame the inhibitory action of glyphosate.In slices pre-incubated with 0.5 µM minocycline, HFS readily induced LTP in the presence of 100 µM glyphosate (131.0 + 4.7%, N=7, P=0.0004 vs 100 µM glyphosate alone, Fig. 3A), supporting a role for microglia in the acute effects of glyphosate.

<Fig.5 around here>
To determine whether effects observed in ex vivo hippocampal slices translate into changes in learning and memory, we also examined the effects of glyphosate on a one-trial inhibitory avoidance task that has been linked previously to hippocampal LTP (Whitlock et al., 2006).Glyphosate was injected at a dose of 16.9 mg/kg, i.p. 24 hours before conditioning.This dose of glyphosate produced no noticeable changes in gait, coordination or body weight.However, glyphosate treatment had marked acute effects on performance in one-trial learning compared to saline-treated controls when tested 24 h after conditioning.
The glyphosate-induced defect in learning was manifest by rats more readily entering the dark chamber where they had been shocked during training, whereas saline-treated controls remained in the lit compartment for the full duration of the 300 second trial (P = 0.0048 by Dunn's test, N=8, Fig. 6).The adverse effects of glyphosate on learning were completely prevented by pretreatment with the TLR4 antagonist, TAK-242.In rats treated with TAK-242 (3mg/kg i.p. twice), glyphosate had no effect on onetrial learning (P=0.0048 vs. glyphosate alone by Dunn's test, N=5, Fig. 6).

