Nolwenn Adam

Marie-Amélie Lachayze

Caroline Parmentier

Hélène Hardin-Pouzet

Sakina Mhaouty-Kodja
email: sakina.mhaouty-kodja@sorbonne-universite.fr

Exposure to environmentally relevant doses of plasticizers alters maternal behavior and related neuroendocrine processes in primiparous and multiparous female mice Authors

Phthalates are organic pollutants frequently detected in the environment. The effects of these substances on male reproduction have been extensively studied but their potential impact on female reproductive behaviors in particular at environmental doses still remains to be documented. In the present study, we examined the effects of chronic exposure to di(2ethylhexyl) phthalate (DEHP) alone at 5 or 50 μg/kg/d, or in an environmental phthalate mixture on maternal behavior of lactating female mice after a first (primiparous) and a second gestation (multiparous). Exposure of DEHP alone or in a phthalate mixture reduced pupdirected behaviors, increased self-care and forced nursing behaviors and altered nest quality for both primiparous and multiparous dams. In pup-retrieval test, primiparous and multiparous dams exposed to DEHP alone or in a phthalate mixture retrieved their pups more rapidly, probably due to a higher emission of ultrasonic vocalizations by the pups. At lactational day 2 following the third and last gestational period, the neural circuitry of maternal behavior was analyzed. A lower number of oxytocin-immunoreactive neurons in the paraventricular and anterior commissural nuclei was found in dams exposed to DEHP alone or in a phthalate mixture, while no changes were observed in the number of arginine-vasopressin immunoreactive cells. In the medial preoptic area, exposure to DEHP alone or in a phthalate mixture reduced ERa-immunoreactive cell number. Dendritic spine density assessed for DEHP at 5 µg/kg/d was also reduced. Thus, exposure to DEHP alone or in a phthalate mixture altered maternal behavior probably through a neuroendocrine mode of action involving oxytocin and estrogen through ERa, key pathways necessary for neuroplasticity and behavioral processing.

Introduction

Phthalates, mainly used as plasticizers, are widely detected in the environment [START_REF] Gao | Phthalate esters in the environment: A critical review of their occurrence, biodegradation, and removal during wastewater treatment processes[END_REF]. Among the phthalate family, butyl benzyl phthalate (BBP), dibutyl phthalate (DBP), dicyclohexyl phthalate (DCHP) and di-isobutyl phthalate (DiBP) were identified as endocrine disrupting compounds for human health, and di(2-ethylhexyl) phthalate (DEHP) was recognized to have endocrine disrupting property for both human health and environment (for review: [START_REF] Adam | Behavioral Effects of Exposure to Phthalates in Female Rodents: Evidence for Endocrine Disruption?[END_REF]. Phthalates have mainly been studied for their ability to alter testosterone production and male urogenital tract development [START_REF] Howdeshell | Mechanisms of action of phthalate esters, individually and in combination, to induce abnormal reproductive development in male laboratory rats[END_REF][START_REF] Rowdhwal | Toxic Effects of Di-2-ethylhexyl Phthalate: An Overview[END_REF], but recent studies have shown that they can also induce female reproductive dysfunctions. Epidemiological studies reported associations between phthalates and the age of puberty in girls, ovarian functions, or development of endometriosis (for reviews: [START_REF] Hlisníková | Effects and Mechanisms of Phthalates' Action on Reproductive Processes and Reproductive Health: A Literature Review[END_REF][START_REF] Mesquita | Update about the disrupting-effects of phthalates on the human reproductive system[END_REF]. Experimental studies performed on rodents described adverse effects on puberty onset, ovarian function and fertility [START_REF] Fletcher | Effects of Phthalate Mixtures on Ovarian Folliculogenesis and Steroidogenesis[END_REF][START_REF] Hannon | The effects of phthalates on the ovary[END_REF][START_REF] Hlisníková | Effects and Mechanisms of Phthalates' Action on Reproductive Processes and Reproductive Health: A Literature Review[END_REF].

In this context, the potential effects of phthalate exposure on the neural control of female reproduction have been less studied. In particular, few studies have addressed the effects of adult chronic exposure to phthalates at environmentally relevant doses [START_REF] Adam | Behavioral Effects of Exposure to Phthalates in Female Rodents: Evidence for Endocrine Disruption?[END_REF]. We recently showed that adult exposure to low doses of DEHP alone or in a phthalate mixture reduced lordosis behavior, olfactory preference towards males, and the ability to attract and stimulate the emission of male ultrasonic vocalizations [START_REF] Adam | Exposure of Adult Female Mice to Low Doses of di(2-ethylhexyl) Phthalate Alone or in an Environmental Phthalate Mixture: Evaluation of Reproductive Behavior and Underlying Neural Mechanisms[END_REF]. These behavioral alterations were associated with reduced progesterone receptor (PR)immunoreactivity in the neural circuitry underlying female sexual behavior. It remains to be investigated whether adult chronic exposure to phthalates, under similar experimental conditions, alters other reproductive behaviors such as maternal behavior.

Maternal behavior includes all the behaviors elicited by the mother to ensure and improve pup survival and growth. Starting from pregnancy, female rodents engage in an intensive nest building and exhibit increased aggression towards intruders [START_REF] Numan | The neurobiology of parental behavior[END_REF]. After birth, dams show a strong interest towards pups by exhibiting licking and nursing behaviors.

Dams also display a retrieving behavior in response to ultrasonic vocalizations (USVs) emitted by pups when they are isolated outside the nest. Sensory information from the pups is integrated in the neural circuitry in which the hypothalamic medial preoptic area plays a key role under the control of a timely regulated hormonal sequence [START_REF] Keller | The parental brain and behavior: A target for endocrine disruption[END_REF]. In particular, the role of the oxytocin signaling pathway induced by estrogens at the end of pregnancy has been largely described [START_REF] Yoshihara | Oxytocin and Parental Behaviors[END_REF].

In a previous study, [START_REF] Lee | Effects of Prenatal Phthalate Exposure and Childhood Exercise on Maternal Behaviors in Female Rats at Postpartum: A Role of Oxtr Methylation in the Hypothalamus[END_REF] reported that developmental exposure of rats to DEHP at 10 mg/kg/d disturbed pup retrieval behavior. The effects of adult exposure to phthalates were addressed in four studies using an exposure period beginning during gestation and lasting until the lactational period. One study reported that exposure to relatively high doses of DBP (50 or 100 mg/kg/d) altered pup retrieval behavior as well as nest quality [START_REF] Lee | Dibutyl phthalate exposure during gestation and lactation in C57BL/6 mice: Maternal behavior and neurodevelopment in pups[END_REF]. In contrast, exposure of rats to high DINP doses (300-900 mg/kg/d) or to a phthalate mixture at low doses ranging from 0.2 to 1 mg/kg/d, or exposure of mice to low DEHP doses (5 to 400 μg/kg/d) was not found to disturb pup retrieval or maternal behavior [START_REF] Boberg | Reproductive and behavioral effects of diisononyl phthalate (DINP) in perinatally exposed rats[END_REF][START_REF] Kougias | Effects of Perinatal Exposure to Phthalates and a High-Fat Diet on Maternal Behavior and Pup Development and Social Play[END_REF][START_REF] Quinnies | Direct and transgenerational effects of low doses of perinatal di-(2-ethylhexyl) phthalate (DEHP) on social behaviors in mice[END_REF]. The causes of this discrepancy are not clear, but they might be related to differences in the experimental conditions (strain, duration of exposure, lactational period of analyses, etc.).

The present study was designed to perform a thorough analysis of the effects induced by chronic exposure to DEHP alone or in an environmental mixture on maternal behavior and potential underlying mechanisms. Adult C57BL/6J female mice were exposed to the vehicle (control group), DEHP alone at the tolerable daily intake (TDI) dose (50 µg/kg/d) or at a dose in the range of the environmental exposure (5 µg/kg/d), or to an environmental phthalate mixture containing DEHP (5 µg/kg/d), DBP (0.5 µg/kg/d), BBP (0.5 µg/kg/d), DiBP (0.5 µg/kg/d) and DEP (0.25 µg/kg/d) as previously described [START_REF] Adam | Exposure of Adult Female Mice to Low Doses of di(2-ethylhexyl) Phthalate Alone or in an Environmental Phthalate Mixture: Evaluation of Reproductive Behavior and Underlying Neural Mechanisms[END_REF]. Dams were exposed for 6 weeks before the first mating and treatment lasted until lactational day (LD) 2 following the third pregnancy and delivery. Primiparous (first litter) or multiparous (second litter) dams were analyzed for spontaneous behaviors including nest quality, pup-directed and self-care behaviors. They were also subjected to pup retrieval testing, and their pups were analyzed for the emission of ultrasonic vocalizations. Brains collected from dams at the end of treatment (LD2 following the third delivery) were analyzed for oxytocin-and arginine vasopressin (AVP)-immunoreactivity in the paraventricular, supraoptic and preoptic nuclei, together with estrogen receptor a (ERa)-immunoreactivity and dendritic spine density in the preoptic area.

Material and methods

Animals and treatments

Studies were performed according to the European legal requirements (Decree 2010/63/UE) and were approved by the "Charles Darwin" Ethical committee (project number 01490-01).

Experiments followed the Animal Research: Reporting in Vivo Experiments (ARRIVE) guidelines.

Mice were obtained from breeding of male and female C57BL/6J mice (Janvier Labs, Le Genest-Saint-Isle, France). They were housed in nest-enriched polysulfone cages with polysulfone bottles, at 22°C under an inverted photoperiod, i.e., dark time began at 1:00 p.m. with a 12:12 h light-dark cycle, with free access to food (A03-10; Safe-diets, Augy, France) and water.

Four treatment groups comprising each 12 adult eight-week old female mice were respectively exposed through the diet to the vehicle, DEHP at 5 µg/kg/d, DEHP at 50 µg/kg/d or DEHP in a phthalate mixture as previously described [START_REF] Adam | Exposure of Adult Female Mice to Low Doses of di(2-ethylhexyl) Phthalate Alone or in an Environmental Phthalate Mixture: Evaluation of Reproductive Behavior and Underlying Neural Mechanisms[END_REF][START_REF] Ahmadpour | Disruption of the blood-brain barrier and its close environment following adult exposure to low doses of di(2ethylhexyl)phthalate alone or in an environmental phthalate mixture in male mice[END_REF][START_REF] Ahmadpour | Effects and underlying cellular pathway involved in the impairment of the neurovascular unit following exposure of adult male mice to low doses of di(2-ethylhexyl) phthalate alone or in an environmental phthalate mixture[END_REF] and illustrated in Figure 1A. The DEHP dose of 50 µg/kg/d corresponds to the tolerable daily intake dose established by the European Food Safety Agency (EFSA, 2019). The DEHP dose of 5 µg/kg/d, within the estimated environmental exposure, and the mixture composition and doses of phthalates in this mixture were based on French and European studies showing the coexposure to these substances [START_REF] Blanchard | Assessment of Adult Human Exposure to Phthalate Esters in the Urban Centre of Paris (France)[END_REF] and the presence of their metabolites in urinary samples [START_REF] Anses | Connaissances relatives aux données de contamination et aux expositions par des composés de la famille des Phtalates[END_REF][START_REF] Dewalque | Estimated daily intake and cumulative risk assessment of phthalate diesters in a Belgian general population[END_REF]. Altered female estrous cyclicity and sexual behavior were observed for these doses of DEHP alone or in a phthalate mixture [START_REF] Adam | Exposure of Adult Female Mice to Low Doses of di(2-ethylhexyl) Phthalate Alone or in an Environmental Phthalate Mixture: Evaluation of Reproductive Behavior and Underlying Neural Mechanisms[END_REF].

