Glyphosate and glyphosate‐based herbicide exposure during the peripartum period affects maternal brain plasticity, maternal behaviour and microbiome

Glyphosate is found in a large array of non‐selective herbicides such as Roundup® (Monsanto, Creve Coeur, MO, USA) and is by far the most widely used herbicide. Recent work in rodent models suggests that glyphosate‐based herbicides during development can affect neuronal communication and result in altered behaviours, albeit through undefined mechanisms of action. To our knowledge, no study has investigated the effects glyphosate or its formulation in herbicide on maternal behaviour and physiology. In the present study, relatively low doses of glyphosate (5 mg kg‐1 d‐1), Roundup® (5 mg kg‐1 d‐1 glyphosate equivalent), or vehicle were administered by ingestion to Sprague‐Dawley rats from gestational day (GD) 10 to postpartum day (PD)22. The treatments significantly altered licking behaviour toward pups between PD2 and PD6. We also show in the dams at PD22 that Roundup exposure affected the maturation of doublecortin‐immunoreactive new neurones in the dorsal dentate gyrus of the hippocampus of the mother. In addition, the expression of synaptophysin was up‐regulated by glyphosate in the dorsal and ventral dentate gyrus and CA3 regions of the hippocampus, and down‐regulated in the cingulate gyrus. Although a direct effect of glyphosate alone or its formulation on the central nervous system is currently not clear, we show that gut microbiota is significantly altered by the exposure to the pesticides, with significant alteration of the phyla Bacteroidetes and Firmicutes. This is the first study to provide evidence that glyphosate alone or in formulation (Roundup) differentially affects maternal behaviour and modulates neuroplasticity and gut microbiota in the mother.


INTRODUCTION
Glyphosate (N-phosphonomethyl-glycine) is the active compound found in over 750 broad-spectrum herbicides and is widely used across the world 1 . In less than 20 years, Glyphosate use has significantly increased, from around 50,000 tons of bioactive compound sold in 1994 to over 825,000 tons in 2014, mostly due to the development of Glyphosateresistant genetically modified corn and soy and pre-harvest application techniques 1,2 .
Glyphosate inhibits the 5-enolpyruvyl shikimate-3-phophate synthase (EPSPS), a key enzyme in the shikimate pathway 3,4 . This metabolic pathway is required for the biosynthesis of aromatic amino acids (tryptophan, tyrosine and phenylalanine) in plants, as well as in bacteria and fungi. Because this pathway is absent in vertebrates, Glyphosate was considered safe for animals and humans 3,5 . However, a few studies link Glyphosate-based herbicide (GBH) exposure to some cancers in human 1,6 while others do not find any association [7][8][9][10] , making risk assessment difficult for regulatory agencies. Indeed in 2015, Glyphosate was classified as "probably carcinogenic for humans" (Group 2A) by the International Agency for Research on Cancer 11 while the European Food Safety Authority 12 and the European chemical agency 13 concluded that Glyphosate is not carcinogenic, mutagen or genotoxic for reproduction. The debate on the safety of Glyphosate and Glyphosate-based herbicide in term of carcinogenicity is currently open [14][15][16][17] .
More recently, an increasing number of studies suggest that GBH exposure might affect vertebrate development and physiology [18][19][20] . More precisely, several laboratories found that Glyphosate exposure could alter brain physiology and behavior, independently of its potential carcinogenic and mutagenic properties. Indeed, GBH induced alteration in neurotransmitter dopamine, norepinephrine, serotonin and glutamate levels and their metabolites in several brain regions such as the hippocampus, substantia nigra and prefrontal cortex in adult male rats 21,22 as well as neurotransmitter binding to their cognate receptor 23 .

