Anne-Charlotte Trouillet

Suzanne Ducroq

Lydie Naulé

Daphné Capela

Caroline Parmentier

Sally Radovick

Hélène Hardin-Pouzet

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

Deletion of neural estrogen receptor alpha induces sex differential effects on reproductive behavior in mice

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Introduction

Sex steroids play a key role in the developmental organization and adult activation of the neural circuitry underlying reproductive behavior. In males, it is largely accepted that perinatal testosterone and its neural metabolite estradiol irreversibly masculinize and defeminize the neural structures underlying behavioral responses [START_REF] Morris | Sexual differentiation of the vertebrate nervous system[END_REF]. During this period, the ovaries are inactive and the a-fetoprotein protects the female brain from these masculinizing effects by selectively binding to maternal and male sibling-derived estradiol [START_REF] Bakker | Alphafetoprotein protects the developing female mouse brain from masculinization and defeminization by estrogens[END_REF]. The ovaries start secreting estradiol around postnatal day 7 [START_REF] Lamprecht | Hormonal stimulation of oestradiol-17 beta release from the rat ovary during early postnatal development[END_REF]. It has been suggested that estradiol, during the time window between postnatal days 15 and 25, is important for a full expression of female sexual behavior during adulthood [START_REF] Bakker | The aromatase knock-out mouse provides new evidence that estradiol is required during development in the female for the expression of sociosexual behaviors in adulthood[END_REF][START_REF] Brock | The development of female sexual behavior requires prepubertal estradiol[END_REF]. In the nervous system, testosterone activates the androgen receptor (AR), while estradiol acts mainly through two nuclear estrogen receptors (ER) α and β encoded by two different genes, with increasing evidence indicating the potential involvement of other non-genomic pathways as well [START_REF] Alexander | Emerging roles for the novel estrogen-sensing receptor GPER1 in the CNS[END_REF]. Conditional genetic mouse models were generated to determine the neural role of nuclear AR and ERs in the expression of reproductive behavior, without interfering with their peripheral functions. We have previously shown that early AR deletion in neuronal and glial cells impaired the expression of sexual and aggressive behaviors in males [START_REF] Marie-Luce | Effects of neural androgen receptor disruption on aggressive behavior, arginine vasopressin and galanin systems in the bed nucleus of stria terminalis and lateral septum[END_REF][START_REF] Raskin | Conditional inactivation of androgen receptor gene in the nervous system: Effects on male behavioral and neuroendocrine responses[END_REF]. In particular, neural AR deletion did not affect the perinatal organization of known sexually dimorphic populations in the preoptic area, the key hypothalamic region controlling male sexual behavior [START_REF] Juntti | The androgen receptor governs the execution, but not programming, of male sexual and territorial behaviors[END_REF][START_REF] Picot | Vulnerability of the neural circuitry underlying sexual behavior to chronic adult exposure to oral bisphenol A in male mice[END_REF], but was found to alter the postnatal organization of the spinal nuclei involved in erection and ejaculation [START_REF] Raskin | Characterization of the spinal nucleus of the bulbocavernosus neuromuscular system in male mice lacking androgen receptor in the nervous system[END_REF][START_REF] Sakamoto | Androgen regulates development of the sexually dimorphic gastrin-releasing peptide neuron system in the lumbar spinal cord: Evidence from a mouse line lacking androgen receptor in the nervous system[END_REF]. Further neural deletion of ERb, using the same transgene, showed that this receptor is not involved in the expression of male or female sexual behavior, but rather mediates estrogen-induced regulation of social and aggressive behaviors in males and mood-related behavior in both sexes [START_REF] Dombret | Effects of neural estrogen receptor beta deletion on social and mood-related behaviors and underlying mechanisms in male mice[END_REF][START_REF] Naulé | Delayed pubertal onset and prepubertal Kiss1 expression in female mice lacking central oestrogen receptor beta[END_REF][START_REF] Naulé | Revisiting the neural role of estrogen receptor beta in male sexual behavior by conditional mutagenesis[END_REF]. In this context, the reproductive phenotype induced by early neural ERa deletion using this same transgene still needs to be determined. Previous studies addressing the effects of ERa deletion in neurons [START_REF] Cheong | Effects of neuronspecific Estrogen Receptor (ER) α and ERβ deletion on the acute estrogen negative feedback mechanism in adult female mice[END_REF][START_REF] Wintermantel | Definition of estrogen receptor pathway critical for estrogen positive feedback to gonadotropin-releasing hormone neurons and fertility[END_REF], or in specific neuronal populations such as glutamatergic and GABAergic neurons [START_REF] Cheong | Expression of ESR1 in glutamatergic and GABAergic neurons is essential for normal puberty onset, Estrogen feedback, and fertility in female mice[END_REF], or Tac2 or kisspeptin neurons [START_REF] Dubois | Positive, but not negative feedback actions of estradiol in adult female mice require estrogen receptor α in kisspeptin neurons[END_REF][START_REF] Dubois | Estradiol restrains prepubertal gonadotropin secretion in female mice via activation of ERα in kisspeptin neurons[END_REF][START_REF] Greenwald-Yarnell | ERα in Tac2 neurons regulates puberty onset in female mice[END_REF][START_REF] Mayer | Timing and completion of puberty in female mice depend on estrogen receptor alpha-signaling in kisspeptin neurons[END_REF] only focused on the regulation of the hypothalamic-pituitarygonadal axis in females. In a male study, it has been shown that postnatal ERa deletion in vesicular GABA transporter (Vgat) neurons altered mating and territorial behaviors without changing hormonal levels [START_REF] Wu | Estrogen receptor alpha is required in GABAergic, but not glutamatergic, neurons to masculinize behavior[END_REF], while its deletion in vesicular glutamate transporter (Vglut) 2 neurons increased testosterone levels but did not affect these behaviors. Other studies conducted on males targeted life stages after the perinatal period. A study using viral-mediated knockdown of ERa in specific brain areas reported that pubertal ERa activation in the medial amygdala is crucial for male behavior expression, while adult ERa expression in the medial preoptic and ventromedial nuclei facilitates this behavior [START_REF] Sano | Differential effects of sitespecific knockdown of estrogen receptor α in the medial amygdala, medial pre-optic area, and ventromedial nucleus of the hypothalamus on sexual and aggressive behavior of male mice[END_REF][START_REF] Sano | Pubertal activation of estrogen receptor α in the medial amygdala is essential for the full expression of male social behavior in mice[END_REF].

The present study was therefore conducted to investigate the effects of neural ERa deletion on male and female behaviors. This mouse model was generated using the nestin-Cre transgene, which triggers gene deletion in neural precursor cells by embryonic day 10.5, before gonadal differentiation, as previously reported for neural deletions of AR and ERb [START_REF] Raskin | Conditional inactivation of androgen receptor gene in the nervous system: Effects on male behavioral and neuroendocrine responses[END_REF][START_REF] Naulé | Delayed pubertal onset and prepubertal Kiss1 expression in female mice lacking central oestrogen receptor beta[END_REF]. For this purpose, the impact of ERa NesCre mutation was assessed in males and females on reproductive parameters including hormonal levels, weight of androgen-or estrogen-dependent organs, sperm count, presence of corpora lutea and fertility. We also assessed the behaviors related to reproduction (mating, olfactory preference, emission of ultrasonic vocalizations), locomotor activity, anxiety-like behavior and aggression. The neuroanatomical organization of sexually dimorphic hypothalamic populations related to male or female sexual behaviors were analyzed. Finally, a double neural knockout for AR and ERa (AR::ERa NesCre ) was generated and the behavioral effects of deletion of both receptors were analyzed in males.

Results

Selective neural ERa deletion and physiological parameters in males and females

The selective neural deletion of ERa is shown by the presence of the deleted ERa allele of 223 bp in the brain but not in the ovary of floxed mice expressing the Cre recombinase (Figure 1a and Supplementary Figure 1). RT-PCR analysis showed comparable ERa mRNA levels in the ovary and testis of control and mutant animals and reduced levels in the cortex, hippocampus and hypothalamus of mutant females (-71%, -71% and -78%, respectively) and males (-75%, -87% and -84%, respectively) in comparison with their control littermates (Figures 1b-c).

Mutant females were found to have unchanged body weight, reduced relative weight of ovaries (-25%) and a higher relative weight of the uterus (+170%), an estrogen-sensitive tissue, as compared with the control group (Supplementary Table 1). In agreement with the latter result, circulating levels of estradiol were significantly increased in mutant females (+61% versus controls).

Histological analysis showed no corpora lutea in the ovaries of mutant females (Supplementary Table 1). In mutant males, body weight was reduced (-5.5%). The relative weight of the seminal vesicles, which are androgen-sensitive, and testosterone levels were increased (+20% and +100%, respectively), while estradiol levels were reduced (-39%) in comparison with control mice (Supplementary Table 1). The other reproductive parameters, such as testis weight or epididymal sperm count, were unchanged between the two genotypes.

Arrested estrous cycle and abolished sexual behavior in neural ERa knockout females

Analysis of the estrous cycle over three weeks showed that mutant females spent more time in the metestrous stage (+174%) and less time (-70 to -93%) in the other stages in comparison with controls (Figure 2a). The estrous cycle arrest was associated with a reduced number of kisspeptinimmunoreactive neurons in the anteroventral periventricular area (AVPV) of mutant females (-78% versus controls at the proestrus-estrus and -68% versus controls at the metestrus-diestrus) (Figure 2b-c). For the other sexually dimorphic population of the AVPV, the number of tyrosine hydroxylase (TH)-expressing neurons is shown for control females regardless of their estrous stage (Figure 2d-e) since no significant differences were seen between the metestrus-diestrus and proestrus-estrus stages as previously reported [START_REF] Clarkson | Dual phenotype kisspeptin-dopamine neurones of the rostral periventricular area of the third ventricle project to gonadotrophin-releasing hormone neurons[END_REF][START_REF] Leite | Alpha-oestrogen and progestin receptor expression in the hypothalamus and preoptic area dopaminergic neurones during oestrous in cycling rats[END_REF]. Neural ERa deletion reduced by 46% the number of TH-immunoreactive cells in mutant females in comparison with controls (Figure 2e).