Females were weighed to adjust phthalate doses every 5 days. Data on the mean body weight throughout the whole study are indicated in Figure S1A.

After 6 weeks of phthalate exposure, females were mated and treatment was maintained during three subsequent pregnancy and delivery periods (Figure 1B). Protocol details are available in Supplemental Methods. The treatment ended two days after the last delivery, and brains were collected and processed for immunofluorescence and dendritic spine density analyses.

Behavioral analyses

Tests were conducted 2 h after the beginning of the dark phase under red-light illumination, except for the nest score evaluation that was performed during the light phase. Dams in their home cages were acclimated for 3 h to the experimental room before the beginning of the behavioral observations by a blind experimenter. Analyses were performed during the lactational period following the first and second mating (Figure 1B) The analyses included spontaneous maternal behavior observation, nest score evaluation and pup retrieval tests. Pup ultrasonic vocalizations were also recorded for multiparous dams.

Spontaneous maternal behavior observation

The protocol of behavioral assessment was adapted from [START_REF] Palanza | Exposure to a low dose of bisphenol A during fetal life or in adulthood alters maternal behavior in mice[END_REF]. Primiparous and multiparous dams were observed daily from LD 2 to 15 in their home cages for 120 min. Each dam was observed every 4 min, for a total of 30 observations per female. The observer recorded the behavior the dam was displaying at the moment of the observation. The observed behaviors included pup directed-behaviors (nursing, pup licking and nest building), self-care behaviors (eating and drinking, grooming and resting outside the nest), and forced nursing as defined in Supplemental Methods. Data were expressed as the sum of behaviors displayed during the fourteen days of observations.

Nest score evaluation

The nest built by each dam was observed during the light phase (10 a.m.) at LD3, LD7, LD11, and LD15 for primiparous and multiparous dams. A nest quality score was given based on the scale of [START_REF] Hess | Home Improvement: C57BL/6J Mice Given More Naturalistic Nesting Materials Build Better Nests[END_REF]. A score of 0 corresponds to undispersed nesting material while 5 corresponds to a complete dome nest.

Pup retrieval test

Pup retrieval tests were performed at LD3, LD7, and LD11 after spontaneous maternal behavior observations. Primiparous and multiparous dams were briefly removed from their home cage and reintroduced after placing four pups in the opposing side of the nest. The cage was recorded for 10 min, and the recordings were analyzed a posteriori. The time taken by the dam from touching the first pup to retrieving the last pup to the nest was measured.

Pup emission of ultrasonic vocalizations

A first recording of emitted ultrasonic vocalizations (USVs) was conducted on PND3 pups born from primiparous females (Figure S2). Analyses of the obtained pup retrieval data motivated further recording of USVs at PND3, PND7 and PND11 for pups born from multiparous dams.

Tests were performed at least 30 min after a pup retrieval test.

One male and one female from each litter were tested. Each pup was put in a beaker placed on a 25°C heating pad and covered with a sound-reducing box, USVs were recorded for 3 min as previously described [START_REF] Adam | Exposure of Adult Female Mice to Low Doses of di(2-ethylhexyl) Phthalate Alone or in an Environmental Phthalate Mixture: Evaluation of Reproductive Behavior and Underlying Neural Mechanisms[END_REF][START_REF] Capela | Effects of pubertal exposure to low doses of di-(2-ethylexyl)phthalate on reproductive behaviors in male mice[END_REF][START_REF] Dombret | Update of the risk assessment of dibutylphthalate (DBP), butyl-benzyl-phthalate (BBP), bis(2-ethylhexyl)phthalate (DEHP), diisononylphthalate (DINP) and di-isodecylphthalate (DIDP) for use in food contact materials[END_REF]. The total number and duration of USVs and of each syllable type were analyzed.

Necropsy and tissue collection

Multiparous dams with a litter after the third mating (vehicle: n = 10; DEHP-5: n = 10; DEHP-50: n = 8; Mix: n = 8) were euthanized at LD2 by pentobarbital injection (120 mg/kg) (Figure 1B). Six females from each treatment group were transcardially perfused with a solution of 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). Brains from 5-6 dams were post-fixed overnight in 4% PFA in PBS and kept in a 0.5% PFA-PBS solution until immunofluorescence analysis was carried out. The remaining brains from 4 dams of the vehicle and the DEHP-5 groups were processed for Golgi-Cox staining; the lower number of lactating females from the DEHP-50 and Mix-groups did not enable us to perform such an analysis.

Immunofluorescence

Brains collected from multiparous females were sliced into 30-μm thick coronal sections in a vibratome (Leica Microsystems VTC1000S, Wetzlar, Germany). Oxytocin-and vasopressinimmunolabeling was conducted as previously described for incubation with primary rabbit antisera against AVP or OT with 1:4000 dilution [START_REF] Vacher | Activation by Serotonin and Noradrenaline of Vasopressin and Oxytocin Expression in the Mouse Paraventricular and Supraoptic Nuclei[END_REF]. ERa immunolabeling using 1/400 diluted polyclonal anti-ERa (Santa Cruz Biotechnology) was detailed in [START_REF] Adam | Exposure of Adult Female Mice to Low Doses of di(2-ethylhexyl) Phthalate Alone or in an Environmental Phthalate Mixture: Evaluation of Reproductive Behavior and Underlying Neural Mechanisms[END_REF]. The detection of the bound primary antibodies was performed for 2 h using the Alexa-Fluor 488-conjugated donkey anti-rabbit secondary antibody (A21206, Life Technologies-Invitrogen) diluted at 1:500. Brain sections were scanned by a high-resolution NanoZoomer Hamamatsu scanner (Hamamatsu Corporation). The number of oxytocin-, arginine vasopressin-and ERa-labeled cells was quantified from anatomically matched sections identified using the Mouse Brain Atlas of [START_REF] Paxinos | The mouse brain in stereotaxic coordinates[END_REF] as shown in Figures S3, S4 and S5, respectively. Immunolabeled cells for oxytocin and arginine-vasopressin were analyzed within the paraventricular and supraoptic nuclei (plates 36-41 and 36-39, respectively) and the anterior commissural nucleus of the dorsal preoptic area in Bregma -0.10 (Yoshihara et al. 2017). Data are expressed as the mean of immunoreactive cells per section. The number of ERa-immunoreactive cells was counted in the medial preoptic area within an area of 0.80 mm 2 (plate 30), in the bed nucleus of the stria terminalis within an area of 0.70 mm 2 (plate 30) and in the medial amygdala within an area of 0.56 mm 2 (plate 46). Data are expressed as the total number of immunoreactive cells per area.

Golgi-Cox staining

Brains collected from multiparous females were fixed for 8 days in Golgi-cox solution and cut into 30-μm thick coronal slices, which were post-impregnated in the same solution as previously described [START_REF] Jean | Revisiting medial preoptic area plasticity induced in male mice by sexual experience[END_REF]. Twelve neurons localized in the medial preoptic area were analyzed for each animal. The analyses were performed for each neuron on a dendritic length of 50 μm localized at 30 μm from the soma. Subtypes of dendritic spines were determined by applying the previously described criteria [START_REF] Harris | Three-dimensional structure of dendritic spines and synapses in rat hippocampus (CA1) at postnatal day 15 and adult ages: implications for the maturation of synaptic physiology and long-term potentiation[END_REF].

Statistics

Data expressed as means ± S.E.M were analyzed using GraphPad Prism 6 (GraphPad Software). Shapiro-Wilk test was performed to analyze the normality of data. A student paired t-test was used to analyze the number of dendritic spines. For data passing the normality test, the one-way analysis of variance (ANOVA) was used to analyze the main effect of exposure, followed by Bonferroni's post hoc tests to determine group differences. For data that did not pass the normality test, the Kruskal-Wallis test was used to analyze the effect of exposure, followed by Dunn's post hoc to determine group differences. Two-way ANOVA was used to analyze the pup USVs sex differences. P values less than 0.05 were considered significant.

Results

Effects of exposure to DEHP alone or in a phthalate mixture on reproductive parameters

Table S1 summarizes the reproductive parameters for primiparous and multiparous dams, including the ratio of pregnant and parturient dams out of the initial total number of mated females, gestation duration, litter size and sex of pups, as well as pup survival. No effects of the treatment to DEHP alone or in a phthalate mixture were found on any of these parameters, despite a tendency to decrease of pup survival for multiparous dams from the DEHP-50 group.

The body weight of females monitored throughout gestation and lactation was also comparable in the four treatment groups (Figure S1A). Their corresponding pups were also weighed during the period of behavioral analyses at postnatal days (PND) 3, 7, 11 and 15. No treatment effects were observed on the mean body weight of pups either (Figure S1B).

Spontaneous behaviors elicited by primiparous and multiparous dams exposed to DEHP alone or in a phthalate mixture

Pup-directed versus selfcare behaviors

Maternal behaviors exhibited by primiparous and multiparous dams were observed in their home cages from LD2 to LD15. Figure 2A shows the effect of treatment on the total number of pup-directed behaviors exhibited by primiparous dams (p = 0. 0002). A lower number of behaviors was observed for DEHP-5 (-26%), DEHP-50 (-34%) and Mix-groups (-21%) compared to the vehicle group. Detailed analyses show an effect of treatment on the number of nursing (p = 0.001), nest building (p = 0.0002) and pup licking behaviors (p = 0.027), with a reduced number of the two first behaviors in all three treatment groups and of the last behavior in the DEHP-5 group (Figure 2B). Figure 2C shows that an effect of treatment was observed on the number of self-care behaviors (p = 0.003), with an increased number of events for DEHP-5 (+25%), DEHP-50 (+23%) and Mix-groups (+21%) compared to the vehicle group. Analyses illustrated in Figure 2D show an effect of treatment on eating-drinking behavior (p = 0.004), with increased behaviors for the three treatment groups (+34%, +33% and +35%, respectively) compared to the vehicle group. Resting outside the nest was also affected by the treatment (p = 0.006), with increased behaviors for the DEHP-50 and the Mix group (+293% and +225% versus the vehicle group, respectively).

In multiparous dams, there was an effect of treatment on pup-directed behaviors (p = 0.029), in particular on nursing (p = 0.021) and nest-building behaviors (p = 0.005) but not on pup licking despite a tendency to decrease (Figure 2E-F). Lower nursing behavior was observed in the DEHP-50 group (-26%), and nest building behavior was reduced in the DEHP-5 and DEHP-50 groups (-35% and -38%, respectively) compared to the vehicle group (Figure 2F). Treatment effect was also seen on self-care behaviors (p = 0.041), with a higher number of behaviors in the DEHP-50 group (+24% versus the vehicle group) (Figure 2G). This was due particularly to the treatment effect on resting behavior (p = 0.004), which was increased for DEHP-5, DEHP-50 and Mix groups (+163%, +225% and 189%, respectively) compared to the vehicle group.

Eating-drinking behavior was no affected in multiparous females (p = 0.324) (Figure 2H).

In both primiparous and multiparous dams, grooming behavior was not affected by the treatment (p = 0.49 and p = 0.63, respectively) as shown in Figure 2D,H.