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. Consequently, these alterations in neurotransmission are likely to be the cause of deficits in locomotor activity and depressive-and anxiety-like symptoms observed in male mice and male and female rats following Glyphosate exposure [24][25][26][27] . Interestingly, while several studies have investigated the effect of perinatal exposure to GBH via the exposure to gestating and lactating females, there is no information about the mother's behavior and brain plasticity and how the dam herself could be affected by the treatment. In general, the vast majority of studies focus on the effects of the chemical environment on neurodevelopmental outcomes during the perinatal period, because of the intense neuroplasticity in the developing brain.
However, the neural circuitry of adult females is also significantly remodeled during pregnancy and lactation to adapt her behavior to her offspring's needs and survival in a variable environment [28][29][30][31] . Amongst the various brain regions remodeled around the peripartum period, including the medial preoptic nucleus, the ventral tegmental area and the bed nucleus of the stria terminalis, the hippocampus has received much attention because of its persistent high level of neuroplasticity, including adult neurogenesis and because of its sensitivity to social and chemical environmental factors [32][33][34] . It should be added that the ventral and dorsal region of the hippocampus present physiological and functional differences, with the agreed consensus that the dorsal hippocampus is involved in spatial cognition and more cognitive performance and memory in general while the ventral part is involved in the regulation of emotions, including stress and anxiety 35,36 . Previous data suggest the GBH exposure affects hippocampal physiology and plasticity 22,26,[37][38][39] . To our knowledge, there is no data on how Glyphosate could affect maternal behavior and the associated neuroplasticity.

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. It should also be noted that most of these effects were observed with relatively high doses of GBH (over 50 mg/kg/day Glyphosate-equivalent) and only a handful of studies investigated the impact of exposure to low dose 40 . Probably as important, there is little work comparing the effects of Glyphosate alone and in GBH on neural and behavioral outcomes. This is important as a few studies suggest that some of the observed adverse effects of GBH are not triggered by Glyphosate itself but by some adjuvants, or "inert" molecules, found in the pesticide formulation, such as the polyethoxylated tallowamine 41,42 .
Although it is hypothesized that the effects of Glyphosate in the brain might be linked to glutamate excitotoxicity and oxidative stress 21,26,38,43 , the molecular target(s) of Glyphosate in the brain is/are not well defined. As introduced above, Glyphosate is best known for its direct inhibitory effects on EPSPS. While this protein is mostly investigated in plants for its clear economical reason, the shikimate enzymatic pathway is also present in most bacteria. Interestingly, every vertebrate (and invertebrate) organism is the host for billions of microorganisms (mostly bacteria), collectively named microbiota. Modification of microbiota by chemical exposure (including antibiotic, diet, pre or probiotics…) is known to be associated to significant alterations of the organism's physiology, including defect in brain neurodevelopment and neuroplasticity 44-46 . Recently, a few studies showed that GBH exposure can affect gut microbiota, a process known as dysbiosis 40,[47][48][49] and such an alteration could result in mental health disorders, including anxiety and depression 24,25 .
Alternatively, or in addition, Glyphosate possess chelating properties and binds divalent cations (e.g. Mg 2+ , Ca 2+ , Fe 2+ , Mn 2+ , Cu 2+ , Zn 2+ ). This chelating effect in vivo is unclear and the outcome poorly investigated 50,51 . A reduction of these elements could lead to a significant alteration of most enzymatic cascades that usually require metallic cofactors but could also indirectly alter the microbiota.

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. Therefore, the aim of the present study was to compare the potential effects of relatively low dose (5 mg/kg/day, 1:10 th of the NOAEL, for "No Observable Adverse Effect Level") of Glyphosate and GBH on maternal behavior and maternal neuroplasticity, focusing on the hippocampus (dorsal and ventral) and cingulate gyrus. To measure changes in neurogenesis and cell structure, we focused on immunohistochemical techniques to investigate cell proliferation (Ki67-immunoreactivity(-ir)), immature neurons (doublecortinir), and pre-and post-synaptic protein densities (synaptophysin-ir and PSD95-ir) in the hippocampus. In the cingulate gyrus pre-and post-synaptic protein densities (synaptophysinir and PSD95-ir) were assessed. In addition, we also investigated signaling pathways that could be affected by Glyphosate exposure and lead to adverse neural outcomes. In parallel to brain and behavior analysis, we investigated whether Glyphosate alone or in formulation could affect the gut microbiota and/or modulate plasma concentration of several cations to affect neuroplasticity in the dam.