Behavioral analyses were performed on ovariectomized females primed with estradiol and progesterone to induce their receptivity under comparable hormonal conditions. Mutant females never exhibited lordosis posture in response to male mounts over three tests, while their control littermates displayed an increased lordosis quotient over tests reaching 0.7 at the last test (Figure 2f). This alteration was not due to an effect of neural ERa deletion on olfactory preference since females investigated males more than females regardless of their genotype (Figure 2g). The impaired lordosis behavior of mutant females was associated with a lower number of progesterone receptor (PR)-immunoreactive neurons (-84% versus controls) in the ventromedial hypothalamus as shown in Figure 2h.

Neural ERa deletion reduced but did not abolish male sexual behavior

All males initiated and achieved sexual behavior in both tests 1 and 2 regardless of their genotype (Figure 3a). However, differences between controls and mutants were seen in the latency to initiate mounting, intromissions and to reach ejaculation at both tests (Figure 3b). In accordance, the mating duration was higher in mutant males (+287% and +420% in Tests 1 and 2, respectively) as shown in Figure 3c. Quantification of the behavioral events showed that mutant males exhibited more mount attempts with or without intromissions and pelvic thrusts (1.8-to 2.4-fold above controls) in both tests (Table 1). These findings together with the increased interval between two consecutive intromissions (+85% and +265% above controls at tests 1 and 2, respectively) suggest a lower behavior of mutant males.

In the olfactory preference test, there was an effect of stimulus but not of genotype, with all males spending more time investigating the female stimulus (Figure 3d). Analysis of the emission of courtship ultrasonic vocalizations in the presence of a sexually receptive female showed a lower number (-21%) and duration (-30%) of ultrasonic vocalizations in mutant males in comparison with the control group (Figure 3e-f, h). Detailed quantification of the nine emitted syllables showed a reduced number and duration of the complex, mixed and frequency-jump syllables (Figure 3g,i).

At the neuroanatomical level, the effects of neural ERa deletion were investigated in two sexually dimorphic neuronal populations known to be organized by perinatal estradiol in the medial preoptic area [START_REF] Gilmore | Effects of blocking developmental cell death on sexually dimorphic calbindin cell groups in the preoptic area and bed nucleus of the stria terminalis[END_REF][START_REF] Simerly | Influence of perinatal androgen on the sexually dimorphic distribution of tyrosine hydroxylase-immunoreactive cells and fibers in the anteroventral periventricular nucleus of the rat[END_REF]. The number of calbindin-immunoreactive neurons was reduced (-40%; Figure 4a-b), while that of TH-immunoreactive neurons was higher (+80%; Figure 4c-d) in mutant males compared to their control littermates. Immunohistochemical analysis of the AR showed a comparable number of AR-immunoreactive neurons in control and mutant males in the medial amygdala, bed nucleus of stria terminalis, medial preoptic area and lateral septum (Figure 4e-f).

The unchanged number of AR-immunoreactive neurons in key regions underlying sexual behavior, together with the increased levels of circulating testosterone may compensate for the sexual dysfunction caused by neural ERa deletion. We thus analyzed sexual behavior of control and mutant males, which were gonadectomized and supplemented with equivalent levels of testosterone. Table 2 shows that under these normalized hormonal conditions, evidenced by the comparable relative weight of seminal vesicles, the latencies to initiate the first mount and intromission and to reach ejaculation were higher in sexually experienced mutants (7.2-, 6-and 3.5fold above controls, respectively). In contrast, no differences were found between the two genotypes in the number of mounts, intromissions and pelvic thrusts.

In continuous fertility tests with control females, neural ERa knockout males were able to produce offspring. The latency to produce the first litter (20.33 ± 0.33 days versus 27.67 ± 0.67 days for controls), the number of litters (7.33 ± 0.29 versus 7.33 ± 0.33 for controls) and litter size (6.26 ± 0.93 versus 6.77 ± 0.76 for controls) were comparable between the two genotypes.

Double neural AR and ERa knockout severely impaired male sexual behavior in mice

In previous studies, we have shown that AR NesCre deletion greatly impaired but did not abolish the expression of male sexual behavior [START_REF] Raskin | Conditional inactivation of androgen receptor gene in the nervous system: Effects on male behavioral and neuroendocrine responses[END_REF][START_REF] Raskin | Characterization of the spinal nucleus of the bulbocavernosus neuromuscular system in male mice lacking androgen receptor in the nervous system[END_REF]. We thus determined the impact of neural deletion of both AR and ERa on male behaviors. In this double knockout model, only 57% of AR::ERa NesCre mutant males versus 100% of controls reached ejaculation in tests 1 and 2 (Figure 5a). In addition, the latencies to initiate the first mount and intromission and to reach ejaculation were greatly increased in test 1 for mutants (65-fold, 58-fold and 14-fold above controls, respectively) (Figure 5b). There was no significant amelioration in Test 2 (48-fold, 51-fold and 24-fold above controls, respectively). In accordance, mating duration was significantly increased in both Tests 1 and 2 (12fold and 28-fold above controls, respectively) (Figure 5c). Quantification of the behavioral events showed reduced number of intromissions and thrusts in test 1, a higher number of mounts in test 2 and a reduced intromission ratio and a higher intromission interval for mutants in both tests (Table 3).

This sexual alteration was associated with a normal olfactory preference towards female stimuli (Figure 5d) but a reduced total number and duration of emitted syllables (-43% and -51 % versus controls, respectively) (Figure 5e). Double neural AR::ERa NesCre males had a lower body weight (-15%) and a higher relative weight of seminal vesicles (+53%) and circulating levels of testosterone (+100%) in comparison with their control littermates (Supplementary Table 2).

Effects of neural ERa or AR::ERa deletion on other behaviors

Male and female mice, subjected to sexual behavior tests, were also analyzed for locomotor activity and anxiety-state level. These latter behaviors are also regulated by estrogens and could interferewith the expression of sexual behavior if altered. Ovariectomized and hormonally primed females carrying the ERa NesCre mutation and gonadally-intact ERa NesCre males displayed a comparable activity to their respective control littermates in the circular corridor over the 120-min test (Figure 6a,c). Similarly, no differences were found between control and mutant ERa NesCre males, which were castrated and supplemented with equivalent testosterone concentrations (Supplementary Figure 2a). In contrast, a higher locomotor activity (+44%) was observed in intact males carrying the double AR::ERa NesCre mutation compared to their control littermates (Figure 6e).

The anxiety-state level assessed in the O-Maze was increased in ERa NesCre females as evidenced by the higher latency to enter the open arms and reduced time spent in the open arms compared to their control littermates (Figure 6b). No changes in the anxiety state level were observed in intact ERa NesCre males (Figure 6d), castrated and testosterone-supplemented ERa NesCre mice (Supplementary Figure 2b), or AR::ERa NesCre animals (Figures 6f) compared to their corresponding control littermates.

Assessment of aggressive behavior showed that it was completely impaired in both neural ERa and AR::ERa knockout males as shown by the very low percentage or absence of mutants showing this behavior towards non-aggressive intruders compared to their corresponding control littermates (Figures 6g-h).

Discussion

The present study shows that neural ERa deletion completely inhibited female sexual behavior and greatly impaired the gonadotropic axis, confirming the predominant neural role of ERa in female reproduction. In comparison, neural ERa deletion impaired the expression of male sexual behavior and organization of sexually dimorphic neuronal populations in the preoptic area.

Nevertheless, mutant males were able to achieve mating and produce offspring. A greater alteration of sexual behavior was observed when both neural ERa and AR were deleted, highlighting the complementary roles of both receptors in male behavior.

Neural ERa knockout female mice had an arrested estrous cycle, with uterine hypertrophy and increased estradiol levels and were infertile as evidenced by the absence of corpora lutea. In mutant females, an altered of pubertal maturation with an advanced time of vaginal opening and first estrus was also observed (Supplementary Figure 3). These data are in agreement with the phenotype reported for females lacking ERa in neurons [START_REF] Cheong | Effects of neuronspecific Estrogen Receptor (ER) α and ERβ deletion on the acute estrogen negative feedback mechanism in adult female mice[END_REF][START_REF] Wintermantel | Definition of estrogen receptor pathway critical for estrogen positive feedback to gonadotropin-releasing hormone neurons and fertility[END_REF], or in Kiss1- [START_REF] Mayer | Timing and completion of puberty in female mice depend on estrogen receptor alpha-signaling in kisspeptin neurons[END_REF] and Tac2expressing cells [START_REF] Greenwald-Yarnell | ERα in Tac2 neurons regulates puberty onset in female mice[END_REF].

At the behavioral level, under normalized hormonal levels between the two genotypes, mutant females were unable to adopt a lordosis posture, despite normal olfactory preference towards males.

The comparable loss of lordosis behavior previously reported for global ERa knockout females [START_REF] Ogawa | Reversal of sex roles in genetic female mice by disruption of estrogen receptor gene[END_REF] can thus be explained at least in part by the altered ERa signaling pathway at the neural level.

The deficient sexual behavior was probably due to the much lower number of PR-expressing neurons in the ventromedial hypothalamus, possibly due to the lack of estrogen-induced upregulation of PR expression in the absence of neural ERa. Indeed, female receptivity during the estrus stage is triggered by estrogen-induced up-regulation of PR in rodents (for review: [START_REF] Mhaouty-Kodja | Sexual behavior: From hormonal regulation to endocrine disruption[END_REF]).

The NesCre transgene used in the present study drives gene excision as early as embryonic day 10.5. It is thus possible that this early ERa deletion resulted in a deficient perinatal organization of brain areas controlling both the expression of sexual behavior and also the gonadotropic axis, thus leading to this infertile phenotype. Indeed, changes in the number of kisspeptin and TH neurons of the medial preoptic area tend to favor of a lack of feminization of this structure in mutant females.