Nest score

The nest quality was analyzed at LD3, LD7, LD11 and LD15 by assigning a nest score, following the scale from [START_REF] Hess | Home Improvement: C57BL/6J Mice Given More Naturalistic Nesting Materials Build Better Nests[END_REF]. For primiparous dams (Figure 3A), an effect of treatment on the nest score was observed at LD7 (p = 0.024) with a significant modification in the DEHP-5 group, and at LD15 (p = 0.017) with a significant change in the DEHP-50 group compared to the vehicle group. For multiparous dams (Figure 3B), an effect of treatment was also observed at LD3 (p = 0.036), LD7 (p = 0.019) and LD15 (p = 0.014), with significant score changes in the DEHP-5 group compared to the control group.

Forced nursing

Exposure to DEHP alone or in phthalate mixture had an effect on forced-nursing behavior performed by pups from both primiparous (p < 0.0001) and multiparous dams (p = 0.0004) (Figure 3C). A higher number of pups exhibited this behavior in the DEHP-50 group of primiparous dams (+582%) and in DEHP-5, DEHP-50 and Mix groups of multiparous dams (+135%, +205% and +187%, respectively) compared to the corresponding vehicle groups.

Pup retrieval by primiparous and multiparous dams exposed to DEHP alone or in a phthalate mixture and emission of ultrasonic vocalizations by their pups

The ability of primiparous and multiparous dams to retrieve pups placed outside the nest was analyzed at LD3, LD7 and LD11 (Figure 4A-B). In primiparous dams, an effect of treatment on the retrieval duration was observed at LD3, LD7 and LD11 (p = 0.011, p = 0.013 and p = 0.036, respectively) (Figure 4A). A reduced duration was seen for DEHP-5 and DEHP-50 groups at LD3 (-29% and -36% versus the vehicle group, respectively) and for DEHP-50 and Mix groups at LD7 (-49% and -43%, respectively) compared to the vehicle group.

In multiparous dams, treatment affected the retrieval duration at LD3, LD7 and LD11 (p = 0.007, p = 0.017 and p = 0.013, respectively) (Figure 4B). A significant decrease was observed at LD3 for DEHP-5 and Mix groups (-30% and -33%, respectively), and for DEHP-50 at LD7 and LD11 (-39% and -49%, respectively) compared to the vehicle group.

Pup retrieval is elicited by dams in response to the USVs emitted by their pups, so USV emission by isolated pups was recorded. The data were grouped for the two sexes since our analysis did not show sex differences in the number of emitted USVs at any of the postnatal days tested (Figure S6). A first analysis conducted on PND3 pups born from primiparous females shows an effect of treatment on the number (p = 0.027) and duration of emitted USVs (p = 0.002), with significantly increased parameters in DEHP-5 and DEHP-50 groups (Figure S2).

Thorough analysis of pups from multiparous dams shows also an effect of treatment on the total number of emitted USVs at PND3 (p = 0.014), PND7 (p = 0.0004) and PND11 (p < 0.0001) (Figure 4C). Comparison with the vehicle group shows a higher number for the DEHP-5 group at PND7 (+265%) and PND11 (+250), for the DEHP-50 group at PND3, PND7 and PND11 (+275%, +238%, +193%, respectively), and for the Mix group at PND7 (+183%). A tendency to decrease was also observed at PND3 for DEHP-50 and Mix-groups as indicated by post hoc analyses (p = 0.0585 and p = 0.0703, respectively, versus the vehicle group).

Similarly, an effect of treatment was observed on the USV duration at PND3, PND7 and PND11 (p = 0.031; p = 0.0002 and p = 0.001, respectively) (Figure 4D). A longer USV duration was observed at PND7 and PND11 for DEHP-5 (+411 % and +263%, respectively), DEHP-50 (+448% and +156%) and Mix groups (+433%, and 170%, respectively) compared to the vehicle group.

A detailed analysis of emitted syllables was performed for pup vocalizations emitted at PND7 (Figure S7A). For the simple category, the treatment affected the total number of short (p = 0.0002) and downward syllables (p = 0.023) (Figure S4B), with a higher number of short syllables emitted by pups from DEHP-5 and DEHP-50 groups (+192% and +241%, respectively), and downward syllables emitted by pups from the DEHP-50 group (+141%) compared to the vehicle group. The treatment also affected the total duration of short (p = 0.0002), flat (p = 0.007) and downward syllables (p = 0.031) (Figure S7E), with a 200% higher duration of short syllables for all treatment groups and a 600% higher duration of flat syllables for the DEHP-50 group.

For the complex category (Figure S7C, F), the treatment affected the number and duration of both modulated (p = 0.011 and p = 0.001, respectively) and complex syllables (p = 0.012 and p = 0.04, respectively), with a higher number of modulated syllables for the DEHP-50 group (+230%) and a higher duration of modulated syllables for all groups (+417%, +533%, +350%, p = 0.005) compared to the vehicle group. For the frequency jump category, the treatment altered both the number and the duration of one-jump syllables (p = 0.002 and 0.011), with a higher number and duration for the Mix group (+624%, and +1250%) compared to the vehicle group (Figure S7D,G). Interestingly, no pups from the control group emitted frequency-jump syllables. These syllables were emitted by pups from the DEHP-and Mix-treated groups. An effect of treatment was observed on both the number and the duration of frequency-jump syllables (p = 0.011 and p = 0.006, respectively).

Neural oxytocin-immunoreactivity in multiparous dams exposed to DEHP alone or in a phthalate mixture

Given the importance of the oxytocinergic system in the expression of maternal behavior, we wished to find out whether the altered behavior of exposed dams was related to modifications in this neuroendocrine system. Analysis of oxytocin immunoreactivity was thus undertaken at LD2 following the third mating/delivery session. Specific cytoplasmic oxytocinimmunoreactivity was detected in the paraventricular nucleus, the supraoptic nucleus and the anterior commissural nucleus (Figures 5A,S3). Quantitative analyses show an effect of treatment on the number of oxytocin-immunoreactive neurons in the paraventricular nucleus (p = 0.009) and the anterior commissural nucleus (p = 0.002), but not in the supraoptic nucleus (p = 0.231) (Figure 5B). Post-hoc analyses showed a lower number of oxytocin-immunoreactive neurons in the paraventricular nucleus of DEHP-5 and DEHP-50 dams (-26% and -27% versus the vehicle group, respectively). A lower number of oxytocin-immunoreactive neurons was also observed in the anterior commissural nucleus of DEHP-50 and Mix dams (-29% and -28 versus the vehicle group, respectively).

Given the similarities in structure and function between oxytocin and arginine-vasopressin (AVP), we investigated whether the treatment also affected the number of AVPimmunoreactive neurons. Specific cytoplasmic AVP-immunoreactivity was detected in the paraventricular nucleus and the supraoptic nucleus (Figures S4,S8A), but quantitative analyses revealed no effect of treatment on AVP immunoreactivity in these regions (Figure S8B).

ERa-immunoreactivity and dendritic spine density in the medial preoptic area of multiparous dams exposed to DEHP alone or in a phthalate mixture

The medial preoptic area, the key region involved in processing of maternal behavior, integrates signals from oxytocin under the control of ERa-mediated regulation by estrogens [START_REF] Champagne | Natural Variations in Maternal Care Are Associated with Estrogen Receptor α Expression and Estrogen Sensitivity in the Medial Preoptic Area[END_REF][START_REF] Young | Estrogen receptor alpha is essential for induction of oxytocin receptor by estrogen[END_REF]. We thus quantified ERa-immunoreactivity in both the medial preoptic area and upstream brain areas including the medial amygdala and bed nucleus of stria terminalis. Figure 6A shows the presence of a specific nuclear signal in these three brain areas.

An effect of treatment was found on the number of ERa-immunoreactive neurons in the medial preoptic area (p = 0.005), with a lower number in DEHP-50 dams (-38% versus the vehicle group) (Figures 6B,S5). No treatment effects were observed in the two other brain regions.

Previous studies have shown that morphological and functional modifications related to dendritic spine density occurring in the medial preoptic area are important in the retention of maternal behavior in experienced dams [START_REF] Okino | The glutamatergic system in the preoptic area is involved in the retention of maternal behavior in maternally experienced female rats[END_REF]. The density and morphology of spines, which are emanating from neuronal dendrites and known to constitute excitatory post synapses, were thus analyzed in the medial preoptic area of multiparous dams from the vehicle and DEHP-5 groups. For this, brains were processed in Golgi-Cox staining and dendritic spines were analyzed and classified in stubby, thin and mushroom types according to their level of maturation as previously described [START_REF] Jean | Revisiting medial preoptic area plasticity induced in male mice by sexual experience[END_REF]. Figure 6C-D shows a lower number of total spines, which seemed to be mainly due to a reduced number of mushroom spines (-39% versus the vehicle group).

Discussion

The present study is the first to show that chronic exposure to low doses of DEHP alone or in an environmental mixture disturbs maternal behavior in both primiparous and multiparous dams. These behavioral alterations were to the observed reduced oxytocin-immunoreactivity in the paraventricular nucleus and ACN, and ERa-immunoreactivity and dendritic spine density in the medial preoptic area, the key brain areas controlling the expression of maternal behavior.

Exposure of females to DEHP alone or in an environmental mixture affected the expression of spontaneous maternal behavior in both naive (primiparous) and maternallyexperienced (multiparous) dams. Behaviors directed toward pups (nursing, nest building and pup licking) were reduced while, conversely, self-care behaviors (eating and drinking, outside the nest) were increased compared to the vehicle group. A global diminished nest quality was also observed in primiparous and multiparous dams exposed to DEHP alone or in a phthalate mixture. Despite these effects on maternal behavior, pup growth did not seem to be affected as evidenced by the comparable body weight measured for pups from mothers of the four treatment groups. This was possibly due to the fact that pups from primiparous or multiparous dams exposed to DEHP alone or in a phthalate mixture displayed more forced-nursing behavior. This kind of compensatory behavior exhibited by pups may then counterbalance the reduced nursing behavior of their corresponding mothers.

When pups were placed outside the nest, dams exposed to DEHP alone or in an environmental mixture retrieved their pups with a shorter latency compared to the vehicle group. This may appear contradictory with the reduced spontaneous behavior reported above.

However, it is important to stress that pup retrieval behavior is induced in response to USVs emitted by isolated pups. Interestingly, analyses of pup USV emission show that those born from dams exposed to DEHP alone or in a phthalate mixture produced a higher number and duration of USVs. First, this may explain, at least in part, why these pups were retrieved more rapidly by their mothers. Second, these data indicate that pup retrieval test, when used alone, cannot account for the effects on maternal behavior. An overall analysis including also spontaneous behaviors and pup emission of vocalizations is required.

The detailed analyses of USVs show that six out of the nine syllables (short, downward, modulated, complex, one-jump, frequency-jump) were disturbed. In particular, pups from the vehicle group did not show any emission of frequency-jump syllables, which were produced by those exposed through their mothers to DEHP alone or in a phthalate mixture. Whether this effect of pup-emitted vocalizations results from direct developmental exposure to phthalates and/or from a reduced maternal behavior remains to be investigated.