Animals
Twenty-one adult pregnant (gestational day (GD) 8) Sprague-Dawley rats (239-340 g) were obtained from Janvier Laboratories, France, and singly housed upon arrival at our animal facility. They had access to rat chow and tap water ad libitum and were kept under standard laboratory conditions in a 12:12 light/dark cycle (light on at 07:00 am to the weaning of the pups at postpartum day (PD) 21. Females were fed 1/9 th of a vanilla wafer cookie (Crousti fondante, Delacre®, Belgium) injected with treatment or vehicle.
Injections were made into the vanilla portion of the wafer. Observations were made to ensure that the biscuit was completely ingested by the dam and not shared with the offspring.
Cookies were consistently eaten in their entirety but the dams. Two females had problems during parturition and were euthanized (1 Control, 1 Glyphosate). Weight was recorded on GD10, PD1 and PD22. Figure 1 provides a timeline of the study.

Maternal behavior
One day after birth (PD1), litter was weighed, sexed and culled to 10 pups per litter (balanced sex ratio). Between PD1 to PD6, maternal behavior was scored during 5 minutes twice a day, directly in the cage without disturbing the animals. Scoring took place in the morning (between 09:30 and 13:30) and in the afternoon (between 14:00 and 16:30) with at least 1 h between the two sessions. An observer blind to the treatment scored the time the dams spent licking the pups, arched-back nursing (ABN), passive nursing, blanket nursing time the dams spent off the nest and the time the dams spent building the nest (NB). Data is reported as percentage of time spent for each maternal behavior. We analyzed maternal behavior on the first day following parturition independently from the other days, because the litter was handled and characterized, causing a short (~15 minutes) maternal separation and

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. potentially affecting behavioral outcome. Therefore, maternal behavior was investigated separately on PD1 and aggregated across PD2-6 for additional analyses.

Microbiome
In addition to standard cage changing, the dams were transferred to new cages with clean bedding on GD9, GD19 and PD22, and feces collected with sterilized tweezer the following day and transferred to sterile 1.5 ml tubes for microbiota analysis. Analysis of the feces prior to treatment (GD9) and at the end of the study (PD22) was done. There were no differences between groups at GD9 and only differences at PD22 are reported. No samples were pooled. Samples from GD19 were not used.

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. the run was checked internally using PhiX, and for further analysis, each pair-end sequence was assigned to its sample using the previously integrated index.
Bioinformatics analysis Sequences were trimmed for adaptors and assembled with Flash1.6.2 53 . PCR primers were removed and sequences with sequencing errors in the primers were excluded (Mothur) 54 . 12000 reads were randomly selected for each sample.
Chimera were removed with UCHIME 55

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Brain histology
Brains were sliced with a cryostat (Microm HM560) at 40 m, in series of 10.
Sections were stored at -16°C in ethylene glycol-based antifreeze solution until immunohistochemical processing.
Immunohistochemistry. Immunohistochemistry was performed as previously published 52,61-63 . Briefly, 1 series of sections was used for each marker (Ki67, Doublecortin, Synaptophysin and PSD95; see Table 1 for details). Sections were brought to room temperature and extensively rinsed in PBS before processing. For PSD95 staining only, sections were incubated in 10 mM sodium citrate buffer (pH 6.0) for 10 min at 80°C for antigen retrieval then rinsed in PBS before further processing. For all markers, endogenous peroxidase activity was blocked in 0.6 % H 2 O 2 for 30 min at room temperature. Sections were then incubated at room temperature in normal donkey serum (NDS, Sigma 5% in PBS containing 0.1% of triton (PBST), for Ki67 and Doublecortin) or in normal goat serum (NGS, 5%, synaptophysin or 2%, PSD95) for 30 min as described in Table 1. Tissue was then incubated at 4°C in primary antibody (Ki67, DCX, synaptophysin, PSD95, as described in Table 1). Sections were rinsed in PBST then incubated in secondary biotinylated antibody.
Signal amplification was performed using avidin-biotin-horseradish peroxydase complex