In comparison, neither a mutation preventing membrane ERa signaling [START_REF] Khbouz | Role for the membrane estrogen receptor alpha in the sexual differentiation of the brain[END_REF] nor neural ERa deletion [START_REF] Naulé | Delayed pubertal onset and prepubertal Kiss1 expression in female mice lacking central oestrogen receptor beta[END_REF] were found to have any effect on the expression of female sexual behavior or the organization of related brain structures. Together, these observations support the idea of the predominant role played by nuclear ERa and its related genomic pathways in the neural control of female sexual behavior and gonadal axis function.

In male mice, neural ERa deletion resulted in increased latencies to initiate and achieve sexual behavior. In particular, sexual experience ameliorated the latency to mount and intromit but not the time needed to reach ejaculation. This moderate deficiency along with normal olfactory preference and slightly impaired emission of ultrasonic vocalizations could explain the fertile phenotype of mutant males. Indeed, neural ERa mutants were able to produce offspring like their control littermates. These results contrast with the previously reported phenotype of global ERα knockout males, which were infertile and exhibited greatly reduced sexual behavior [START_REF] Ogawa | Behavioral effects of estrogen receptor gene disruption in male mice[END_REF][START_REF] Ogawa | Modifications of testosterone-dependent behaviors by estrogen receptor-α gene disruption in male mice[END_REF][START_REF] Wersinger | Masculine sexual behavior is disrupted in male and female mice lacking a functional estrogen receptor α gene[END_REF]. It is possible that these huge changes in global knockout males were due to cumulative effects of ERα deletion in neural and peripheral sites; ERa plays a key function in the male urogenital tract.

Sex steroid hormones permanently organize the sexually dimorphic neuronal populations of the preoptic nucleus during the perinatal period. The lower number of calbindin-immunoreactive neurons and higher number of TH-immunoreactive neurons in neural ERα knockout males are in favor of an altered perinatal masculinization/defeminization processes in mutant males. It is possible that the consequences of altered brain organization and resulting sexual dysfunctions were partly compensated by the increased levels of testosterone, due to the altered negative feedback exerted by testosterone and its neural estradiol metabolite and unchanged number of ARimmunoreactive neurons in the neural circuitry controlling sexual behavior. When testosterone levels were normalized between the two genotypes, ERa NesCre males still exhibited an altered sexual behavior but the behavioral differences as compared with controls were reduced.

We have previously reported a greater impairment of sexual behavior in males lacking the neural AR as evidenced by the observed higher latencies to initiate and achieve sexual behavior and a more important reduction in the emission of ultrasonic vocalizations together with an hypofertile phenotype [START_REF] Raskin | Conditional inactivation of androgen receptor gene in the nervous system: Effects on male behavioral and neuroendocrine responses[END_REF][START_REF] Picot | Vulnerability of the neural circuitry underlying sexual behavior to chronic adult exposure to oral bisphenol A in male mice[END_REF][START_REF] Raskin | Characterization of the spinal nucleus of the bulbocavernosus neuromuscular system in male mice lacking androgen receptor in the nervous system[END_REF][START_REF] Dombret | Neural mechanisms underlying the disruption of male courtship behavior by adult exposure to di(2ethylhexyl) phthalate in mice[END_REF]. In these mutant males, no effect of neural AR deletion was observed on sexually dimorphic populations of the preoptic area [START_REF] Picot | Vulnerability of the neural circuitry underlying sexual behavior to chronic adult exposure to oral bisphenol A in male mice[END_REF][START_REF] Mhaouty-Kodja | Role of the androgen receptor in the central nervous system[END_REF] but changes in cell number, soma size or dendritic arborization were observed in spinal nuclei involved in erection and ejaculation [START_REF] Raskin | Characterization of the spinal nucleus of the bulbocavernosus neuromuscular system in male mice lacking androgen receptor in the nervous system[END_REF][START_REF] Sakamoto | Androgen regulates development of the sexually dimorphic gastrin-releasing peptide neuron system in the lumbar spinal cord: Evidence from a mouse line lacking androgen receptor in the nervous system[END_REF][START_REF] Mhaouty-Kodja | Role of the androgen receptor in the central nervous system[END_REF]. Together, these observations support complementary roles of AR and ERa in the control of sexual behavior in male mice. Accordingly, double knockout males lacking both the AR and ERa in the nervous system displayed a more important sexual deficiency than neural ERa knockouts. AR::ERa NesCre males took much longer to initiate mounts and exhibited reduced emission of ultrasonic vocalizations, suggesting an impaired motivational state. In addition, only half of them reached ejaculation and those achieving mating took a very long time in comparison with control littermates with no behavioral improvement after a first sexual experience although olfactory preference remained unchanged. Altogether, these data strongly suggest that they possibly also displayed an impaired erectile activity and organization and activation of the spinal nucelus of the bulbocavernosus as we have previously shown for male mice lacking the neural AR [START_REF] Raskin | Conditional inactivation of androgen receptor gene in the nervous system: Effects on male behavioral and neuroendocrine responses[END_REF][START_REF] Raskin | Characterization of the spinal nucleus of the bulbocavernosus neuromuscular system in male mice lacking androgen receptor in the nervous system[END_REF]. Further studies will address the reproductive phenotype of AR::ERa NesCre males in more detail to determine whether the ejaculating males are able to produce litters in continuous fertility tests.

In chemosensory areas located downstream of the olfactory bulb, study is still required of whether AR and ERa operate in identical or different neuronal populations. In this context, a previous study showed that ERα deletion in inhibitory Vgat neurons impaired mating [START_REF] Wu | Estrogen receptor alpha is required in GABAergic, but not glutamatergic, neurons to masculinize behavior[END_REF]. In addition, a mutation preventing membrane ERa signaling triggered altered behavior and reduced number of calbindinand kisspeptin-ir neurons [START_REF] Khbouz | Role for the membrane estrogen receptor alpha in the sexual differentiation of the brain[END_REF], thus suggesting the involvement of different ERa signaling pathways in these processes.

Neural ERa deletion had no effect on locomotor activity in females. In contrast, it increased their anxiety-related behavior compared to their control littermates. This may participate to their inhibited sexual behavior. Comparison with the previous effects for neural ERb on mood behavior [START_REF] Dombret | Effects of neural estrogen receptor beta deletion on social and mood-related behaviors and underlying mechanisms in male mice[END_REF][START_REF] Naulé | Delayed pubertal onset and prepubertal Kiss1 expression in female mice lacking central oestrogen receptor beta[END_REF] suggests that estradiol acts through both ERa and ERb signaling pathways to modulate this behavior.

In males, assessment of the effects of neural ERa or AR::ERa deletion on locomotor activity showed genotype effects only on the activity of AR::ERa NesCre males, which was elevated maybe due to the higher sex steroid levels of these mutants compared to their control littermates. Indeed, mutant AR NesCre male mice displayed also a high activity that was restored to control levels by gonadectomy and supplementation with equivalent concentration of testosterone [START_REF] Raskin | Conditional inactivation of androgen receptor gene in the nervous system: Effects on male behavioral and neuroendocrine responses[END_REF]. No effects of neural ERa or AR::ERa deletion were observed on anxiety-related behavior. Estrogens may thus regulate differently the basal anxiety state level between males and females. In males, if estrogens seem to be less involved in the modulation of mood behavior, they regulate together with androgens the expression of social behavior such as aggression. Indeed, suppression of one of the three neural estrogen or androgen pathways (ERa, ERb or AR) has been found to be sufficient to greatly impair or completely inhibit the expression of aggressive behavior ( [START_REF] Marie-Luce | Effects of neural androgen receptor disruption on aggressive behavior, arginine vasopressin and galanin systems in the bed nucleus of stria terminalis and lateral septum[END_REF][START_REF] Raskin | Conditional inactivation of androgen receptor gene in the nervous system: Effects on male behavioral and neuroendocrine responses[END_REF][START_REF] Dombret | Effects of neural estrogen receptor beta deletion on social and mood-related behaviors and underlying mechanisms in male mice[END_REF], present study).

In summary, the present data show that neural ERa deletion triggers sex differential effects on reproductive behavior. In females, neural ERa is mandatory for the organization and activation of structures underlying sexual behavior. In males, although ERa plays a role in the early neuroanatomical organization of brain areas involved in sexual behavior, both AR and ERa are required for an efficient behavior. These observations were confirmed by the sexual phenotype of double knockout males lacking both receptors in the nervous system. under non-stressful basal conditions. The data suggest a participation of neural ERa signaling pathway, as for neural ERb [START_REF] Dombret | Effects of neural estrogen receptor beta deletion on social and mood-related behaviors and underlying mechanisms in male mice[END_REF][START_REF] Naulé | Delayed pubertal onset and prepubertal Kiss1 expression in female mice lacking central oestrogen receptor beta[END_REF], in estrogen-dependent modulation of anxiety-related behavior.

In males, while the neural contribution of ERa pathway seems less important in the regulation of anxiety-related behavior, both pathways together with ERb [START_REF] Dombret | Effects of neural estrogen receptor beta deletion on social and mood-related behaviors and underlying mechanisms in male mice[END_REF] are important for the expression of aggressive behavior. It would be interesting for further studies to identify the neuronal versus glial contribution of these signaling pathways across life stages.

Methods

Animals.

The ERα NesCre mouse line was obtained, on a mixed C57BL/6 x CD1 background, by crossing floxed ERα females in which the exon 3 of ERα was flanked by loxP sites [START_REF] Chen | Generation and characterization of a complete null estrogen receptor α mouse using Cre/LoxP technology[END_REF] with floxed ERα males expressing the Cre recombinase under the control of the rat Nestin promoter (NesCre transgene; [START_REF] Tronche | Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety[END_REF]). Mutant mice (ERα fl/fl , NesCre: ERα NesCre ) and their control littermates (ERα fl/fl ) were used. To obtain the double AR::ERα NesCre mouse line, we first generated animals carrying floxed ERα and AR genes by crossing ERα fl/fl and AR fl/fl mouse lines. Homozygous floxed females (ERα fl/fl ::AR fl/fl ) were then crossed with ERα NesCre males to generate mutant males (ERα fl/fl ::AR fl/Y , NesCre: AR::ERα NesCre ) and their control littermates (ERα fl/fl ::AR fl/Y ; AR::ERα fl/fl ). After weaning, mice were kept at 22°C under an inverted light schedule, i.e. the dark time began at 1:30 PM with a 12:12 hour light-dark cycle and fed a standard diet with free access to food and water.