We recently reported reduced number and duration of USV emission during mating in adult males exposed under similar experimental conditions to DEHP alone or in a phthalate mixture [START_REF] Capela | Effects of pubertal exposure to low doses of di-(2-ethylexyl)phthalate on reproductive behaviors in male mice[END_REF][START_REF] Dombret | Update of the risk assessment of dibutylphthalate (DBP), butyl-benzyl-phthalate (BBP), bis(2-ethylhexyl)phthalate (DEHP), diisononylphthalate (DINP) and di-isodecylphthalate (DIDP) for use in food contact materials[END_REF]. To our knowledge, this is the first study showing an alteration of pup vocalizations following maternal exposure to phthalates.

Altered pup emission of ultrasonic vocalizations is widely used as a marker of autism-like behaviors in rodents [START_REF] Fischer | Ultrasonic vocalizations in mouse models for speech and socio-cognitive disorders: insights into the evolution of vocal communication[END_REF]. It is possible that these offspring exhibited deficits in social interaction. In agreement with this hypothesis, previous studies reported altered social behavior following perinatal exposure to phthalates in male and female rats and mice [START_REF] Kougias | Effects of Perinatal Exposure to Phthalates and a High-Fat Diet on Maternal Behavior and Pup Development and Social Play[END_REF][START_REF] Morová | A Mixture of Diethylhexyl, Diisononyl and Dibutyl Phthalate Decreased Anogenital Distance, Postnatal Testosterone Levels, and Changed Social Behavior in Wistar Rats[END_REF][START_REF] Quinnies | Direct and transgenerational effects of low doses of perinatal di-(2-ethylhexyl) phthalate (DEHP) on social behaviors in mice[END_REF][START_REF] Quinnies | Transgenerational Effects of Di-(2-Ethylhexyl) Phthalate (DEHP) on Stress Hormones and Behavior[END_REF][START_REF] Zhang | Prenatal exposure to di (2-ethylhexyl) phthalate causes autism-like behavior through inducing Nischarin expression in the mouse offspring[END_REF].

Overall, the present studies indicate an impact of exposure to low doses of DEHP alone or in a phthalate mixture on maternal behavior. To our knowledge, this is the first study addressing maternal behavior of both primiparous and multiparous dams. The comparable data obtained for primiparous and multiparous dams indicate that the deleterious effects exerted by phthalate exposure were maintained and did not change with maternal experience.

The lower spontaneous behavior of dams reported in this study contrasts with the unaffected behavior observed at relatively low doses of DEHP in mice [START_REF] Kougias | Effects of Perinatal Exposure to Phthalates and a High-Fat Diet on Maternal Behavior and Pup Development and Social Play[END_REF] and of a phthalate mixture in rats [START_REF] Quinnies | Direct and transgenerational effects of low doses of perinatal di-(2-ethylhexyl) phthalate (DEHP) on social behaviors in mice[END_REF]. This could be related to differences in the treatment duration, since in our study the exposure started 6 weeks before the first mating session and lasted for a long period until LD2 following the third delivery, with analyses performed on both primiparous and multiparous dams.

The expression of maternal behavior is regulated by oxytocin neurons located in the paraventricular nucleus [START_REF] Champagne | Naturally occurring variations in maternal behavior in the rat are associated with differences in estrogen-inducible central oxytocin receptors[END_REF][START_REF] Pedersen | Oxytocin activates the postpartum onset of rat maternal behavior in the ventral tegmental and medial preoptic areas[END_REF]. These neurons project to the medial preoptic area, where oxytocin acts on its oxytocin receptor to activate the behavior [START_REF] Okabe | Pup exposure facilitates retrieving behavior via the oxytocin neural system in female mice[END_REF]. This circuitry is highly sensitive to estrogens, with a high expression of ERa in the medial preoptic area [START_REF] Champagne | Natural Variations in Maternal Care Are Associated with Estrogen Receptor α Expression and Estrogen Sensitivity in the Medial Preoptic Area[END_REF][START_REF] Fang | A Hypothalamic Midbrain Pathway Essential for Driving Maternal Behaviors[END_REF][START_REF] Ribeiro | siRNA silencing of estrogen receptor-α expression specifically in medial preoptic area neurons abolishes maternal care in female mice[END_REF]. It is of great interest that exposure to DEHP alone or in a phthalate mixture induced changes in this circuitry in multiparous dams. Oxytocin-immunoreactivity was selectively reduced in the paraventricular nucleus, while no effects were found on AVP-immunoreactivity.

Reduced oxytocin-immunoreactivity was also found in the anterior commissure nucleus, a brain locus containing oxytocin neurons thought to modulate parental care in mammals [START_REF] Yoshihara | Oxytocin and Parental Behaviors[END_REF]. This shows that the oxytocinergic system can be also impacted by adult exposure to phthalates. A previous study reported changes in the expression of hypothalamic oxytocin following developmental exposure of rats to DEHP at 10 mg/kg/d [START_REF] Lee | Effects of Prenatal Phthalate Exposure and Childhood Exercise on Maternal Behaviors in Female Rats at Postpartum: A Role of Oxtr Methylation in the Hypothalamus[END_REF].

In parallel to these modifications, ERa-immunoreactivity was reduced in the medial preoptic area of dams exposed to DEHP alone or in a phthalate mixture. The behavioral effects and related changes in oxytocin-and ERa-immunoreactivity induced by exposure to DEHP alone or in a phthalate mixture were observed in maternally-experienced dams. This suggests that these chemicals may impact neuronal connectivity and underlying neuroplasticity processes, which occur in the medial preoptic area and are necessary for behavioral maintenance.

Quantification of dendritic spine density using Golgi staining in the medial preoptic area of multiparous dams showed a reduced total number of dendritic spines. In particular, an effect was mainly found on mushroom spines, which represent the most stable and mature spines. It is largely reported that maturation of dendritic spines towards mushroom characterizes learning processes and synaptic strength in the hippocampus and medial preoptic area and are associated with behavioral improvement [START_REF] Jean | Revisiting medial preoptic area plasticity induced in male mice by sexual experience[END_REF][START_REF] Leuner | New Spines, New Memories[END_REF][START_REF] Mahmmoud | Spatial and Working Memory Is Linked to Spine Density and Mushroom Spines[END_REF]. This suggests that exposure to DEHP alone or in a phthalate mixture interfered with neuroplasticity processes possibly involved in long-term maintenance of maternal behavior in the medial preoptic area. Previous studies showed that exposure of to DBP altered adult hippocampal neurogenesis in mice [START_REF] Lee | Dibutyl phthalate impairs neural progenitor cell proliferation and hippocampal neurogenesis[END_REF], while MEHP inhibited excitability and synaptic plasticity of CA3 hippocampal neurons [START_REF] Lu | Distinct Role of Mono-2-ethylhexyl Phthalate in Neuronal Transmission in Rat CA3 Hippocampal Neurons: Involvement of Ion Channels[END_REF]. The present study indicates that neural processes may be also impacted by phthalate exposure in other brain regions including the hypothalamus.

In conclusion, the present data describing disturbed maternal behavior, together with our previous study showing impaired estrous cyclicity and sexual behavior [START_REF] Adam | Exposure of Adult Female Mice to Low Doses of di(2-ethylhexyl) Phthalate Alone or in an Environmental Phthalate Mixture: Evaluation of Reproductive Behavior and Underlying Neural Mechanisms[END_REF], indicate that exposure to DEHP alone or in an environmental mixture may have an important impact on female reproduction in mice. For the majority of analyzed parameters and behaviors, comparable effects were observed for DEHP alone or in a phthalate mixture, suggesting that DEHP drove the effects possibly because it was more abundant than the other phthalates in this mixture.

The behavioral and neural effects of DEHP were observed at doses equivalent to or below the TDI of 50 μg/kg/d, established on the basis of reduced fetal testosterone production EFSA, 2019EFSA, , 2005)). This is added evidence in favor of a high brain sensitivity to exposure to environmental doses of phthalates. In particular, neuroendocrine pathways involving progesterone receptor [START_REF] Adam | Exposure of Adult Female Mice to Low Doses of di(2-ethylhexyl) Phthalate Alone or in an Environmental Phthalate Mixture: Evaluation of Reproductive Behavior and Underlying Neural Mechanisms[END_REF] and oxytocin and estrogen-dependent signaling (present study) were altered by exposure to environmental doses of DEHP alone or in a phthalate mixture. It is possible that these described effects and modes of action in mice may also occur in other mammalian species including humans, given the conserved hormonal regulation of reproductive processes.

Figure 1 .

 1 Figure 1. Schematic representation of the experimental design. A. Adult females were

Figure 2 .

 2 Figure 2. Effects of adult exposure to DEHP alone or in a phthalate mixture on pup-

Figure 3 .

 3 Figure 3. Effects of adult exposure to DEHP alone or in phthalate a mixture on forced-

Figure 4 .

 4 Figure 4. Effects of exposure to DEHP alone or in a phthalate mixture on pup-retrieval

Figure 5 .

 5 Figure 5. Effects of exposure to DEHP alone or in a phthalate mixture on oxytocinergic

Figure 6 .

 6 Figure 6. Effects of exposure to DEHP alone or in a phthalate mixture on estrogen

Figure S1 .

 S1 Figure S1. Effects of exposure to DEHP alone or in a phthalate mixture on body weight

Figure S2 .

 S2 Figure S1

  -immunoreactivity in the paraventricular nucleus (PVN), supraoptic nucleus (SON) and anterior commissural nucleus (ACN) of multiparous dams exposed to the vehicle (Vehicle), DEHP at 5 (DEHP-5) or 50 μg/kg/d (DEHP-50), or to a phthalate mixture (Mix) at lactational day 2. The insert shows the cytoplasmic immunostaining. Scale bar = 250 μm. ac, anterior commissure; f, fornix; opt, optic chiasma; 3V, 3 rd ventricle.

  Figure S5

  Figure S7

  Figure S6

  Figure S3

  Figure S4

Acknowledgments

This work was supported by Sorbonne Université, Centre National de la Recherche Scientifique (CNRS) and Institut National de la Santé et de la Recherche Médicale (Inserm). We thank the IBPS platform for taking care of the animals. and DEHP-5 groups. B-G. Total number (B-D) and duration (E-G) of syllables of the simple (B, E), complex (C, F) and frequency jump (D, G) categories. Data are presented as means ± S.E.M. Kruskal-Wallis tests for the treatment effect (#p < 0.05, ##p < 0.01, ###p < 0.001) followed by Dunn's multiple comparisons tests (*p < 0.05, **p < 0.01, ***p < 0.001 versus the vehicle group) are indicated.

CFID declaration

The authors declare they have no actual or potential competing financial interests.

Supplemental Methods

Figure S1

Methods

Gestational and lactational parameters

After 6 weeks of exposure, females were checked each morning for their estrous cycle stage.

Females at the proestrus stage were paired overnight with control C57BL/6J males. They were checked the next day for plugs and the day of the plug was gestational day (GD) 1. Females were then weighed every five days to adjust the phthalate doses. Females that did not gain weight during the first five days were paired with another male. If they did not get pregnant after 4 mating attempts, they were excluded from analyses. Females had a total of three pregnancy and delivery sessions as described in Figure 1B. The gestation duration, corresponding to the number of days from the observed plug until parturition was scored.

On postnatal day 1 (PND 1), litter size (number of pups per dam), pup survival (number of live pups divided by the total litter size), and sex of animals were reported. Sex was determined based on [START_REF] Wolterink-Donselaar | A method for gender determination in newborn dark pigmented mice[END_REF]. Litters were culled to 6 pups per dam, with a sex ratio of 1 when possible. Dams and their pups were weighed on lactational days (LD) 3, LD7, LD11, and LD15.