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Plasticity measures
Hippocampal volume. Cresyl-stained sections were observed under 2X magnification using an Olympus AX70 TRF and a DP71 digital camera with Cell^F software (Olympus).
Areas of the granular cell layer and subgranular zone (GCL/SGZ) throughout the dentate gyrus rostro-caudal extent were measured using ImageJ software 64 . Areas were summed and multiplied by the sampling interval (10) to derive an estimate of volume.  .
Optical densities (OD) were quantified with ImageJ (NIH) software. The relative optical density was calculated as the difference between optical density (grey level after calibration) of the area of interest and the background of an adjacent region 52,61 .

Statistical analysis
Data were analyzed using the software Statistica 13 (Dell Inc.) and GraphPad Prism Probabilities were considered significant when p < 0.05.

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General toxicity
Several parameters were measured to define potential general toxic effects of Glyphosate or Roundup. As expected, the weight of the dams was significantly different during pregnancy and the postpartum period (F (2,32) = 13.24, p < 0.0001;  Table 2).

Hippocampal Plasticity
Hippocampal volume. There were no significant effects of treatment by Glyphosate or Roundup on the volume of the dentate gyrus (F (2,16) = 0.2633, p = 0.7718, Table 3).
Therefore, total number of cell counts were used throughout.

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. Synaptophysin. There was a significant main effect of the treatment on synaptophysin OD in the dorsal region of the dentate gyrus (F (2,16) = 9.807, p = 0.0017, Figure 5A) with a significantly greater density of synaptophysin in Glyphosate and Roundup-treated dams compared to the Controls (p < 0.02). There was also a significant main effect of treatment on synaptophysin density in the ventral dentate gyrus (F (2,16) = 7.064, p = 0.0063, Figure 5B) with Glyphosate-treated dams having significantly greater synaptophysin density compared to both Control and Roundup treated dams (respectively p = 0.0025 and 0.0121). In the CA3

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. region, there was also a significant main effect of treatment on synaptophysin density (F (2,16) = 5.817, p = 0.0126, Figure 5C) with Glyphosate dams having a significantly greater synaptophysin density compared to Control and Roundup groups (p's < 0.02).
When looking at synaptophysin density in the cingulate gyrus there was also a significant main effect of treatment (F (2,15) = 4.908, p = 0.0229, Figure 5D) Table 4).

Microbiome
We also evaluated the impact of the Glyphosate and Roundup on the gut microbiota.
Composition of the gut microbiota did not display significant differences between groups at GD9, i.e before treatment (data not shown). At PND22, we found several significant differences at phylum, family and genus level between groups ( Figure 6). We found a significant effect of the treatment on Bacteroidetes (p = 0.009) and Firmicutes (p = 0.006), with respectively an increase and a decrease in Roundup group compared to the Control and Glyphosate (p's < 0.04; Figure 6B). We further analyzed the gut microbiota variation at the family level and showed a significant effect of the treatment on Lachnospiraceae (Phylum: Firmicutes, p = 0.033) which were less abundant in Roundup compared to Glyphosate dams (p = 0.013), with a trend in Roundup group compared to Control (p = 0.065; Figure 6C and D). We also found a significant effect of the treatment on Erysipelotrichaceae (Phylum: Firmicutes, p = 0.039), which were conversely more abundant in Roundup-treated dams

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. compared to Glyphosate (p = 0.017), and with a strong tendency in Roundup dams compared to Controls (p = 0.056; Figure 6C  Control and Glyphosate groups (p's < 0.05; Figure 6E). Turicibacter were more abundant in Roundup-treated dams compared to Glyphosate (p = 0.017), and there was strong tendency in Roundup compared to Control group (p = 0.056). In contrast Butyricicoccus genus decreased in Glyphosate group comparatively to Control and Roundup groups (p's < 0.04; Figure 6E).