All experiments were conducted on adult animals of 3 to 6 months of age, in compliance with the French and European legal requirements (Decree 2010/63/UE) and were approved by the "Charles Darwin" Ethical committee (project number 01490-01).

PCR and quantitative RT-PCR.

The selective neural ERα deletion was assessed by PCR and qRT-PCR as previously described [START_REF] Raskin | Conditional inactivation of androgen receptor gene in the nervous system: Effects on male behavioral and neuroendocrine responses[END_REF][START_REF] Chen | Generation and characterization of a complete null estrogen receptor α mouse using Cre/LoxP technology[END_REF]. For PCR, control and mutant females were sacrificed and brain and ovaries were collected.

Detection of the Cre transgene and the floxed or truncated ERα allele was carried out using the following primers: ERa forward: 5'-AGG-CTT-TGT-CTC-GCT-TTC-C-3'; ERa reverse: 5'-GAT-CAT-TCA-GAG-AGA-CAA-GAG-GAA-CC-3'; NesCre forward: 5'-GCC-TGC-ATT-ACC-GGT-CGA-TGC-AAC-GA-3'; reverse: 5'-GTG-GCA-GAT-GGC-GCG-GCA-ACA-CCA-TT-3'. Amplification conditions were: 5 min at 95°C for denaturation, 40 cycles comprising 30 s at 95°C for denaturation, 30 s at 58°C for annealing and 1 min at 72°C for elongation and then 10 min at 72°C for final elongation.

For semi-quantitative RT-PCR, ovaries, testes and three brains areas (cortex, hippocampus, hypothalamus) were collected and rapidly frozen with dry ice. Total RNAs were extracted using TRIzol reagent (Invitrogen, Carlsbad, USA) and 1 µg was reverse transcribed into cDNA using the Superscript III First-Strand Synthesis System (Invitrogen Carlsbad, USA) and random hexamers.

PCR reactions were performed using the resulting cDNA (4 µl), dNTPs (10 nM each), primers (100 µM, Eurogentec, Seraing, Belgium) and Taq DNA pol (Invitrogen) in a MyCycler Thermal Cycler (Bio Rad, Marne la Coquette, France). Primers for ERa (forward: 5'-CGT-GTG-CAA-TGA-CTA-TGC-CTC-T-3'; reverse: 5'-TGG-TGC-ATT-GGT-TTG-TAG-CTG-G -3') and GADPH (forward: 5'-TGC-ACC-ACC-AAC-TGC-TTA-GC -3'; reverse: 5'-GGC-ATG-GAC-TGT-GGT-CAT-GAG -3') were used. Amplification conditions were: 5 min at 95°C for denaturation, 40 cycles comprising 30 s at 95°C for denaturation, 30 s at 56°C for annealing and 1 min at 72°C for elongation and then 10 min at 72°C for final elongation. The amplified cDNA fragments were separated by electrophoresis in a 1.5% agarose gel and stained by ClearSight DNA stain (Euromedex-BM, Souffelweyersheim).

Reproductive physiology.

Prepubertal females were monitored daily for visual evidence of vaginal opening from postnatal day 15 and vaginal smears flushed with physiological saline were taken daily and colored with hematoxylin-eosin to identify the estrus stage based on cell types [START_REF] Naulé | Delayed pubertal onset and prepubertal Kiss1 expression in female mice lacking central oestrogen receptor beta[END_REF][START_REF] Naulé | Neuroendocrine and behavioral effects of maternal exposure to oral bisphenol A in female mice[END_REF]. Adult females were also examined daily for the estrous cycle for 3 weeks using the same protocol, and were sacrificed at the diestrus phase to collect trunk blood and weigh ovaries and uteri. Ovaries were fixed in Bouin's buffer, washed in ethanol, paraffin-embedded and sliced into 5 µm sections [START_REF] Naulé | Neuroendocrine and behavioral effects of maternal exposure to oral bisphenol A in female mice[END_REF]. The sections were mounted and deparaffinized, stained with hematoxylin-eosin and the number of corpus lutea was analyzed.

At sacrifice of males, blood was collected and body, testis and seminal vesicles were weighed. The epididymis was dissected and sperm collected, incubated in PBS for 10 minutes at 37°C, and centrifuged 5 minutes at 100 x g. The supernatant was removed and 10-fold diluted [START_REF] Raskin | Conditional inactivation of androgen receptor gene in the nervous system: Effects on male behavioral and neuroendocrine responses[END_REF] and spermatozoa were counted in a hemocytometer with a Normaski microscope. Fertility was assessed in continuous mating for 5 months. Each male was paired with two age-matched control females. The interval from mating to the first litter and the number and size of litters were reported.

Sera obtained by decantation and centrifugation (2500 x g for 15 minutes) were used to measure by radioimmunoassay circulating levels of estradiol using DSL-4800 kit (Beckman-Coulter), and testosterone by the platform of the laboratory of Behavioral and Reproductive Physiology (UMR

INRAE-CNRS-IFCE-University of Tours).

Experimental conditions for behavioral tests.

Assays were conducted under red-light illumination 2 hours after lights-off and were videotaped for later analyses. The devices used in the tests, with the exception of animal home cages, were cleaned with 10% ethanol between trials. The analyses were performed by blind observation.

Stimulus C57BL/6J females used for the analyses of male behaviors and control (ERα fl/fl ) and mutant (ERα NesCre ) females were ovariectomized under general anesthesia (xylazine 10 mg/kg, ketamine 100 mg/kg), and implanted with 1 cm sc SILASTIC implants (3.18 mm outer diameter X 1.98 mm inner diameter; Dow Corning, Midland, MI) filled with 50 μg of estradiol-benzoate (Sigma-Aldrich) diluted in 30 μl of sesame oil. Females were subcutaneously injected with 1 mg of progesterone (Sigma-Aldrich) in 100 μl of sesame oil 4 to 5 hours before the tests to experimentally induce their receptivity. The stimulus C57BL/6J females used for the analyses of male behaviors were all sexually experienced twice and screened before each mating test for their lordosis behavior with stud males.

Control (ERα fl/fl ) and mutant (ERα NesCre ) males were gonadectomized under similar experimental conditions and implanted with a subcutaneous SILASTIC tubes containing 10 mg of testosterone (Sigma-Aldrich). Stimulus C57BL/6J males used for the analyses of female behaviors were sexually experienced and selected for showing a strong mounting behavior in less than 5 minutes after the presentation of a female (Supplementary Figure 4).

Sexual behavior.

Sexually naive females were tested for their sexual receptivity toward male mounting as previously described [START_REF] Naulé | Delayed pubertal onset and prepubertal Kiss1 expression in female mice lacking central oestrogen receptor beta[END_REF]. Test began when the female was introduced into the home cage of a sexually experienced C57BL/6J stud male and lasted for 20 minutes or until the female received 20 mounts.

Females were tested three times with an interval of 1 week between tests. The lordosis quotient (number of lordosis responses/number of mounts) was reported.

Sexually naive males were housed in individual cages 4 days before the beginning of the tests. Each male was tested in its home cage for 10 hours after the introduction of a sexually experienced stimulus female. Males were tested twice in tests 1 (naive) and 2 (sexually-experienced) with a time interval of one week. Male sexual behavior was analyzed by scoring the latency to initiate mounts, intromissions and to reach ejaculation, and the frequency of mounts, intromissions, thrusts as previously described [START_REF] Raskin | Conditional inactivation of androgen receptor gene in the nervous system: Effects on male behavioral and neuroendocrine responses[END_REF]. Mating duration was defined as the time from the first mount to ejaculation and the ratio intromission as the proportion of mounts with successful intromission. A latency of 600 minutes was given to males that did not initiate the behavior.

Olfactory preference.

Sexually experienced males and females were tested for odor preference in a Y-maze apparatus as previously described [START_REF] Picot | Vulnerability of the neural circuitry underlying sexual behavior to chronic adult exposure to oral bisphenol A in male mice[END_REF][START_REF] Naulé | Delayed pubertal onset and prepubertal Kiss1 expression in female mice lacking central oestrogen receptor beta[END_REF][START_REF] Trouillet | Sensory detection by the vomeronasal organ modulates experiencedependent social behaviors in female mice[END_REF]. Female mice were allowed to become familiar with the maze for 10 minutes over two consecutive days. On the experimental test day, each Y-arm randomly received either an anesthetized stimulus male or female. Subjects were free to investigate the stimuli for 9 min. Time spent in investigation by the tested animals was recorded and expressed as a percentage of the total time of investigation.

Ultrasonic vocalizations.

Each sexually-experienced male was tested in its home cage in the presence of a receptive female.

The vocalizations were recorded for 4 minutes, after the introduction of a female, with an Ultrasound gate microphone CM16/CMPA (Avisoft Bioacoustics), connected to an Ultrasound recording interface plugged into a computer equipped with the Avisoft-SASLab Pro 5.2.09 recording software. The recordings were analyzed using SASLab Pro (Avisoft Bioacoustics). A cutoff frequency of 30 kHz was used and elements were separated based on an automatic threshold with a hold time of 15 ms. [START_REF] Dombret | Neural mechanisms underlying the disruption of male courtship behavior by adult exposure to di(2ethylhexyl) phthalate in mice[END_REF][START_REF] Capela | Adult male mice exposure to nonylphenol alters courtship vocalizations and mating[END_REF]. The total number and duration of total ultrasonic vocalizations and syllables were reported.

Locomotor activity.

Activity was analyzed in a computed circular corridor made up of two concentric cylinders and crossed by four diametrically opposite infrared beams (Imetronics) as previously described [START_REF] Raskin | Conditional inactivation of androgen receptor gene in the nervous system: Effects on male behavioral and neuroendocrine responses[END_REF][START_REF] Naulé | Delayed pubertal onset and prepubertal Kiss1 expression in female mice lacking central oestrogen receptor beta[END_REF].

The locomotor activity was counted when animals interrupted two successive beams and had thus traveled a quarter of the circular corridor. The cumulative activity was reported.