Spontaneous maternal behavior observation

The observed behaviors included: a)

Nursing: the dam allowed at least one pup to suckle.

b)

Licking pups: the dam was licking or grooming at least one pup. c) Nest building: the dam was rearranging her nest from being inside or outside the nest.

d)

Eating and drinking: the dam was eating its pellet or drinking from the bottle.


Suzanne Tran

Bertrand Mathon

Elise Morcos-Sauvain

Julie Lerond

Vincent Navarro

Franck Bielle
email: franck.bielle@aphp.fr

en français La neuropathologie de l'épilepsie vise à caractériser les lésions cérébrales à l'origine d'une épilepsie, identifiées lors d'une chirurgie curative ou plus rarement d'une biopsie ou d'une autopsie. Les principales caractéristiques histologiques et immunohistochimiques de plusieurs entités sont décrites : la sclérose hippocampique, les tumeurs associées à l'épilepsie à long terme, les malformations corticales, les malformations vasculaires, les cicatrices gliales, les encéphalites et la lipofuscinose neuronale focale. La démarche diagnostique, le diagnostic différentiel et les outils immunohistochimiques sont détaillés afin de fournir au pathologiste un référentiel synthétique pour aborder le large éventail des lésions épileptogènes.

Résumé en anglais

The neuropathology of epilepsy aims at diagnosing the cerebral lesions underlying epilepsy that are obtained from epilepsy surgery, or rarely from biopsy or autopsy. The main histopathological and immunohistochemical characteristics of several entities are described: epilepsy-associated hippocampal sclerosis, long term epilepsy-associated tumours, cortical malformations, vascular malformations, glial scars, encephalitides, and focal neuronal lipofuscinosis. The diagnostic approach, the differential diagnosis and the histochemical and immunohistochemical tools are detailed in order to provide the pathologist with a summarized toolkit to handle the broad range of epileptogenic lesions.

Introduction

L'épilepsie concerne 600 000 patients en France et peut se compliquer de traumatismes, d'un handicap et d'une surmortalité [START_REF] Baulac | Un ensemble de maladies complexe, encore mal compris 2018[END_REF]. En cas de résistance aux médicaments antiépileptiques (épilepsie pharmacorésistante), la chirurgie curative de l'épilepsie vise à reséquer la région cérébrale initiant les crises d'épilepsie, et donne accès au tissu cérébral.

Plus rarement, un prélèvement cérébral peut provenir d'une biopsie cérébrale, réalisée pour obtenir un diagnostic étiologique. Enfin des prélèvements peuvent être obtenus lors d'une autopsie.

L'analyse de 9500 échantillons de chirurgie de l'épilepsie a permis de montrer la fréquence des différents aspects histologiques qui seront détaillés dans les chapitres suivants (1): la sclérose hippocampique (36%), les tumeurs associées à l'épilepsie (24%), les malformations corticales (20%), les malformations vasculaires (6%), les cicatrices gliales (5%), les encéphalites (2%), l'absence de lésion (8%).

Sclérose Hippocampique associée à l'épilepsie

La sclérose hippocampique associée à l'épilepsie (SHAE) est la cause la plus fréquente d'épilepsie pharmacorésistante [START_REF] Blumcke | Histopathological Findings in Brain Tissue Obtained during Epilepsy Surgery[END_REF][START_REF] Semah | Is the underlying cause of epilepsy a major prognostic factor for recurrence?[END_REF]. La physiopathologie en reste mal comprise et repose probablement sur des mécanismes différents selon les patients [START_REF] Goldberg | Mechanisms of epileptogenesis: a convergence on neural circuit dysfunction[END_REF]. Les crises fébriles d'épilepsie de l'enfant sont un des principaux facteurs de risque de développer cette lésion.

La survenue de lésions de l'hippocampe chez l'enfant serait liée à l'excès d'activation neuronale (excitotoxicité) responsable d'une perte neuronale [START_REF] Klein | Commonalities in epileptogenic processes from different acute brain insults: Do they translate?[END_REF]. Une gliose astrocytaire se développe en parallèle du fait de la libération de nombreuses cytokines pro-inflammatoires, et est responsable de la consistance anormalement ferme du tissu hippocampique décrite par le terme de « sclérose » [START_REF] Thom | Review: Hippocampal sclerosis in epilepsy: a neuropathology review[END_REF]. Après un bilan préopératoire multidisciplinaire, la prise en charge neurochirurgicale consiste en l'ablation de l'amygdale et de l'hippocampe, parfois associée à la résection du pôle temporal [START_REF] Mathon | Surgical treatment for mesial temporal lobe epilepsy associated with hippocampal sclerosis[END_REF][START_REF] Mathon | Evolution of ideas and techniques, and future prospects in epilepsy surgery[END_REF]. L'examen macroscopique repose sur des tranches de section coronales de l'hippocampe [START_REF] Blumcke | International recommendation for a comprehensive neuropathologic workup of epilepsy surgery brain tissue: A consensus Task Force report from the ILAE Commission on Diagnostic Methods[END_REF]. Le diagnostic est fait dès la coloration standard par hématoxyline et éosine (H&E). Les immunomarquages anti NeuN (Neuronal nuclear antigen pour Antigène nucléaire neuronal) et anti GFAP (Glial Fibrillary Acidic Protein pour Protéine Acide Fibrillaire Gliale) facilitent l'évaluation de la perte neuronale et de la gliose.

Les étapes du diagnostic sont (i) le diagnostic de SHAE, (ii) la catégorisation selon la classification proposée par ILAE (International League Against Epilepsy) [START_REF] Blumcke | International consensus classification of hippocampal sclerosis in temporal lobe epilepsy: a Task Force report from the ILAE Commission on Diagnostic Methods[END_REF][START_REF] Calderon-Garciduenas | New clinicopathological associations and histoprognostic markers in ILAE types of hippocampal sclerosis[END_REF][START_REF] Deleo | Short-and longterm surgical outcomes of temporal lobe epilepsy associated with hippocampal sclerosis: Relationships with neuropathology[END_REF][START_REF] Na | Long-term seizure outcome for international consensus classification of hippocampal sclerosis: a survival analysis[END_REF][START_REF] Thom | Reliability of patterns of hippocampal sclerosis as predictors of postsurgical outcome[END_REF], et (iii) la recherche d'une deuxième lésion épileptogène associée (on parle alors de pathologie duale). Le diagnostic de SHAE repose sur la mise en évidence d'une perte neuronale dans la corne d'Ammon avec préservation des neurones du subiculum (Figure 1). La SHAE se démarque ainsi de la sclérose hippocampique associée aux maladies neurodégénératives dans laquelle la perte neuronale affecte également le subiculum [START_REF] Thom | Review: Hippocampal sclerosis in epilepsy: a neuropathology review[END_REF][START_REF] Miyata | Surgical pathology of epilepsy-associated nonneoplastic cerebral lesions: a brief introduction with special reference to hippocampal sclerosis and focal cortical dysplasia[END_REF]. La SHAE présente typiquement une gliose astrocytaire caractérisée par un très dense réseau de prolongements astrocytaires, et souvent une modification de la couche des grains du gyrus denté : perte neuronale, dispersion ou duplication (Figure 1) [START_REF] Thom | Review: Hippocampal sclerosis in epilepsy: a neuropathology review[END_REF][START_REF] Miyata | Surgical pathology of epilepsy-associated nonneoplastic cerebral lesions: a brief introduction with special reference to hippocampal sclerosis and focal cortical dysplasia[END_REF][START_REF] Blumcke | Towards a clinico-pathological classification of granule cell dispersion in human mesial temporal lobe epilepsies[END_REF]. Les neurones persistant dans le champ ammonien CA4 peuvent être hypertrophiques et ne doivent alors pas être confondus avec les neurones dysmorphiques de la dysplasie corticale focale de type II [START_REF] Thom | Hippocampal sclerosis with hypertrophy of end folium pyramidal cells[END_REF]. La classification selon ILAE dépend de l'importance de la perte neuronale dans les champs ammoniens CA1 et CA4. Le type 1, le plus fréquent, correspond à une perte sévère dans CA1 et CA4, le type 2 à une perte prédominant dans CA1, le type 3 à une perte prédominant dans CA4, le type « gliose isolée » à l'absence de perte neuronale significative (Tableau 1).

Cette classification corrèle avec des complications cognitives différentes et pourrait correspondre à des étiologies différentes [START_REF] Coras | Differential influence of hippocampal subfields to memory formation: insights from patients with temporal lobe epilepsy[END_REF][START_REF] Glover | ILAE type 3 hippocampal sclerosis in patients with anti-GAD-related epilepsy[END_REF]. La recherche d'une pathologie duale, c'est-à-dire la présence d'une deuxième lésion épileptogène doit être systématiquement faite dans l'hippocampe ou le tissu cérébral associé à l'hippocampe : gyrus parahippocampique voire le pôle temporal en fonction du type de résection. Cette seconde lésion peut appartenir aux différentes catégories de lésions épileptogènes citées en introduction, comme par exemple une dysplasie corticale focale.

Tumeurs associées à l'épilepsie à long terme

Bien que toute tumeur au contact du cortex cérébral puisse se compliquer d'épilepsie, on désigne par tumeurs associées à l'épilepsie à long terme (Long Term Epilepsy-associated Tumours) certaines tumeurs cérébrales où le pronostic est conditionné par l'épilepsie plutôt que par le risque oncologique [START_REF] Thom | Long-term epilepsy-associated tumors[END_REF][START_REF] Slegers | Low-grade developmental and epilepsy associated brain tumors: a critical update 2020[END_REF]. Il s'agit de tumeurs de volume stable pour lesquelles l'indication neurochirurgicale repose sur le contrôle de l'épilepsie et non sur celui de l'évolutivité tumorale. Les types tumoraux seront abordés succinctement ici (Tableau 2 et Le principal diagnostic différentiel à rechercher est un gliome diffus, particulièrement si l'apparition de l'épilepsie est récente (l'évolutivité volumétrique de la tumeur est alors inconnue) ou s'il existe une croissance du volume tumoral au cours du suivi radiologique.

Le gangliogliome et la tumeur neuroépithéliale dysembryoplasique (DNET) sont les plus fréquents. Il s'agit de tumeurs glioneuronales situées le plus souvent dans le lobe temporal.