DISCUSSION
Main findings of the present study show that peripartum exposure to a low dose of Glyphosate or Roundup affect maternal behavior, neuroplasticity and induce dysbiosis in dam rats. More specifically, we showed that perinatal exposure to Glyphosate or Roundup significantly altered maternal licking behavior without affecting other characteristics of the maternal behavior. In addition to the behavior, we also showed that Glyphosate alone or in formulation affected aspects of neurogenesis in the maternal brain postpartum. Indeed, there were more post-mitotic immature neurons in the dorsal dentate gyrus of Roundup dams, while cell proliferation and the total number of immature neurons were not affected.
Moreover, synaptophysin was modified following Glyphosate and Roundup perinatal exposure in dorsal and ventral dentate gyrus and CA3 of the hippocampus, as well as in the Accepted Article . cingulate gyrus, albeit with different profiles depending on the region of interest. It is important to highlight here that the behavioral and physiological outcomes following exposure to the active compound alone (Glyphosate) or to the formulation (Roundup) were also significantly different, confirming previous observations that the effects of formulations could be linked to adjuvants within these formulations 41,42 .

Absence of general toxicity.
In the present study, Glyphosate and Roundup had no significant effect on dam weight throughout pregnancy and the postpartum period. Similarly, the litter characteristics such as length, weight, sex ratio were not altered by the treatment. This observation is in agreement with recent studies that investigated dams weight during pregnancy 18,27 and litter characteristics 18,38,68 following exposure to relatively low dose of GBH. Only higher doses were found to reduce weight in juvenile or in pregnant rat (500 and 1000 mg/kg BW/d) peripartum exposure in dams 68 . We also confirm the absence of visible toxicity at the biochemical level, reflected by the low plasma levels of AST and ALT, two hepatic transaminases released in the vascular system upon liver aggression 60 . These results are in line with those obtained in adult male rats exposed for 5 weeks to 5, 50 and 500 mg/kg BW/day of Glyphosate, where only the highest dose led to a significant increase in AST and ALT circulating levels 69 .

Glyphosate and maternal behaviors.
In the present study, Control dams tended to spend more time licking their pups on the day following parturition, compared to Glyphosate or Roundup-treated animals. Additional maternal behaviors were not different between groups and time spent licking in these untreated Control dams was significantly reduced the following days. It is important to

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. highlight here that on the day following parturition the offspring were handled and separated from their mother for up to 10 minutes to record offspring characteristics and cull the litter.
Previous studies show that pup handling or isolation after parturition disrupts basal maternal behaviors, with most changes being associated with an increase in licking and grooming behaviors following the reintroduction of the pups with their mother [70][71][72][73][74][75]  little is known about the outcome on the mother and the underlying physiology. It is, however, likely that separation causes some form of anxiety, not only for the pup, but also for the mother. Interestingly, previous work suggests that early or adult exposure to Glyphosatebased herbicide increases anxiety and depression-like behavior in rodents 24,27,78 . Therefore, we can speculate that the altered interpretation of the social environment associated with anxiety or depressive-like behavior will result in modification in maternal care.
Rather surprisingly, the maternal behavior was later affected in the Roundup-treated animal between PD2 to 6, with an increase in licking behavior compared to dams from Control and Glyphosate groups. It could be that one of the adjuvant, such as polyethoxylated tallowamine, present in the Roundup formulation might be responsible for the effect as suggested by Mesnage and colleagues 41,42,79 , but this hypothesis remains to be tested.
It is also interesting to note that the offspring influences the amount of parental care given by the mother through direct tactile contact, olfactory cues, and ultrasonic vocalizations. For example in rodents, the male is more likely to initiate contact with the mother and in turn, licking and grooming from the dam toward their male offspring is usually more important [80][81][82] . A misinterpretation of these cues by the mother exposed to Glyphosate or Roundup could result in different amounts of licking as observed in the present study. It