Anxiety-related behavior.

The anxiety state was assessed in the elevated O-maze paradigm as previously described [START_REF] Naulé | Delayed pubertal onset and prepubertal Kiss1 expression in female mice lacking central oestrogen receptor beta[END_REF]. The test lasted 9 min and began when the animal was placed in a closed arm. The latency to enter into an open arm (with the four paws out) and the time spent in the open arms were analyzed.

Aggressive behavior.

Each sexually experienced male was tested in the resident-intruder test, after one week without bedding change, as previously described [START_REF] Marie-Luce | Effects of neural androgen receptor disruption on aggressive behavior, arginine vasopressin and galanin systems in the bed nucleus of stria terminalis and lateral septum[END_REF][START_REF] Raskin | Conditional inactivation of androgen receptor gene in the nervous system: Effects on male behavioral and neuroendocrine responses[END_REF]. Tests lasted 10 minutes and began when a sexually inexperienced gonadally intact adult A/J male (The Jackson Laboratory, USA) was introduced into the home-cage of the tested resident male. Each resident male was exposed to a different intruder on three consecutive days. Aggressive behavior was defined as lunging, biting, wrestling and kicking.

The proportion of males showing aggressive behavior was reported.

Tissu preparation.

Brains from perfused animals were removed, postfixed overnight in 4% PFA, cryoprotected in 0.1 M phosphate buffer containing 30% sucrose and stored at -80 °C. Coronal sections (30 µm) were then processed for immunolabeling as previously described [START_REF] Picot | Vulnerability of the neural circuitry underlying sexual behavior to chronic adult exposure to oral bisphenol A in male mice[END_REF][START_REF] Naulé | Delayed pubertal onset and prepubertal Kiss1 expression in female mice lacking central oestrogen receptor beta[END_REF].

AR, TH and Calbindin immunostainings.

Endogenous peroxidases were blocked for 30 minutes in PBS containing 3% H 2 O 2 and sections were incubated in blocking solution (PBS containing 0.2% Triton X-100 and 1% bovine serum albumin for AR or 5% horse serum for TH and Calbindin) 2 h at room temperature (RT). Sections were incubated at 4°C in the blocking solution supplemented with 1:200 diluted rabbit anti-AR (sc-816, Santa Cruz Biotechnology) for 36 hours, or with 1:2000 diluted mouse anti-TH antibody (MAB318, Millipore) or 1:1000 diluted mouse anti-Calbindin antibody (C9848, Sigma-Aldrich) overnight. Sections were incubated in PBS-0.2%Triton solution supplemented with 1:1000 diluted biotinylated secondary antibody (goat anti-rabbit BA1000, Vector; or horse anti-mouse BA2000, Vector) for 2 hours at RT. Signals were amplified with VECTASTAIN ® ABC kit (Vector) 1:1000 diluted in PBS for 1 hour at RT and visualized with 3,3'-diaminobenzidine tetrahydrochloride (DAB 0.02%, 0.01% H 2 O 2 in 0.05 M Tris, pH 7.4). Sections were mounted on slides, dehydrated and coversliped using Permount (Sigma-Aldrich). The slices were scanned using an automatic slice scanner (NanoZoomer, Hamamatsu). Regions of interest were drawn based on the Mouse Brain Atlas [START_REF] Paxinos | The Mouse Brain in Stereotaxic Coordinates[END_REF] and immunoreactive cells were manually counted using the NDPview software. AR positive nuclei quantification was performed bilaterally on one representative section of each area (MeA: plate 43; BNST-MPOA: plate 30; LS: plate 27). TH positive cells quantification was performed bilaterally on five sections covering the entire AVPV/PeN (plates 28 to 33).

Quantification of calbindin-positive cells was performed on one representative section of the SDN-POA (plate 33).

Kisspeptin and PR labelings.

Sections were incubated in a blocking solution (PBS containing 0.3% Triton X-100 and 3% donkey serum) 2 hours at RT and then at 4°C for 36 hours in the blocking solution supplemented with 1:10 000 diluted sheep anti-kisspeptin (AC053, generous gift of I. Franceschini [START_REF] Franceschini | Immunohistochemical evidence for the presence of various kisspeptin isoforms in the mammalian brain[END_REF]) or 1:100 rabbit anti-PR (A0098, Dako) antibodies. The sections were incubated in PBS-0.3%Triton solution supplemented with 1:500 donkey anti-sheep-Alexa 488 (A11015, Life Technologies) or 1:1000 biotinylated horse anti-rabbit (BA1100, Vector) secondary antibodies for 2 hours at RT. The signal was amplified with a Cy3-Streptavidin (s32355, Life Technologies

Statistics and Reproductibility.

Analyses were performed using GraphPad Prism 8.0 (GraphPad Software, Inc.) Assumption of normality (Shapiro-Wilk test) was tested before conducting the following statistical analyses. Twoway repeated measures ANOVA was used to analyze the effect of genotype and stimulus or genotype and time on olfactory preference, lordosis quotient for ERα NesCre males and females and AR::ERα NesCre males. One-way ANOVA with Tukey's post hoc was used to analyze group effect on kisspeptin-immunoreactivity for ERα NesCre females. Mann-Whitney test was used to compare estrous cycle duration, the latency, number of entries and time spent in the O-maze open arm for ERα NesCre females. It was also used to compare in males the latencies to mount and intromit, the number of mounts and pelvic thrusts in sexually naive and experienced ERα NesCre males; the number of pelvic thrusts in sexually naive ERα NesCre males and the interval between intromissions in sexually experienced ERα NesCre males, the number of short, downward, modulated and one-jump USV syllables, the duration of short, upward, downward, modulated, mixed and one-jump USV syllables, the levels of plasma testosterone and estradiol, the number of spermatozoa, the body weight and relative testis weight in ERα NesCre males, as well as the latencies to mount, intromit and ejaculate, the mating duration and the interval between intromissions in sexually naive and experienced

  ) 1:1000 diluted in PBS for 1 hour at RT. The sections were counterstained in a 2 μg/ml Hoechst solution (Invitrogen) for 5 min, mounted on slides and coverslipped with Mowiol. Three sections covering the AVPV/PeN (plates 28-31 of the Paxinos and Franklin Mouse Brain Atlas) were imaged using an inverting microscope equipped with an apotome (Axiovert 200M coupled to the AxioCam MRm camera, Zeiss). Z-stacks (3 µm) were carried out on the entire thickness of the sections. Kisspeptin positive cells were manually counted on using the ImageJ software (NIH). PR positive nuclei quantification was performed bilaterally on one representative section of the VMHvl (plates 45 of the Paxinos and Franklin Mouse Brain Atlas).

  AR::ERα NesCre males, the latency to enter, the number of entries and the time spent in the O-maze open arm, the body weight and the relative testis weight in AR::ERα NesCre males. The remaining data were analyzed by a two-tailed unpaired Student t-test. The probability of error level (alpha) was chosen to be 0.05 (* < 0.05, **p < 0.01 and ***p < 0.001). Results are presented as means ± SEM, except for the percentage of ejaculating ERα NesCre and AR::ERα NesCre males and the percentage of aggressive ERα NesCre and AR::ERα NesCre males, which were analyzed by Fisher's exact test.

Figures captionsFigure 1 .

 1 Figures captions

Figure 2 .

 2 Figure 2. Neural ERα deletion induced severe deficit of female reproductive function and

Figure 3 .

 3 Figure 3. The effects of neural ERα NesCre deletion on sexual behavior in males. a. Percentage of

Figure 4 .

 4 Figure 4. Neural ERα deletion altered the organization of sexually dimorphic populations. a.

Figure 5 .

 5 Figure 5. The behavioral effects of double AR::ERα deletion in males. a. Percentage of control

Figure 6 .

 6 Figure 6. Effect of neural ERα or AR::ERα deletion on locomotor activity, anxiety-related and

Table 1 . Sexual behavior of gonadally intact ERα NesCre males

 1 Quantification of sexual behavior components displayed by intact sexually naive and experienced control (ERα fl/fl ) and mutant (ERα NesCre ) males. Data are means ± S.E.M. for 10 males per genotype. *p < 0.05, ***p < 0.01; ***p < 0.001 versus control littermates.

	Behavioral events	Sexually naive (Test 1) ERα fl/fl ERα NesCre	Sexually experienced (Test 2) ERα fl/fl ERα NesCre
	Number of Mounts (M)	6.4 ±1.7	15.2 ±3.2 *	5.7 ±1.8	13.3 ±2.8 *
	Number of Mounts with	26.6 ±4.7	52.5 ±7.1 **	25.6 ±4.7	45.2 ±7.1 *
	intromissions (MI)
	Intromission ratio	78 ±5	76 ±4	82 ±5	78 ±3
	MI/M+MI (%)
	MI duration (sec)	17.9 ±2.1	17.6 ±1.2	18.6 ±1.7	19.6 ±1.5
	MI interval (sec)	43.7 ±7.9	80.9 ±7.7 **	22.8 ±2.7	83.4 ±26.1 ***
	Pelvic thrusts	531.2 ±83.2	1026.2 ±150.5 *	560.3 ±100.6	995.1 ±129.8 *

Table 2 . Sexual behavior of castrated and testosterone supplemented ERα NesCre males

 2 The mean weight ± S.E.M of seminal vesicles is indicated as percentage of body weight (bw) for control (ERα fl/fl ) and mutant (ERα NesCre ) males, which were gonadectomized and normalized for their testosterone levels (9-10 males per genotype). The latencies and behavioral events are indicated for sexually experienced males that reached ejaculation (7-8 males per genotype). Data are means ± S.E.M. *p < 0.05 versus control littermates.

	ERα fl/fl	ERα NesCre

Acknowledgements

We thank the Institut Biologie Paris-Seine platform for taking care of the animals. This work was funded by the Agence Nationale de la Recherche (ANR-09-CESA-006 and ANR-programme blanc SVSE 7-2012), CNRS, Inserm and Sorbonne Université.

Data Availability statement

Source data is provided as Supplementary Data 1.