Le gangliogliome présente une composante gliale, souvent astrocytaire piloïde, et une composante neuronale faite de cellules ganglionnaires [START_REF] Prayson | Cortical architectural abnormalities and MIB1 immunoreactivity in gangliogliomas: a study of 60 patients with intracranial tumors[END_REF]. La mise en évidence de cellules ganglionnaires binucléées permet d'affirmer leur caractère tumoral de façon objective. Des cellules ganglionnaires mononuclées sont possibles mais posent le problème du diagnostic différentiel avec des neurones résiduels. Ces tumeurs comportent typiquement des corps granuleux éosinophiles et un infiltrat inflammatoire lymphocytaire périvasculaire. 70% de ces tumeurs expriment CD34, souvent sous forme de cellules étoilées extravasculaires [START_REF] Prabowo | BRAF V600E mutation is associated with mTOR signaling activation in glioneuronal tumors[END_REF]. L'altération moléculaire la plus fréquemment retrouvée est la mutation BRAF V600E (40% des cas) [START_REF] Capper | Assessment of BRAF V600E mutation status by immunohistochemistry with a mutationspecific monoclonal antibody[END_REF][START_REF] Chappe | Dysembryoplastic neuroepithelial tumors share with pleomorphic xanthoastrocytomas and gangliogliomas BRAF(V600E) mutation and expression[END_REF][START_REF] Schindler | Analysis of BRAF V600E mutation in 1,320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma, ganglioglioma and extra-cerebellar pilocytic astrocytoma[END_REF]. La DNET se distingue par une architecture dénommée « l'élément glioneuronal spécifique » : celui-ci est formé de cellules pseudo-oligodendrogliales régulières associées à une substance fibrillaire et disposées autour d'axes parallèles séparés entre eux par une substance myxoïde contenant des neurones flottants [START_REF] Daumas-Duport | Dysembryoplastic neuroepithelial tumours[END_REF][START_REF] Daumas-Duport | Dysembryoplastic neuroepithelial tumor: a surgically curable tumor of young patients with intractable partial seizures. Report of thirty-nine cases[END_REF][START_REF] Thom | One hundred and one dysembryoplastic neuroepithelial tumors: an adult epilepsy series with immunohistochemical, molecular genetic, and clinical correlations and a review of the literature[END_REF]. Les altérations moléculaires les plus fréquentes sont les mutations faux sens de FGFR1 N546K et K656E (40%), la duplication du domaine tyrosine kinase de FGFR1 (40%), et plus rarement la mutation BRAF V600E (10%) [START_REF] Chappe | Dysembryoplastic neuroepithelial tumors share with pleomorphic xanthoastrocytomas and gangliogliomas BRAF(V600E) mutation and expression[END_REF][START_REF] Fina | Droplet digital PCR is a powerful technique to demonstrate frequent FGFR1 duplication in dysembryoplastic neuroepithelial tumors[END_REF][START_REF] Qaddoumi | Genetic alterations in uncommon low-grade neuroepithelial tumors: BRAF, FGFR1, and MYB mutations occur at high frequency and align with morphology[END_REF]. Le xanthoastrocytome pléomorphe (PXA) comporte des cellules aux noyaux pléomorphes et aux cytoplasmes épithélioïdes, abondants et lipidisés, entourées par une riche trame de réticuline réalisant des encorbellements monocellulaires [START_REF] Giannini | Immunophenotype of pleomorphic xanthoastrocytoma[END_REF][START_REF] Im | Pleomorphic xanthoastrocytoma: a developmental glioneuronal tumor with prominent glioproliferative changes[END_REF]. Ces tumeurs présentent souvent un infiltrat inflammatoire lymphocytaire et une expression de CD34. L'altération moléculaire la plus fréquente est la mutation BRAF V600E [START_REF] Capper | Assessment of BRAF V600E mutation status by immunohistochemistry with a mutationspecific monoclonal antibody[END_REF][START_REF] Chappe | Dysembryoplastic neuroepithelial tumors share with pleomorphic xanthoastrocytomas and gangliogliomas BRAF(V600E) mutation and expression[END_REF][START_REF] Schindler | Analysis of BRAF V600E mutation in 1,320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma, ganglioglioma and extra-cerebellar pilocytic astrocytoma[END_REF][START_REF] Phillips | The genetic landscape of anaplastic pleomorphic xanthoastrocytoma[END_REF]. Le gliome angiocentrique est constitué de cellules astrocytaires fusiformes réalisant des pseudo-rosettes périvasculaires : l'immunophénotype des cellules périvasculaires est Olig2 négatif et EMA positif punctiforme [START_REF] Lehman | Cortical ependymoma. A case report and review[END_REF][START_REF] Lellouch-Tubiana | Angiocentric neuroepithelial tumor (ANET): a new epilepsy-related clinicopathological entity with distinctive MRI[END_REF][START_REF] Wang | Monomorphous angiocentric glioma: a distinctive epileptogenic neoplasm with features of infiltrating astrocytoma and ependymoma[END_REF]. L'altération moléculaire constante est le gène de fusion MYB-QKI [START_REF] Bandopadhayay | MYB-QKI rearrangements in angiocentric glioma drive tumorigenicity through a tripartite mechanism[END_REF]. Récemment, des entités plus rares ont été décrites. La tumeur neuronale multinodulaire et vacuolisante (MVNT pour Multinodular Vacuolating Neuronal Tumour, assimilée aux gangliocytomes selon OMS 2016) est organisée en nodules dans la substance blanche et en moindre partie dans le cortex cérébral [START_REF] Louis | WHO classification of tumours of the central nervous system[END_REF][START_REF] Huse | Multinodular and vacuolating neuronal tumors of the cerebrum: 10 cases of a distinctive seizure-associated lesion[END_REF][START_REF] Nagaishi | Localized overexpression of alpha-internexin within nodules in multinodular and vacuolating neuronal tumors[END_REF][START_REF] Thom | Multinodular and vacuolating neuronal tumors in epilepsy: dysplasia or neoplasia?[END_REF][START_REF] Pekmezci | Multinodular and vacuolating neuronal tumor of the cerebrum is a clonal neoplasm defined by genetic alterations that activate the MAP kinase signaling pathway[END_REF]. Les cellules tumorales sont des neurones au cytoplasme vacuolisé séparés par un fond évoquant le neuropile du cortex cérébral. L'immunophénotype des neurones est anormal : NeuN négatif, Chromogranine A souvent négatif, Olig2 positif, ELAVL2/HuB positif (pour ELAV like neuron-specific RNA binding protein 2/Hu-antigen B, qui est un marqueur exprimé dans le lignage neuronal dès le stade de précurseur neuronal). Le fond fibrillaire est fortement positif pour Internexine-alpha. Les altérations moléculaires sont variables et activent la voie des MAP kinases : mutation BRAF G469S ou L597R, délétion de l'exon 2 ou 3 de MAP2K1, mutation MAP2K1 Q56P, gènes de fusion FGFR2-INA ou FGFR2- ZMYND11 [START_REF] Huse | Multinodular and vacuolating neuronal tumors of the cerebrum: 10 cases of a distinctive seizure-associated lesion[END_REF][START_REF] Pekmezci | Multinodular and vacuolating neuronal tumor of the cerebrum is a clonal neoplasm defined by genetic alterations that activate the MAP kinase signaling pathway[END_REF][START_REF] Choi | Clinicopathological and molecular analysis of multinodular and vacuolating neuronal tumors of the cerebrum[END_REF]. Les tumeurs neuroépithéliales polymorphes de bas grade du sujet jeune (PLNTY) présentent des cellules monomorphes pseudo-oligodendrogliales ou au noyau ovoïde et qui réalisent de vagues pseudorosettes autour d'une vascularisation endocrinoïde. Elles sont souvent fortement calcifiées et expriment souvent le CD34 [START_REF] Huse | Polymorphous low-grade neuroepithelial tumor of the young (PLNTY): an epileptogenic neoplasm with oligodendroglioma-like components, aberrant CD34 expression, and genetic alterations involving the MAP kinase pathway[END_REF][START_REF] Johnson | Plenty of calcification: imaging characterization of polymorphous low-grade neuroepithelial tumor of the young[END_REF].

Les altérations moléculaires les plus fréquentes sont des gènes de fusions impliquant FGFR2 et FGFR3 (par exemple FGFR3-TACC3) et les mutations BRAF V600E [START_REF] Huse | Polymorphous low-grade neuroepithelial tumor of the young (PLNTY): an epileptogenic neoplasm with oligodendroglioma-like components, aberrant CD34 expression, and genetic alterations involving the MAP kinase pathway[END_REF]. [START_REF] Taylor | Focal dysplasia of the cerebral cortex in epilepsy[END_REF]. L'immunomarquage anti-SMI32 (l'épitope non phosphorylé de la chaîne lourde du neurofilament) est positif de façon modérée dans le corps cellulaire des neurones pyramidaux de plus grande taille du cortex cérébral (il s'agit des neurones de projection longue dans les couches III, V et VI). Les neurones dysmorphiques présentent un marquage intense comblant leur large cytoplasme [START_REF] Blumcke | clinicopathologic spectrum of focal cortical dysplasias: a consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission[END_REF][START_REF] Knerlich-Lukoschus | Clinical, imaging, and immunohistochemical characteristics of focal cortical dysplasia Type II extratemporal epilepsies in children: analyses of an institutional case series[END_REF][START_REF] Liu | Early lipofuscin accumulation in frontal lobe epilepsy[END_REF]. SMI32 est ainsi un outil utile pour les détecter sur des prélèvements fragmentés ou de grande taille. Le cortex cérébral en regard des sillons nécessite une attention particulière lors de l'examen microscopique car c'est la localisation fréquente des petites FCD de type II. Les cellules ballonisées sont des cellules gliales immatures dont le noyau ovoïde ou réniforme est excentré dans un cytoplasme abondant d'aspect pâle et laqué [START_REF] Lamparello | Developmental lineage of cell types in cortical dysplasia with balloon cells[END_REF]. Un immunomarquage anti-Vimentine ou anti GFAP les identifie mais n'est pas spécifique par rapport aux astrocytes réactionnels qui sont cependant de plus petite taille [START_REF] Garbelli | Taylor's cortical dysplasia: a confocal and ultrastructural immunohistochemical study[END_REF][START_REF] Urbach | Focal cortical dysplasia of Taylor's balloon cell type: a clinicopathological entity with characteristic neuroimaging and histopathological features, and favorable postsurgical outcome[END_REF].

Les malformations du cortex cérébral

Le type III correspond aux malformations du cortex cérébral associées à une autre lésion épileptogène : une SHAE (type IIIa), une tumeur (type IIIb), une malformation vasculaire (type IIIc), ou une cicatrice gliale (type IIId). Lorsque les anomalies du cortex cérébral ne correspondent à aucun des types de FCD et qu'il n'existe pas d'autres lésions épileptogènes spécifiques, on peut utiliser le terme de malformations mineures du cortex cérébral (mild forms of Malformations of Cortical Development, mMCD) selon la classification de l'ILAE: soit mMCD de type I correspondant à un nombre augmenté de neurones dans la couche I du cortex cérébral, soit mMCD de type II correspondant à un nombre augmenté de neurones dans la substance blanche [START_REF] Blumcke | clinicopathologic spectrum of focal cortical dysplasias: a consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission[END_REF][START_REF] Emery | White matter neuronal heterotopia in temporal lobe epilepsy: a morphometric and immunohistochemical study[END_REF][START_REF] Liu | Highthroughput, automated quantification of white matter neurons in mild malformation of cortical development in epilepsy[END_REF][START_REF] Rojiani | Distribution of heterotopic neurons in normal hemispheric white matter: a morphometric analysis[END_REF][START_REF] Thom | Microdysgenesis in temporal lobe epilepsy. A quantitative and immunohistochemical study of white matter neurones[END_REF].