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. should be kept in mind that in utero exposure of the offspring could have affected the sensory cues emitted by the offspring 83 . For example, perinatal exposure to organochloride pesticides such as chlorpyryfos or lindane significantly affected the ultrasonic vocalization emitted by pups 84,85 . The modification of the pup behavior following in utero exposure to Glyphosate would alter the care provided by the mother but this hypothesis remains to be investigated.
While most studies, including the present one, investigated only one part of the mother/offspring dyad (the mother), the behavior results from the interaction and constant feedback between the mother and the pups. Indeed, the polychlorinated biphenyl congener PCB 77 affected maternal behavior when both the mother and offspring were exposed, as confirmed by cross-fostering experiments 86 . This is an important reminder that the effects of many substances on offspring outcomes may be due, in part, to their effects on maternal caregiving behaviors. Future research needs to acknowledge the importance of maternal caregiving on offspring outcomes and investigate both parts of the mother/offspring dyad.

Neurogenesis in the dentate gyrus of the hippocampus.
Two brain regions, the sub-ventricular zone of the lateral ventricles and the subgranular zone in the dentate gyrus of the hippocampus are known to produce a large number of new neurons throughout adulthood [87][88][89][90] . Importantly, hippocampal neurogenesis is strongly modulated during pregnancy and the postpartum period in rodents 33,[91][92][93][94][95] . Research has also shown that neuroplasticity and neurogenesis in the hippocampus is strongly affected by the chemical environment [96][97][98][99] including medication 66,100,101 . In the present work we

Synaptic plasticity in the hippocampus
Findings of the present study also show that peripartum exposure to Glyphosate and Roundup increased synaptophysin expression in the dorsal region of the dentate gyrus and interestingly, decreased synaptophysin expression in the cingulate gyrus, an area of the medial prefrontal cortex. Glyphosate alone increased synaptophysin expression in both ventral dentate gyrus and CA3 regions of the hippocampus. This suggests region specific effects of Glyphosate and Roundup on synaptic proteins throughout the brain.
Synaptophysin is an membrane protein expressed at the surface of synaptic vesicles and can be found in most neurons within the central nervous system 105,106 . To our knowledge, the precise function of this protein is still not clear but studies suggest that synaptophysin is involved in calcium binding 107 , chanel formation 108,109 , exocytosis 110,111 , as well as synaptic vesicle recycling 112 . The change in synaptophysin expression following

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. peripartum exposure to Glyphosate is perhaps not surprising as several studies have demonstrated a change in neurotransmitter release and metabolism in various regions of the rat brain after exposure to Glyphosate or Glyphosate-based herbicides [21][22][23]26,27 . For example, high doses of Glyphosate lead to an increase in serotonin release and turnover in the striatum and hypothalamus and in dopamine turnover in prefrontal cortex and hippocampus 22 . The change in dopamine is likely to be related to the reduction of tyrosine hydroxylase expression 24 , the key enzyme responsible for dopamine synthesis. The modulation of dopamine levels can also be associated to the reduction in D1-dopamine specific receptor binding in the nucleus accumbens in adult male Sprague-Dawley rats exposed to Glyphosate 23 . It should be noted that the nucleus accumbens is a fundamental brain region involved in the motivation aspect of maternal behavior and modulation of activity within this brain region only can alter proper maternal care toward the offspring [113][114][115] . In addition to monoamines, acetylcholine esterase, the enzyme responsible for the breakdown of acetylcholine in the synaptic cleft to terminate synaptic transmission, is inhibited in the prefrontal cortex, striatum and hippocampus of male and female adult offspring after a perinatal exposure to Glyphosate 26,38 . Extracellular glutamate concentration and the reuptake of amino acids are also hindered in the hippocampus following exposure to Glyphosate-based herbicide 21,26 .
Whether or not the effects of glyphosate on the maternal brain reported in this study are unique to motherhood remain to be determined. Previous research shows that pregnancy and the postpartum period both affect plasticity in the hippocampus 33 . How exposure to this herbicide interacts with the normative changes in the female brain during pregnancy and the postpartum period is needed to understand the complexities of these relationships and how they may affect behavioral outcomes.