Author contributions

SMK and ACT designed the study. SR provided the floxed ERa mouse line. ACT, SD, LN, DC and CP carried out the experiments. ACT, SD, HHP and SMK analyzed the data. SMK and ACT wrote the manuscript and all authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

SUPPLEMENTARY INFORMATION Content:

Supplementary Figure 1, relative to Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Table 1 Supplementary Table 2 Supplementary Figure 1,relative


MD, MSc Lucie Jurek
email: lucie.jurek@ch-le-vinatier.fr

MD, PhD Jean-Michel Dorey

MD Mikaïl Nourredine

MD, PhD Filipe Galvao

PhD Jérome Brunelin

Impact of vascular risk factors on clinical outcome in elderly patients with depression receiving electroconvulsive therapy

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Major depressive disorder is a common mental disorder, occurring across all world regions and at any age. With a worldwide prevalence of 4.4%, depressive disorders are ranked as the single largest contributor to non-fatal health loss in the world [START_REF] Who | WHO | Depression and Other Common Mental Disorders[END_REF]. Typically, the first line treatment consists of pharmacological medications using selective serotonin reuptake inhibitors. Antidepressant treatments are moderately effective, and only 30% of patients achieve remission to an initial antidepressant trial prescribed at the appropriate dose and duration and symptoms remain after a second line treatment in 50% of the cases [START_REF] Fava | Diagnosis and definition of treatment-resistant depression[END_REF]. In the elderly, depression is one of the most common psychiatric disorder [START_REF] Panza | Late-life depression, mild cognitive impairment, and dementia: possible continuum?[END_REF].

Late-life depression (LLD) is associated with significant morbidity and mortality with loss in functioning, dependence [START_REF] Ferrari | Burden of Depressive Disorders by Country, Sex, Age, and Year: Findings from the Global Burden of Disease Study[END_REF] and high rate of suicide [START_REF] Raue | Suicide risk in primary care: identification and management in older adults[END_REF]. For several reasons, such as low tolerance to pharmacological treatments, drug-drug interactions, high rates of treatment-resistant depressive symptoms are observed in LLD [START_REF] Mulsant | Treatment-resistant depression in late life[END_REF].

Whatever the age, treatment-resistant depression is frequent in clinical settings. However, the number of treatment-resistant elderly depressive patients is estimated to one-third of the elderly depressed patients. In case of severe and/or treatment-resistant episode, the use of electroconvulsive therapy (ECT) is common in LLD [START_REF] Socci | Electroconvulsive therapy and age: Age-related clinical features and effectiveness in treatment resistant major depressive episode[END_REF]. Although ECT is a safe, highly effective intervention with a response rate of more than 74% [START_REF] Bahji | ECT beyond unipolar major depression: systematic review and meta-analysis of electroconvulsive therapy in bipolar depression[END_REF] in patients with major depressive disorder, in the geriatric population, there is a large variability in response rates across individuals ranging from 55% to 92% (Antosik-Wójcińska and Święcicki, 2016; [START_REF] Kellner | A Novel Strategy for Continuation ECT in Geriatric Depression: Phase 2 of the PRIDE Study[END_REF][START_REF] Socci | Electroconvulsive therapy and age: Age-related clinical features and effectiveness in treatment resistant major depressive episode[END_REF][START_REF] Van Diermen | Prediction of electroconvulsive therapy response and remission in major depression: meta-analysis[END_REF]. Imaging studies report that MRI-defined vascular changes, such as deep white matter hyperintensities (WMHI), or cerebral atrophy could explain this heterogeneity, but the results remains inconsistent [START_REF] Dols | Early-and Late-Onset Depression in Late Life: A Prospective Study on Clinical and Structural Brain Characteristics and Response to Electroconvulsive Therapy[END_REF][START_REF] Hickie | Subcortical hyperintensities on magnetic resonance imaging: Clinical correlates and prognostic significance in patients with severe depression[END_REF][START_REF] Oudega | Are Apathy and Depressive Symptoms Related to Vascular White Matter Hyperintensities in Severe Late Life Depression?[END_REF][START_REF] Oudega | White Matter Hyperintensities and Cognitive Impairment During Electroconvulsive Therapy in Severely Depressed Elderly Patients[END_REF][START_REF] Oudega | White Matter Hyperintensities, Medial Temporal Lobe Atrophy, Cortical Atrophy, and Response to Electroconvulsive Therapy in Severely Depressed Elderly Patients[END_REF].

In LLD, apathy syndrome, defined as diminished motivation for physical, cognitive or emotional activity, is frequently described [START_REF] Groeneweg-Koolhoven | Apathy in early and late-life depression[END_REF][START_REF] Oudega | Are Apathy and Depressive Symptoms Related to Vascular White Matter Hyperintensities in Severe Late Life Depression?[END_REF] and could be associated with poor prognosis and treatment response of depression [START_REF] Husain | Neuroscience of apathy and anhedonia: a transdiagnostic approach[END_REF]. It has also been proposed that response rate was related to medical comorbidities observed in the elderly. Among them, vascular risk factors (VRF), a common comorbidity in the elderly raised a particular interest [START_REF] Allan | Does the Framingham Stroke Risk Profile predict white-matter changes in late-life depression[END_REF][START_REF] Sheline | Support for the Vascular Depression Hypothesis in Late-Life Depression: Results of a 2-Site, Prospective, Antidepressant Treatment Trial[END_REF][START_REF] Spaans | Vascular risk factors in older patients with depression: outcome of electroconvulsive therapy versus medication: VRF in depression: ECT versus medication[END_REF].

To the best of our knowledge, only few studies have been designed to investigate the relationship between response rate to ECT and VRF in LDD. Recently, Spaans et al. explored the impact of VRF on response to treatment in depressed participants. Comparing pharmacological treatment to ECT, they underlined the superior efficacy of ECT over pharmacotherapy in elderly patients with MDD, regardless the presence of VRF. Post-hoc analysis described non-statistical difference in ECT response rate between participants with one VRF compared to control group [START_REF] Spaans | Vascular risk factors in older patients with depression: outcome of electroconvulsive therapy versus medication: VRF in depression: ECT versus medication[END_REF].

However, in another study, a significant difference was found when focusing on pharmacological treatment in LLD with or without VRF, with a lower response rate in patients with VRF [START_REF] Bingham | Relationship Between Cerebrovascular Risk, Cognition, and Treatment Outcome in Late-Life Psychotic Depression[END_REF].

If the hypothesis of vascular depression is well described in the scientific literature, the impact of vascular burden on response rate to depression treatment remains unclear. The aim of this study was therefore to compare response rates to ECT in patients with LLD presenting with or without VRF.

We hypothesized that the presence of VRF would predict a poorer treatment outcome to ECT. As a secondary objective, we evaluated the relationship between the severity of brain vascular burden measured with the Framingham score and the clinical improvement.

METHOD

Participants

Patients were recruited in a specialized ECT unit located in a psychiatric hospital, Hospital "Le Vinatier", Lyon, France, between March 2016 and May 2018. All patients referred to our ECT unit with an age superior or equal to 55 years-old were considered for inclusion. Patients should present with a DSM-5 (American Psychiatric Association, 2013) unipolar or bipolar major depressive episode as assessed during a structural interview using the Mini International Neuropsychiatric Interview -MINI 7.0.2. The severity of the episode should be rated moderate to severe defined as a 10-item Montgomery-A ̊sberg Depression Rating Scale (MADRS10) score > 20 [START_REF] Leucht | What does the MADRS mean? Equipercentile linking with the CGI using a company database of mirtazapine studies[END_REF].

Exclusion criteria were: schizophrenia, schizoaffective disorder, delusional ideation, neurodegenerative disease or clinical conditions preventing the evaluation of the cognitive functioning (such as severe catatonia).

Assessment

For each patient, a global assessment was realized before the first session of ECT and after the last one. The severity of depression was assessed by the MADRS10 and the Beck Depression Inventory (BDI) scores. Maudsley Staging Method was used to define the resistance degree of depression [START_REF] Fekadu | A multidimensional tool to quantify treatment resistance in depression: the Maudsley staging method[END_REF]. Cognitive functioning was assessed with the Montreal Cognitive Assessment (MoCA) [START_REF] Moirand | Usefulness of the Montreal Cognitive Assessment (MoCA) to monitor cognitive impairments in depressed patients receiving electroconvulsive therapy[END_REF][START_REF] Nasreddine | The Montreal Cognitive Assessment, MoCA: A Brief Screening Tool For Mild Cognitive Impairment: MOCA: A BRIEF SCREENING TOOL FOR MCI[END_REF]. Other demographic and clinical characteristics were also collected such as age, sex, duration of the illness, pharmacological treatments (table 1). Duration of the illness was calculated based on the medical records and reflects the duration between the first acute mood disorder and the beginning of the ECT course.

Patients were separated into two groups based on the presence of a high vascular risk. To be consistent with the scientific literature, a Risk Factor Composite Score were calculated and a score of 2 or more were considered to be reflecting a high vascular risk (Valkanova and Ebmeier, 2013a).

Patients were thus included in the VRF group when 2 or more VRF were observed in their medical records among: hypercholesterolemia (LDL cholesterol > 4.91mmol/L), hypertension (systolic arterial pressure > 130 mmHg), tobacco smoking (current or in the last 6 months), diabetes mellitus (diagnosis), cardiovascular disease, and cerebral vascular accident/transient ischemic attack (diagnosis). Information in the medical records were considered only before the ECT course.

Repartition of vascular risk factors in both groups is presented in Table 2.

Framingham score was calculated for each patient using the Framingham algorithm [START_REF] D'agostino | General Cardiovascular Risk Profile for Use in Primary Care: The Framingham Heart Study[END_REF]. The Framingham risk score was calculated at baseline and represents a weighted sum of age, sex, antihypertensive treatment (yes or no), systolic blood pressure (millimeters of mercury), body mass index, history of diabetes (yes or no), and current cigarette smoking status (yes or no). It provides a 10-year probability of future cardiovascular events. Framingham score is correlated to cerebral vascular region and provide information about brain vascular burden [START_REF] Rabin | Interactive Associations of Vascular Risk and β-Amyloid Burden With Cognitive Decline in Clinically Normal Elderly Individuals[END_REF].