Malformations vasculaires

La malformation vasculaire la plus fréquemment associée à l'épilepsie est le cavernome ou malformation vasculaire cérébrale caverneuse [START_REF] Gray | Escourolle & Poirier's Manual of Basic Neuropathology[END_REF]. Il s'agit de lumières vasculaires béantes et anfractueuses bordées d'un simple endothélium et séparées entre elles de façon plus ou moins complète par des cloisons fibro-hyalines (Figure 4) [START_REF] Akers | Synopsis of Guidelines for the Clinical Management of Cerebral Cavernous Malformations: Consensus Recommendations Based on Systematic Literature Review by the Angioma Alliance Scientific Advisory Board Clinical Experts Panel[END_REF][START_REF] Caton | Cerebral Cavernous Malformations[END_REF][START_REF] Rigamonti | Cerebral cavernous malformations. Incidence and familial occurrence[END_REF]. La présence de tissu nerveux interposé ne doit pas faire exclure le diagnostic même si les lumières vasculaires du cavernome ont tendance à être davantage contiguës et coalescentes par rapport aux vaisseaux des malformations artério-veineuses. La chirurgie repose sur l'exérèse du cavernome lui-même mais aussi de la gliose périlésionnelle qui est chargée de dépôts d'hémosidérine et le siège d'un infiltrat inflammatoire lymphocytaire [START_REF] Baumann | Seizure outcome after resection of cavernous malformations is better when surrounding hemosiderin-stained brain also is removed[END_REF][START_REF] Dammann | Should we resect peri-lesional hemosiderin deposits when performing lesionectomy in patients with cavernoma-related epilepsy (CRE)?[END_REF]. En effet, le point de départ des crises d'épilepsie n'est pas dans le cavernome mais dans les réseaux neuronaux dysfonctionnels à son contact. L'examen microscopique de ce tissu nerveux périlésionnel peut montrer comme dans un gangliogliome un aspect piloïde avec des fibres de Rosenthal, un infiltrat inflammatoire lymphocytaire et des corps granuleux éosinophiles.

En revanche, dans un cavernome, il n'existe pas de population tumorale gliale, ni de cellules ganglionnaires binucléées permettant ainsi le diagnostic différentiel avec un gangliogliome à vascularisation pseudo-angiomateuse.

Les malformations artério-veineuses sont plus rarement impliquées dans la chirurgie de l'épilepsie, leurs complications principales étant hémorragiques [START_REF] Gray | Escourolle & Poirier's Manual of Basic Neuropathology[END_REF]. Elles sont la conséquence d'un shunt artério-veineux et sont constituées d'artères et de veines dilatées à paroi artérialisée séparées par un tissu nerveux gliotique.

Cicatrices gliales

Il s'agit de lésions focales séquellaires du tissu nerveux faisant suite, par exemple, à un accident vasculaire cérébral ou à un traumatisme crânien grave [START_REF] Gray | Escourolle & Poirier's Manual of Basic Neuropathology[END_REF]. Elles se caractérisent par une gliose astrocytaire qui peut être piloïde avec des fibres de Rosenthal et, de façon inconstante, par des macrophages ou sidérophages résiduels, et par des corps granuleux éosinophiles témoins d'une perte neuronale.

Encéphalites

L'absence de lésions

L'absence de lésion histologique sur un prélèvement de chirurgie de l'épilepsie est une situation courante, évaluée à 8% dans la série de Blumcke et al [START_REF] Blumcke | Histopathological Findings in Brain Tissue Obtained during Epilepsy Surgery[END_REF]. La corrélation des aspects microscopiques avec la clinique, l'imagerie et le geste chirurgical est alors essentielle. Si une lésion radiologique épileptogène était décrite, la résection peut ne pas avoir intéressé la lésion. Si la lésion radiologique est réséquée, la totalité du matériel n'a peut-être pas été adressée pour examen anatomopathologique. Par exemple, le neurochirugien peut réséquer la lésion avec un bistouri à ultrasons -aussi appelé cavitron ultrasonique-qui fragmente et aspire le tissu : les copeaux produits par cet instrument ne sont pas systématiquement adressés en anatomopathologie.

Enfin, certaines chirurgies de l'épilepsie sont réalisées en l'absence de lésion radiologique : la zone épileptogène est alors définie par l'intégration de données cliniques, d'examens de médecine nucléaire (hypométabolisme entre les crises sur la tomographie par émission de positons couplée à l'IRM et hypermétabolisme pendant les crises sur la tomographie par émission monophotonique) et par les enregistrements électrophysiologiques obtenus par l'implantation neurochirurgicale d'électrodes profondes intracérébrales. En l'absence de lésion radiologique, l'examen histologique peut révéler une dysplasie corticale focale qui n'avait pas de traduction radiologique ou bien peut ne révéler aucune lésion [START_REF] Lagarde | Can histologically normal epileptogenic zone share common electrophysiological phenotypes with focal cortical dysplasia? SEEG-based study in MRI-negative epileptic patients[END_REF]. En effet la zone épileptogène a été définie pendant le bilan préopératoire par des anomalies

Le point de vue du neurochirurgien

Obtenir un diagnostic histologique est essentiel dans la prise en charge neurochirurgicale d'un patient atteint d'épilepsie pharmacorésistante. En effet, l'examen neuropathologique permet de s'assurer, dans un grand nombre de cas, que le tissu cérébral réséqué pendant la chirurgie contient une lésion épileptogène. A fortiori, pour certaines entités comme la sclérose hippocampique, l'analyse neuropathologique permet de classer celle-ci en différents types, ce qui, à défaut d'avoir une valeur pronostique consensuelle pour le patient [START_REF] Deleo | Short-and longterm surgical outcomes of temporal lobe epilepsy associated with hippocampal sclerosis: Relationships with neuropathology[END_REF][START_REF] Mathon | Predictive factors of long-term outcomes of surgery for mesial temporal lobe epilepsy associated with hippocampal sclerosis[END_REF] Dans le futur, l'examen neuropathologique pourrait apporter une aide supplémentaire au neurochirurgien, en peropératoire, par un examen extemporané confirmant le caractère lésionnel de la pièce de résection, à l'instar de ce qui est fait en routine en neurochirurgie oncologique (78) mais le développement de nouvelles technologies anatomopathologiques adaptées est nécessaire. Le transfert de ces technologies pour la visualisation sous microscope opératoire, à l'aide de marqueurs cellulaires spécifiques, des lésions épileptogènes invisibles en IRM, (comme certaines dysplasies corticales focales ou lipofuscinoses neuronales focales), pourrait révolutionner la chirurgie de l'épilepsie [START_REF] Erkkila | Surgical microscope with integrated fluorescence lifetime imaging for 5-aminolevulinic acid fluorescence-guided neurosurgery[END_REF].

Le point de vue du neurologue

L'examen neuropathologique est fondamental pour une prise en charge optimale des patients ayant une épilepsie pharmaco-résistante. Il va permettre de confirmer l'étiologie de l'épilepsie. Les progrès de l'imagerie cérébrale, couplés aux données cliniques et électrophysiologiques permettent aujourd'hui d'évoquer de nombreuses étiologies différentes, mais seule l'étude histologique et immunohistochimique du prélèvement cérébral permet de confirmer la nature de la lésion. L'identification de la lésion va également apporter des informations pronostiques : certaines lésions, comme la sclérose hippocampique liée à l'épilepsie, de type 1, sont associées à un pourcentage très élevé de guérison postopératoire, c'est-à-dire un arrêt définitif des crises, et vont donc permettre une simplification des médicaments antiépileptiques, dans les années qui suivent la chirurgie. A l'inverse, l'identification d'autres lésions moins bien délimitées, comme par exemple, les tumeurs neuronales multinodulaires et vacuolisantes doit conduire à un allégement plus prudent des médicaments anti-épileptiques. Enfin, l'examen neuropathologique est crucial pour appréhender la physiopathologie de ces lésions épileptogènes, et donc l'identification de nouvelles stratégiques thérapeutiques, et pourrait permettre à l'avenir le développement de thérapies personnalisées. Par exemple, l'identification de perturbations spécifiques dans la voie métabolique de mTOR, dans les dysplasies corticales focales, pourrait permettre le recours à des inhibiteurs de cette voie, comme c'est déjà le cas dans la sclérose tubéreuse de Bourneville [START_REF] Marsan | Review: Mechanistic target of rapamycin (mTOR) pathway, focal cortical dysplasia and epilepsy[END_REF]. L'identification précise des différents contingents inflammatoires d'une encéphalite auto-immune va également guider le clinicien en lui permettant de choisir le traitement immuno-suppresseur adapté.

Conclusion

La neuropathologie de l'épilepsie rassemble un éventail très large, encore incomplet, de lésions d'étiologies variées (Figure 7). Les outils immunohistochimiques et moléculaires pour caractériser ces lésions s'enrichissent pour assister le pathologiste. L'intégration de l'examen anatomopathologique avec le contexte clinique, les données radiologiques et les constatations peropératoires aide l'interprétation des aspects histopathologiques. L'identification et la classification des lésions épileptogènes permettent de confirmer que l'exérèse neurochirurgicale a bien intéressé la cause de l'épilepsie. Elles sont également nécessaires au progrès de la compréhension et du traitement des causes de l'épilepsie pour un objectif principal : le patient libre de crise. Références bibliographiques : [START_REF] Slegers | Low-grade developmental and epilepsy associated brain tumors: a critical update 2020[END_REF][START_REF] Louis | WHO classification of tumours of the central nervous system[END_REF][START_REF] Wang | Monomorphous angiocentric glioma: a distinctive epileptogenic neoplasm with features of infiltrating astrocytoma and ependymoma[END_REF][START_REF] Bandopadhayay | MYB-QKI rearrangements in angiocentric glioma drive tumorigenicity through a tripartite mechanism[END_REF][START_REF] Huse | Multinodular and vacuolating neuronal tumors of the cerebrum: 10 cases of a distinctive seizure-associated lesion[END_REF][START_REF] Nagaishi | Localized overexpression of alpha-internexin within nodules in multinodular and vacuolating neuronal tumors[END_REF][START_REF] Thom | Multinodular and vacuolating neuronal tumors in epilepsy: dysplasia or neoplasia?[END_REF][START_REF] Pekmezci | Multinodular and vacuolating neuronal tumor of the cerebrum is a clonal neoplasm defined by genetic alterations that activate the MAP kinase signaling pathway[END_REF][START_REF] Choi | Clinicopathological and molecular analysis of multinodular and vacuolating neuronal tumors of the cerebrum[END_REF] * : Les cellules ganglionnaires tumorales peuvent être mononucléées mais il est alors difficile de les distinguer de neurones résiduels. La détection de cellules ganglionnaires binucléées permet de mettre en évidence une composante tumorale neuronale de façon objective.

Légendes des Figures

Figure 2 )

 2 Figure 2) et sont présentés en détails dans la classification selon l'OMS 2016 (22) à l'exception des PLNTY (pour Polymorphous Low-Grade Neuroepithelial Tumour of the Young) qui sont cependant reconnues dans l'actualisation n°6 par le comité cIMPACT-NOW (consortium to inform molecular and practical approaches to CNS tumor taxonomy pour consortium visant à notifier les méthodes moléculaires et pratiques de taxonomie des tumeurs du système nerveux central) (23). L'identification des tumeurs associées à l'épilepsie à long terme peut être aidée par la mise en évidence d'altérations moléculaires.