Discussion
The primary mechanism of glyphosate in plants involves disruption of the shikimate pathway.Because this pathway is absent in animal cells, it has long been held that glyphosate is not harmful to animals.However, in 2015, the World Health Organization's International Agency for Research on Cancer identi ed glyphosate as a probable human carcinogen (Guyton et al., 2015).Although the carcinogenicity of glyphosate is still debated (Benbrook, 2019), there are other concerns with this environmental agent.In particular, there are now concerns that the CNS is one of the targets of glyphosate (Costas-Ferreira et al., 2022).Parkinson's disease (PD) was observed in a woman after chronic occupational exposure to GBH (Wang et al., 2011), and in rats even shorter exposure to glyphosate alters dopaminergic systems (Hernández-Plata et al., 2015; Martinez & Al-Ahmad, 2019).Additionally, a possible link with autism is speculated based on epidemiological data (von Ehrenstein et al., 2019), and maternal exposure to glyphosate results in autism-spectrum disorder (ASD)-like behaviors in murine offspring (Pu et al., 2020(Pu et al., , 2020;;von Ehrenstein et al., 2019).Excitotoxicity in the CNS is also possible because CSF levels of aspartate and glutamate double within a day after a single oral dose of GBH in rats (Limberger et al., 2020).In mice, intranasal exposure to GBH results in anxiogenic behaviors (Baier et al., 2017).Moreover, oral administration of 250-500 mg/kg GBH results in memory impairment in mice observed as decreased latency in a passive avoidance test (Bali et al., 2019).
In the present study, we rst tested if GBH itself alters basal synaptic function and found that EPSPs were acutely depressed by 200 ppm GBH, a level that is equivalent to 82 ppm or about 500 µM glyphosate.We also found that LTP, a mechanism of learning and memory, was completely disrupted by 4 ppm GBH.This level is higher than concentrations detected in healthy human uids.For example the maximal concentration in urine from young individuals in Germany is 11.1 µg/L (Lemke et al., 2021).However, the levels we examined in hippocampal slices may be observed in individuals who are exposed to Roundup accidentally or following a suicide attempt.Sato et al. ( 2011) described a semi-comatose woman in whom glyphosate was 122.5 µg/ml in CSF (and 1294.5 µg/ml in the serum) two days after ingestion.Given that 4 ppm GBH inhibits LTP induction and 200 ppm depresses synaptic transmission (Fig. 1), this level in the CSF is likely su cient to impair consciousness and cognitive function.
In our studies, we hypothesized that glyphosate in GBH causes neuroin ammation to impair cognitive function.It has been recently shown that oral administration of glyphosate to mice (125, 250 and 500 mg/kg for 14 days) elevates glyphosate levels (10-50 ng/mg) and tumor necrosis factor-(TNFα) in the brain (Winstone et al., 2022).This study has two important implications: orally administered glyphosate in ltrates the CNS and elevates pro-in ammatory cytokines in the CSF.The aforementioned case of GBH ingestion (Sato et al., 2011) also suggests that GBH may trigger in ammation in the CNS.Microglia are major contributors to neuroin ammation.Consistent with this, LTP was successfully induced in the presence of glyphosate when hippocampal slices were pretreated with minocycline, an inhibitor of microglia.The inhibitory effect of glyphosate on LTP induction at least partially shares mechanisms with LPS and acrylamide, both of which induce neuroin ammation (Izumi, Cashikar, et al., 2021;Izumi et al., 2022).The ability of LPS-RS to overcome effects of glyphosate suggests that glyphosate behaves like LPS in the CNS.Moreover, we observed that TAK-242, a selective TLR4 antagonist, clearly overcomes the effects of glyphosate in both LTP and behavioral experiments, suggesting that activation of TLR4 is pivotal for glyphosate to disrupt the CNS.
Although LTP induction was impaired by acute administration of 10 µM glyphosate, we used 100 µM glyphosate for our experiments because it consistently and completely blocked LTP induction, allowing us to determine mechanisms underlying its neurotoxicity.With this experimental paradigm, we observed that TAK-242 e ciently allows LTP induction in the presence of glyphosate.However, 100 µM glyphosate could be excessive and obscure other possible effective treatments.Consistent with this, we were surprised that MCC-950, a reliable NLRP3 inhibitor, failed to overcome the inhibitory effect of glyphosate on LTP induction because MCC-950 effectively overcomes the LTP inhibiting effects of acrylamide, another environmental toxin (Izumi, Cashikar, et al., 2021;Izumi et al., 2022).The failure of MCC-950 does not necessarily preclude a role for NLRP3 but the discrepancy may imply that glyphosate activates proin ammatory pathways in a manner different from other toxins.For example, proteasome inhibitors, which are used for the treatment of multiple sclerosis, induce in ammasome activation independent of NLRP3 (Ullrich et al., 2022).Similarly, in gout-related arthritis triggered by phagocytosis of monosodium urate crystals, caspase-1 activation, which is a fundamental mechanism for IL-1β secretion, occurs independently of NLRP3 (Chuang et al., 2020).
Even if the activation of pro-in ammation is independent from NLRP3, it can be attenuated by other cellular stress inhibitors.Because the ISR contributes to the pathogenesis of memory impairment and neurodegeneration accompanied by in ammation, systemic inhibition of ISR by ISRIB can reverse memory de cits (Chou et al., 2017).Consistent with this, ISRIB successfully overcame the inhibitory effect of glyphosate on LTP induction in the current study.Quercetin, a avonoid, attenuates in ammation by inhibition of endoplasmic reticulum stress (Feng et al., 2019).Interestingly, hepatotoxicity induced by sub-chronic administration of glyphosate in rats is reportedly attenuated by simultaneous administration of quercetin (Soudani et al., 2019).Moreover, it has been reported that quercetin overcame the decrease of reduced glutathione levels and increase in reactive oxygen species in the mouse hippocampus after sub-chronic exposure to a GBH (Bicca et al., 2021).Consistent with these reports, quercetin was effective in allowing LTP in the presence of glyphosate in our study.Regular dietary intake of quercetin in vegetables such as onions could help prevent neuroin ammation triggered by GBH if these vegetables are not contaminated with the herbicide.Although it is di cult to prevent GBH exposure as evidenced by that observation that glyphosate is detected in the urine of nearly all (99.8%) of the French population in one study (Grau et al., 2022), it is important to identify measures to prevent its neurotoxcity.These measures may include inhibitors of integrated stress responses, or modulators of endoplasmic reticulum stress.
In this study, we focused on direct neurotoxic aspects of glyphosate and found that glyphosate activates microglia via TLR4 and triggers cellular stress to impair hippocampal plasticity and learning.However, the neurotoxicity of GBH may not be limited to the direct actions of glyphosate.The GBH, Roundup, uses polyethoxylated tallow amine (POEA) as a surfactant and POEA can also contribute to toxicity (Brausch et al., 2007) because POEA is a strong inducer of ER stress (Mesnage et al., 2022).Gut microbiota dysbiosis by glyphosate also may result in neuronal impairment (Samsel & Seneff, 2013) because block of the shikimate pathway impacts microbiota.Furthermore, aminomethylphosphonic acid (AMPA), one of glyphosate's main metabolites, may have additional actions.Thus, the neurotoxicity of GBH is likely more complicated and perhaps more severe than the results observed in the present study.

Figure 4 An
Figure 4