The presence or absence of white matter intensities was clinically determined by a radiologist on a T1-weighted magnetic resonance imaging (MRI). The MRI were acquired in a specialized hospital and only the clincal conclusions from the specialized radiologist were available. When the FAZEKAS score [START_REF] Fazekas | CT and MRI Rating of White Matter Lesions[END_REF] was specified, the presence of WHI was determine as a score equal or superior to one.

Administration of ECT

ECT sessions were administered twice weekly using a Mecta Spectrum 5000Q (Mecta Corporation, Tualatin, OR, USA) and followed the NICE recommendations [START_REF] Nice | Guidance on the use of electroconvulsive therapy[END_REF]. Anesthetic agents were either propofol (1-1.5 mg/kg) or etomidate (0.15-0.2 mg/ kg). All patients were treated with bitemporal (BT) stimulation, some of them being switched to right unilateral (RUL) in the case of major cognitive effects (e.g., disorientation or confusion). The seizure threshold (ST) was defined as the minimal electrical stimulus charge eliciting a generalized seizure lasting at least 20 s as measured with electroencephalography (EEG). ST was individually determined according to a titration schedule during the first session, and treatment was administered at 2 times the ST for BT stimulation and at 6 times the ST for RUL stimulation [START_REF] Sackeim | Seizure Threshold in Electroconvulsive Therapy: Effects of Sex, Age, Electrode Placement, and Number of Treatments[END_REF]. The pulse duration was brief (1ms) during BT stimulation and ultra-brief (0.3ms) during RUL stimulation. A specialized psychiatrist determined the duration of the ECT course according to mood assessments after the sixth ECT session and then every other 2 ECT sessions using the MADRS10. ECT was delivered until patients achieved remission or until there was no decrease in MADRS10 scores between two assessments. A maximum of 20 sessions was delivered.

Response and remission to ECT

Response was defined as an at least fifty percent decrease of the MADRS10 score between the baseline assessment and the score obtained after the ECT course [START_REF] Trivedi | Remission, response without remission, and nonresponse in major depressive disorder: impact on functioning[END_REF]. Remission was considered for patients with a MADRS score < 10 [START_REF] Hawley | Defining remission by cut off score on the MADRS: selecting the optimal value[END_REF].

Ethical issues

All patients signed an informed consent to receive ECT. They received individual information folder and opposition rights to record data. The database was approved and registered (record number MR-003-2017-002) by the French national commission for information technology, data processing and civil liberties (CNIL).

Statistical analysis

Statistical analyses were processed with R (version 4.0.2). Statistical level of significance was set at p < 0.05 (two-tailed). Descriptive statistics of baseline demographic and clinical characteristics of participants were calculated, and differences between the VRF and the non-VRF groups were investigated using chi-squared tests or Fisher's exact tests for categorical variables, two-sided Student's t-tests for continuous variables in cases of normal distribution, Mann-Whitney U-tests in cases of non-normal distribution.

Differences in response rate between the VRF and non-VRF groups were calculated using a Fisher's exact test.

Spearman correlation test was undertaken to evaluate the relationship between Framingham score and MADRS scores changes (measured as the difference between before and after ECT course).

Repeated-measures mixed ANOVA (rm-ANOVA) were conducted for MADRS scores with "time" (before and after treatment) as a within-subject factor and "VRF" (group with or without) as a between-subject factor. The assumption of sphericity was assessed using Mauchly's test.

RESULTS

Demographics and clinical characteristics

Among the 95 patients who received ECT between March 2016 and May 2018 in our unit, 59 were included according to our inclusion criteria. Among them, 56 completed the study and 3 patients were lost at the end of the ECT course and didn't show up for testing, therefore their data were not available for analysis. We excluded 4 patients that received only unilateral stimulation as they were most probably different from the rest of the population to be indicated for unilateral stimulation at the beginning of the ECT course. Among the 52 included patients, 20 were considered having a vascular burden (VRF≥2). There was no significant difference between the two groups regarding age, sex, illness duration, MADRS scores, cognitive functions and white matter hyperintensities (Table 1) at baseline. A non-significant difference was found for BDI scores: patients with VRF declared lower scores on this auto-evaluation scale (20.5 /+-9.1 in the non-VRF-group versus 14.0 +/-6.5 in the VRF-group; p=0.04)). No difference was found between the groups regarding the number of ECT sessions delivered, the switch of electrode positioning during the course (i.e., RUL or BT), the used anesthetic agent or the duration of the EEG seizures. Regarding concomitant pharmacological treatments, there was no significant difference between groups in terms of antidepressant, antipsychotic and benzodiazepine use. Mood stabilizer intake was more frequent in the VRF group.

Insert table 1 about here.

Patient with VRF versus without

According to our hypothesis, response rate was significantly lower in patients presenting with a vascular burden than in patients without VRF. While 30 out of 32 non-VRF patients (94%) responded to ECT, only 12 out of 20 (60%) were responders in the VRF group (p = 0.004) (see Figure 1).

Considering remission to ECT, 24 out of 32 non-VRF patients (75%) were in remission after the ECT course, while only 10 out of 20 (50%) were in remission in the VRF group, although this result is non-significant (p=0.065).

Insert figure 1 about here.

The rm-ANOVA revealed a significant interaction between the group (VRF, non VRF) and the decrease of MADRS before and after ECT (F(1,50) = 10.22, p = 0.002); no effect of group F(1,50) = 0.319, p = 0.575); but a significant effect of time F(1,55) = 279.1, p < 0.001). Post-hoc analysis found a significant difference in MADRS decrease between the non-vascular group (-28.9 ± 9.5) and the VRF-group ( -19.4 ± 12.2; p = 0.003) with a significant smaller decrease in the VRF-group than in the non-VRF group.

As observed with the MADRS10 scores, the rm-ANOVA with BDI scores revealed a significant interaction between the group and the decrease of BDI before and after ECT (F(1,17) = 8.08, p = 0.011); no effect of group (F(1,17) = 0.027, p = 0.870); and a significant effect of time (F(1,17) = 16.97, p < 0.001).

Although BDI scores seems lower at baseline in the VRF-group (14 ± 6.50) than in the non-VRF group (20.53 ± 9.07), the opposite results were found after ECT regimen. Patients from the VRFgroup displayed significant higher BDI scores at endpoint (10.8 ± 5.2) than patients from the non-VRF group (5.4 ± 3.4; p=0.003).

Correlation between Framingham and MADRS score

A significant negative correlation between the Framingham score and the MADRS differences before and after ECT course was found, rho = -0.42 (p = 0.0039) (see Figure 2).

Insert figure 2 about here.

Sensibility analysis

To determine the impact of important explicative variables on response to ECT a logistic regression model was conducted (Table 3). Independently to sex, age and depression type (bipolar or unipolar), response is decreased for patients presenting with a vascular burden with an OR = 0.06 (IC95%[0.01;0.034], p=0.004). This result was similar when considering mood stabilizer instead of type depression (see Supplemental).

DISCUSSION

The aim of the study was to compare response rate to ECT in elderly depressed patients presenting or not with VRF. According to our hypothesis, response to ECT was lower in patients with vascular burden compared to patients without vascular burden. It also seems that, based on Framingham score, the higher was the vascular burden, the lower was the MADRS change after ECT course. Our results are in line with those from [START_REF] Spaans | Vascular risk factors in older patients with depression: outcome of electroconvulsive therapy versus medication: VRF in depression: ECT versus medication[END_REF] reporting that, although the difference did not reach significance, remission rate to ECT decreased from 80 to 58 in patients with at least one VRF as compared with patients without VRF [START_REF] Spaans | Vascular risk factors in older patients with depression: outcome of electroconvulsive therapy versus medication: VRF in depression: ECT versus medication[END_REF].

To the best of our knowledge, the current study is among the first studies designed to explore the impact of VRF on ECT outcome in LLD. Because of a high prevalence of major depressive disorder but also the increase of VRF in the elderly population, this subject is a major clinical concern. Our population is representative of severe elderly depressed participants, in terms of age and depression severity [START_REF] Spaans | Vascular risk factors in older patients with depression: outcome of electroconvulsive therapy versus medication: VRF in depression: ECT versus medication[END_REF][START_REF] Steffens | Severity of Subcortical Gray Matter Hyperintensity Predicts ECT Response in Geriatric Depression[END_REF]. We included both unipolar and bipolar depression as there is no difference in response rate to ECT between these two diagnoses described in the scientific literature [START_REF] Daly | ECT in bipolar and unipolar depression: differences in speed of response[END_REF][START_REF] Dierckx | Efficacy of electroconvulsive therapy in bipolar versus unipolar major depression: a meta-analysis: ECT in bipolar versus unipolar depression[END_REF][START_REF] Narayanaswamy | Efficacy of ECT in bipolar and unipolar depression in a real life hospital setting[END_REF][START_REF] Pinna | Clinical and biological predictors of response to electroconvulsive therapy (ECT): a review[END_REF]. Results from the regression model suggests that the depression type did not influenced the impact of VRF on the response to ECT in our population. ECT protocols were realized according to guidelines with a sufficient duration of seizure. Patients received mostly bitemporal ECT stimulation, which is described as more efficient [START_REF] The | Efficacy and safety of electroconvulsive therapy in depressive disorders: a systematic review and meta-analysis[END_REF]. The high response rates observed in the current study (94% in non-VRF group versus 60% in VRF group) are in line with response rates reported in other studies on ECT, notably in older population, with a high proportion of women with unipolar disorder [START_REF] Medda | Response to ECT in bipolar I, bipolar II and unipolar depression[END_REF].