  Les malformations du cortex cérébral relevant d'une chirurgie curative de l'épilepsie correspondent le plus souvent aux dysplasies corticales focales (FCD pour Focal Cortical Dysplasia) (48). La classification proposée par l'ILAE en 2011 distingue trois catégories (15, 49) (Tableau 3). Le type I correspond aux anomalies de l'architecture du cortex cérébral en l'absence de neurones dysmorphiques et en l'absence d'autres lésions décrites dans le type III. Le type Ia se caractérise par l'alignement anormal des neurones en colonne radiaire, le type Ib par une anomalie des couches du cortex, et le type Ic par une association des anomalies Ia et Ib. Le diagnostic du type I reste subtil et sa signification clinique est discutée. Le type II correspond à une malformation du cortex cérébral comportant des neurones dysmorphiques soit dépourvus de cellules ballonisées (IIa), soit associés à des cellules ballonisées (type IIb auparavant dénommé « dysplasie de Taylor »). Le neurone dysmorphique est défini -comme son nom ne l'indique pas-par une taille très augmentée du noyau (15 à 28 µm versus une normale de 10 à 18 µm) et du corps cellulaire (16 à 43 µm versus une normale de 12 à 25 µm). Il s'y associe de nombreux corps de Nissl et une forme souvent anormalement globuleuse, « dysmorphique », du corps cellulaire par rapport à la silhouette triangulaire des neurones pyramidaux normaux du cortex cérébral (Figure 3) (50,

Certaines

  encéphalites autoimmunes (EAI) sont des causes de crises d'épilepsie transitoires et d'épilepsie à long terme (67-69). Leur diagnostic et leur classification reposent sur la présentation clinico-radiologique et sur la détection d'auto-anticorps. Néanmoins, une biopsie cérébrale peut parfois être pratiquée pour écarter un diagnostic différentiel ou consolider le diagnostic en vue d'un traitement immunosuppresseur. Les EAI sont divisées en deux catégories selon que les auto-anticorps visent des antigènes intracellulaires ou de surface. Les EAI liées à des auto-anticorps dirigés contre les antigènes intracellulaires sont médiées par des lymphocytes T cytotoxiques infiltrant de façon marquée le tissu cérébral et réalisant des figures d'apposition contre les neurones : ces contacts lymphocyte/neurone et la perte neuronale à plus long terme traduisent l'agressivité des lymphocytes envers les neurones (70, 71). Les cellules microgliales sont activées et se regroupent en amas sans neurone appelés nodules microgliaux ou en amas centrés par un neurone en voie de résorption appelés nodules de neuronophagie. Les auto-anticorps auraient ici un rôle secondaire dans la physiopathologie. De façon différente, les EAI liées à des auto-anticorps dirigés contre les antigènes de surface présentent peu d'infiltrat lymphocytaire. Les autoanticorps ont un rôle principal dans la physiopathologie. Il existe, dans l'EAI associée aux auto-anticorps dirigés contre le complexe du canal potassium contrôlé par le voltage (voltage-gated potassium channel-complex) des dépôts de complément à la surface des neurones (71). L'encéphalite de Rasmussen, décrite par Théodore Rasmussen en 1958 (72), est une maladie rare, sporadique, d'étiologie inconnue, survenant le plus souvent chez l'enfant, et touchant un hémisphère cérébral par extension de proche en proche dite « en tâche d'huile ». Elle évolue en trois phases : la phase prodromale (durée des symptômes de 0 à 8 ans, médiane de 7 mois) avec des crises d'épilepsie rares et des déficits neurologiques inconstants, la phase aiguë (durée de 4 à 8 mois) avec des crises fréquentes et l'installation de déficits neurologiques sensitivomoteurs ou cognitifs, et la phase résiduelle avec des déficits neurologiques permanents et une épilepsie à long terme (69). Les aspects histologiques correspondent aux mêmes lésions élémentaires que les EAI associées aux anticorps contre des antigènes intracellulaires : un infiltrat lymphocytaire T cytotoxique CD3+ CD8+ Granzyme B+ réalisant des figures d'apposition contre les neurones, des nodules microgliaux et/ou de neuronophagie, une perte neuronale et une gliose (Figure 5). L'infiltration lymphocytaire et les nodules microgliaux prédominent à la phase aiguë et ont pour corrélat radiologique un hypersignal cortical et sous-cortical tandis que la perte neuronale et la gliose prédominent à la phase résiduelle et ont pour corrélat radiologique une atrophie limitée à un hémisphère cérébral (73). Lipofuscinose Neuronale Focale Récemment, Liu et al ont identifié un nouvel aspect histologique au sein de six prélèvements de chirurgie d'épilepsie frontale (53). Il s'agissait d'un contexte clinique similaire à celui des dysplasies corticales focales : les patients ont bénéficié d'une résection d'une zone épileptogène identifiée par des études électrophysiologiques alors que l'aspect radiologique était normal ou proche de la normale. L'étude microscopique a mis en évidence des neurones de taille anormalement grande en raison d'un cytoplasme dilaté par l'accumulation d'un large et unique amas de lipofuscine. Cet amas est positif avec les colorations de Noir Soudan, de PAS ou du bleu de luxol ; il est faiblement positif pour les immunomarquages anti ubiquitine et anti p62. De tels aspects ne sont pas observés dans les dysplasies corticales focales. De plus, bien que les neurones normaux contiennent de la lipofuscine qui s'accumule au cours de la vie, la quantité de lipofuscine est excessive dans les lésions de lipofuscinose neuronale focale. L'analyse protéomique par microdissection puis spectrométrie de masse a montré une quantité augmentée de protéines constituant la lipofuscine et de protéines impliquées dans le trafic vésiculaire et l'autophagie : cette accumulation serait ainsi le résultat d'un excès d'autophagie (53).

  fonctionnelles et non par l'existence d'une lésion anatomique. Ce bilan préopératoire long et méticuleux repose souvent sur l'implantation d'électrodes profondes intracérébrales pour l'enregistrement de l'activité neuronale des régions cérébrales lors des crises et pour préciser ainsi le point de départ anatomique des crises (75). Un piège consiste à interpréter les remaniements induits par les électrodes comme une lésion épileptogène : l'inflammation n'est alors pas en rapport avec une encéphalite, les astrocytes au large cytoplasme immunopositif pour la Vimentine sont alors des astrocytes réactionnels et non les cellules ballonisées d'une FCD type IIb. Quand aucune lésion n'est identifiée histologiquement sur la coloration standard, des techniques complémentaires doivent être réalisées de façon systématique afin d'augmenter la sensibilité de détection des étiologies les plus fréquentes. Un exemple de panel de techniques est donné dans le tableau 4.

  , permet d'affiner l'identification du syndrome épileptique. Dans les cas où le patient n'est pas libre de crise après une chirurgie de l'épilepsie, l'absence de lésion épileptogène dans la pièce opératoire représente un argument fort pour éventuellement proposer au patient une seconde intervention chirurgicale. Dans certains cas d'encéphalites épileptiques de classification difficile ou avant d'introduire un traitement immunosuppresseur, une biopsie cérébrale peut être réalisée (77). Également, certaines tumeurs bénignes épileptogènes (gangliogliomes, DNETs) sont parfois difficilement accessibles à une chirurgie d'exérèse et peuvent être diagnostiquées à l'aide d'une biopsie cérébrale stéréotaxique. Le rôle du neuropathologiste s'en trouve ainsi être décisif pour guider la prise en charge thérapeutique du patient dans les suites de ce geste neurochirurgical à visée exclusivement diagnostique.

Figure 1 .

 1 Figure 1. Aspects histopathologiques de la sclérose hippocampique associée à l'épilepsie

Figure 2 .

 2 Figure 2. Aspects histopathologiques des tumeurs associées à l'épilepsie à long terme

Figure 3 .

 3 Figure 3. Aspects histopathologiques des dysplasies corticales focales

Figure 4 .

 4 Figure 4. Aspects histopathologiques de cavernome.

Figure 5 .

 5 Figure 5. Aspects histopathologiques de l'encéphalite de Rasmussen sur une biopsie du cortex cérébral.

Figure 6 .

 6 Figure 6. Aspects histopathologiques de la lipofuscinose neuronale focale.

Figure 7 .

 7 Figure 7. Les principaux diagnostics de la neuropathologie de l'épilepsie et leurs diagnostics différentiels

Table 1 . Classification of epilepsy-associated hippocampal sclerosis.

 1 1. Focal cortical dysplasia / Dysplasie corticale focale. 2. Encephalitis / Encéphalite. 3. Glial scar / Cicatrice gliale. 4. Long term epilepsy-associated 29 tumours / Tumeurs associées à l'épilepsie à long terme. 5. Cavernoma / Cavernome. 6. Epilepsy-associated hippocampal sclerosis / Sclérose hippocampique associée à l'épilepsie. Main differential diagnoses / Principaux diagnostics différentiels. Focal neuronal lipofuscinosis / Lipofuscinose neuronale focale. Inflammation related to deep brain electrodes / Inflammation associée à l'implantation d'électrodes profondes intracérébrales. Gliosis related to deep brain electrodes / Gliose associée à l'implantation d'électrodes profondes intracérébrales. Diffuse gliomas if recent onset of epilepsy or if growth of the tumour / Gliomes diffus si apparition récente de l'épilepsie ou si croissance de la tumeur. Glioneuronal tumours with a cavernomatoid vascularization / Tumeurs glioneuronales avec vascularisation pseudo-cavernomateuse. Dual pathology: association of Hippocampal Sclerosis/HS with tumours (dysembryoplastic neuroepithelial tumours/DNET, multinodular and vacuolating neuronal tumours/MVNT, ganglioglioma) / Pathologie duale : association d'une sclérose hippocampique avec une tumeur (tumeur neuroépithéliale dysembryoplasique/DNET, tumeur neuronale multinodulaire et vacuolisante/MVNT, gangliogliome).La classification a été proposée en 2013 sous l'égide de l'ILAE[START_REF] Baulac | Un ensemble de maladies complexe, encore mal compris 2018[END_REF]. La perte neuronale sévère désigne une perte de la moitié ou plus des neurones. La perte neuronale modérée désigne une perte de moins de la moitié des neurones. Les scléroses de type 2 et 3 sont caractérisées par une perte neuronale prédominant respectivement dans CA1 et CA4 : cette perte neuronale peut être sévère ou modérée.

	Classification selon ILAE	Perte neuronale dans CA1	Perte neuronale dans CA4
	Type 1	Sévère	Sévère
	Type 2	celle dans CA4 Prédominante par rapport à	Absente ou modérée
	Type 3	Absente ou modérée	Prédominante par rapport à celle dans CA1
	Gliose isolée	Absente	Absente
	Tableau 1. Classification des scléroses hippocampiques associées à l'épilepsie

Principales caractéristiques immunohistochimiques et moléculaires des tumeurs associées à l'épilepsie au long terme.Table 2 . Main immunohistochemical and molecular characteristics of long term epilepsy-associated tumours.

 2 Abréviations. DNET, tumeur neuroépithéliale dysembryoplasique ; INA, internexine alpha ; MVNT, tumeur neuronale multinodulaire et vacuolisante ; PLNTY, tumeurs neuroépithéliales polymorphes de bas grade du sujet jeune ; PXA, xanthoastrocytome pléomorphe.

Remerciements

FB est soutenu par un financement Emergences (Sorbonne Université) et par un financement Emergences du SiRIC CURAMUS, qui est un programme conjoint de l'Institut National du Cancer, du Ministère des Solidarités et de la Santé et de l'Inserm (INCA-DGOS-Inserm_12560).

Conflits d'intérêt

FB déclare l'emploi d'un apparenté par Bristol-Myers Squibb, la détention d'actions de Crossject, des financements de recherche reçus par l'institution de la part de Sanofi et Abbvie, des frais de voyages, de séjours et d'inscriptions aux congrès par Bristol-Myers Squibb. Les autres auteurs ne déclarent pas de conflits d'intérêt.

Classification des dysplasies corticales focales (FCD)