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Molecular target of Glyphosate
As introduced earlier, the molecular target of Glyphosate is the protein EPSPS, an enzyme found in plants and bacteria. This mode of action suggests that vertebrates are relatively safe if exposed to Glyphosate-based herbicides. However, the increased number of First, as mentioned in the introduction, the inhibition of the enzyme EPSPS not only impacts the growth and survival of plants, but also that of bacteria. Multicellular organisms are colonized with hundreds of species of bacteria, collectively known as microbiota and they exert a profound influence on key physiological processes. More precisely, microbiota present in the digestive system is known to exert major influence on neuroinflammation, activation of the stress axes, neurotransmission, and neurogenesis to name a few, resulting in the modulation complex behaviors, such as sociability and anxiety [116][117][118][119][120] . Gut microbiota influences the central nervous system through its ability to modulate the immune system, to provide bioactive metabolites such as fatty acids and synthesize neurotransmitters including GABA, noradrenaline, and dopamine 121,122 . Any dysregulation of the gut microbiome, or dysbiosis, is known to significantly affect the central nervous system and the resulting behavior, leading to increased susceptibility to psychiatric disorders 120,123

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. investigated the effect of the pesticide on microorganism ecology. While the precise role of each of these bacteria phyla for brain physiology is, to our knowledge, far from understood, a few studies have suggested that a dysregulation of Bacteroidetes or Firmicutes populations is associated to mental health disorders, including autism [129][130][131] and Alzheimer's disease 132,133 .
To support our findings, other studies, in rats but also in bees, report similar changes in Bacteroidetes and/or Firmicutes following Glyphosate or Glyphosate-based herbicide exposure 40,48,127,128 . Similarly, the population of the genera Alloprevotella is increased in our study, while studies have found an association between Parkinson and Alzheimer's disease and reduced population of the family of Prevotellaceae [134][135][136][137]  spleen and kidney in a dose dependent-way in adult male rat 69 . In addition, they found a significant increase of Mg 2+ in the brain following exposure for 35 days to 50 and 500 mg/kg Glyphosate. There was no information about the plasma levels. It is clear that additional studies should investigate this potential effect of Glyphosate on divalent cations and define whether the observed changes could lead to alter physiology in the organism.

Future Directions
This is the first study to investigate how glyphosate may affect the maternal brain and behavior. However, we acknowledge the much research is needed to determine the extent of these effects on the mother and offspring. Of particular importance in the present study would be to investigate how changes in the maternal brain are causally related to changes in the Accepted Article . maternal care-giving behaviors. In the present study we focused on hippocampal plasticity at the time of weaning, but future work would benefit from investigating brain changes in areas playing a key role in maternal care-giving behaviors (i. e. MPOA) at the time the care-giving is at its peak, early in the postpartum period.  Funding sources had no involvement in study design, in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the article for publication.

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.    There was a significant interaction between the cell morphology and the treatment in the dorsal GCL. * denotes significance (p < 0.05). Scale bar is 20 µm.  Data are presented as box plots. Significant difference (* p < 0.05 and ** p < 0.01, respectively