To explain the difference in response rate observed between the VRF and the non-VRF groups, one can hypothesize that vascular damages would alter affective brain networks that are involved in ECT response [START_REF] Steffens | Severity of Subcortical Gray Matter Hyperintensity Predicts ECT Response in Geriatric Depression[END_REF]. It has indeed been reported that micro-damage to small cerebral vessels may compromise the integrity of the frontal-subcortical circuits involved in mood regulation (Valkanova and Ebmeier, 2013b) and could precipitate, perpetuate, or predispose one to specific geriatric depressive syndromes [START_REF] Aizenstein | Vascular depression consensus report -a critical update[END_REF][START_REF] Alexopoulos | Vascular depression" hypothesis[END_REF]) also these results are inconsistent [START_REF] Oudega | White Matter Hyperintensities, Medial Temporal Lobe Atrophy, Cortical Atrophy, and Response to Electroconvulsive Therapy in Severely Depressed Elderly Patients[END_REF]. Defect in left superior longitudinal fascicle, cingulum bundle, and frontal projections to the corpus callosum, disrupting frontal and frontal-to-limbic white matter tracts have been shown in case of late-life vascular depression [START_REF] Aizenstein | Vascular depression consensus report -a critical update[END_REF]. These white matter damages have been related to inflammatory mechanisms with a role of cytokines and other pro-inflammatory markers [START_REF] Aizenstein | Vascular depression consensus report -a critical update[END_REF][START_REF] Alexopoulos | Mechanisms and treatment of late-life depression[END_REF]. Reduction of gray matter in frontal, hippocampal and limbic regions have also been implied, as well as altered default-mode network following white and gray matter damages [START_REF] Aizenstein | Vascular depression consensus report -a critical update[END_REF][START_REF] Jellinger | Organic bases of late-life depression: a critical update[END_REF].

Recent meta-analyses showed that LLD increases the risk of Alzheimer's disease and vascular dementia compared to non-depressed older adults, therefore, resistant cognitive symptoms could also explain the poorer treatment response in LLD [START_REF] Aizenstein | Vascular depression consensus report -a critical update[END_REF].

Limitations:

We acknowledge some limitations of the current study. First, the study design is retrospective, based on collected data, with a moderate sample of patients with moderate to severe depression (N = 52, baseline MADRS10 > 20). However, our population is quite homogeneous with similar baseline characteristics in both groups. The observed difference could thus not be explained by difference between groups regarding age, gender, type of depression (bipolar versus unipolar), severity of depression (symptoms or cognition), illness duration, or the occurrence of comorbidities such as obstructive sleep apnea or alcohol use disorder (table 1). BDI scores at baseline seems different between the two groups, with a higher score in non-VRF group, but this difference were not significant. To explain this difference, one can hypothesize that patients in the VRF-group could displayed a more severe level of apathy than patients from the non-VRF group resulting in a reduced auto-evaluation of depressive symptoms. This is supported by evidence suggesting that apathy symptoms are well represented in LLD and are associated to vascular burden [START_REF] Groeneweg-Koolhoven | Apathy in early and late-life depression[END_REF][START_REF] Hollocks | Differential relationships between apathy and depression with white matter microstructural changes and functional outcomes[END_REF][START_REF] Ligthart | Association of vascular factors with apathy in communitydwelling elderly individuals[END_REF]. Moreover, a lack of insight has been described in vascular depression and may also be taken into account to explain this difference [START_REF] Aizenstein | Vascular depression consensus report -a critical update[END_REF]. ECT treatment have an impact on apathy symptoms in the context of depression which could explain the BDI decrease observed post-ECT [START_REF] Carlier | The course of apathy in late-life depression treated with electroconvulsive therapy; a prospective cohort study[END_REF]. It is also important to note that a large porportion of patients from the current study were not able to rate their mood with the BDI before receiving ECT. Thus, the BDI seems not to be a reliable tool to evaluate depression in our sample of patients with severe depression.

As we did not use a cut-off score at the MoCA scale to exclude patients with a pre-clinical stage of dementia in, we cannot rule out to have including some of such patients in the current study.

Cognitive status based on rapid scales tends to improve after the ECT course, it is therefore not recommended to withhold ECT in older patients with severe depression in presence of pretreatment cognitive impairment (Obbels et al., 2019).

Add-on pharmacological treatment could also constitute a limitation. However, no significant difference was observed between groups regarding psychotropic medication intake in terms of antidepressant, benzodiazepine, first and second generation of antipsychotic. The observed difference between groups seems thus not linked with medication. It is important to note that a significant difference was observed for mood stabilizer with a higher consumption of mood stabilizer in the VRF group at baseline. This difference is not explained by the number of bipolar depressions in VRF group as both groups are not significantly different on this point. Moreover, VRF still decrease response to ECT when adjusting on depression type in the regression model. Patients with vascular depression could present atypical clinical features [START_REF] Aizenstein | Vascular depression consensus report -a critical update[END_REF]. Some recent reviews propose that lithium could be used in association to antidepressant in this clinical context [START_REF] Knöchel | Treatment-resistant Late-life Depression: Challenges and Perspectives[END_REF], which could explain the difference for mood stabilizer in our study. Mood stabilizer such as lithium and anti-epileptic drugs can interact with ECT outcomes. Combination of ECT and lithium is known to increase the risk of confusion and seizures length, but do not affect ECT outcome [START_REF] Zolezzi | Medication management during electroconvulsant therapy[END_REF]. In our study, the duration of seizure was comparable between the two groups.

Confusion was not higher in VRF group (estimate by the use of unilateral stimulation in both groups).

Concerning anti-epileptic drugs, combination with ECT could lead to difficulty eliciting seizure [START_REF] Zolezzi | Medication management during electroconvulsant therapy[END_REF] but do not affect ECT outcome [START_REF] Rubner | Frequency of and rationales for the combined use of electroconvulsive therapy and antiepileptic drugs in Austria and the literature[END_REF]. As described in table 1, sufficient seizure length was obtained in both groups. The supplemental analysis also confirm that adjusting on mood stabilizer did not influence our results.

Findings from neuroimaging studies have been inconsistent regarding the impact of MRI-defined vascular changes, such as deep white matter hyperintensities (WMHI), and response to ECT [START_REF] Dols | Early-and Late-Onset Depression in Late Life: A Prospective Study on Clinical and Structural Brain Characteristics and Response to Electroconvulsive Therapy[END_REF][START_REF] Hickie | Subcortical hyperintensities on magnetic resonance imaging: Clinical correlates and prognostic significance in patients with severe depression[END_REF][START_REF] Sheline | Support for the Vascular Depression Hypothesis in Late-Life Depression: Results of a 2-Site, Prospective, Antidepressant Treatment Trial[END_REF][START_REF] Spaans | Vascular risk factors in older patients with depression: outcome of electroconvulsive therapy versus medication: VRF in depression: ECT versus medication[END_REF]. In the current study, there is a lack of longitudinal precise neuroimaging to investigate this point. WMHI were investigated through a binary criterion ("WMHI" or "no-WMHI") based on the radiologist's conclusion. With this criterion, we observed no difference between VRF group and non-VRF group. These results were not surprising as low WMHI are very common in mid-life and older people (d'Arbeloff et al., 2019).

Although each patient performed a cerebral imaging prior to ECT course, we were not able to measure standardized WMHI or hippocampus size scores and data where analyzed by several radiologists in multiple laboratories with different protocols. Further studies are needed to establish the relationship between WHMI, VRF and response rate to ECT. Strikingly, althought the response rate was significantly different between the groups, we did not observed a significant difference in regards of remission rate between the VRF-group and the non-VRF group. One may hypothesized that a study with a larger number of patients achieving remission is needed to detect a significant difference in remission rate.

Consideration for future studies:

Regarding limitations in our methodology, some advice should be considered for future studies. As none are found on this topic in literature, impacts of VRF on ECT outcome in LLD should be assessed with a prospective design. Association of well-validated VRF scores such as Framingham score [START_REF] D'agostino | General Cardiovascular Risk Profile for Use in Primary Care: The Framingham Heart Study[END_REF] with depression characteristics, cognitive features and imaging data could be useful to complete lacking answers of available literature. Impact of modifiable and nonmodifiable VRF could offer some perspectives for therapeutic improvement. Also, prospective studies with survival analysis could monitor the impact of VRF duration on depression and treatment outcome. Automatically computed imaging rating scales should be used to determine anatomical cerebral impairment, as their visual measurement are poorly reproducible [START_REF] De Guio | Reproducibility and variability of quantitative magnetic resonance imaging markers in cerebral small vessel disease[END_REF][START_REF] Koikkalainen | Automatically computed rating scales from MRI for patients with cognitive disorders[END_REF][START_REF] Scheltens | Qualitative assessment of cerebral atrophy on MRI: inter-and intra-observer reproducibility in dementia and normal aging[END_REF]. Moreover, as it can act as a confusion factor, apathy should be assessed at baseline [START_REF] Pavlovic | Baseline characteristic of patients presenting with lacunar stroke and cerebral small vessel disease may predict future development of depression[END_REF][START_REF] Yuen | Neuroanatomical correlates of apathy in late-life depression and antidepressant treatment response[END_REF]. It has been proposed that the brains of older depressed patients may take longer to respond to the antidepressant effects of ECT.

Improvement may not be detected immediately after an acute course of ECT. Future studies may evaluate improvement at distance of the ECT course [START_REF] Steffens | Severity of Subcortical Gray Matter Hyperintensity Predicts ECT Response in Geriatric Depression[END_REF].

Prevention and treatment option in resistant late-life depression:

Facing the lower response rate in the VRF group, we should discuss other therapeutic strategies that can be proposed to these patients. One option is to promote primary prevention, in general population but also in at-risk subgroups, as addressing modifiable risk factors since early mid-life may reduce the risk of vascular depression [START_REF] Whyte | Depression in late-life: shifting the paradigm from treatment to prevention[END_REF]. Also, sufficient brain reserve, characterized by educational attainment, may counterbalance the effect of cerebrovascular burden with respect to depressive symptoms, thereby preserving mood in late life [START_REF] Aizenstein | Vascular depression consensus report -a critical update[END_REF].

Moreover, as secondary prevention measure, in addition to the appropriate treatment for depression, screening and optimized management of risk factors for cardiovascular and cerebrovascular disease is necessary [START_REF] Alexopoulos | Mechanisms and treatment of late-life depression[END_REF].

Neurobiologically based psychotherapies has also been proposed, focusing on barriers to "reward exposure" in three behavioral domains, known to be impacted in LLD, i.e., "negativity bias" (negative valence system dysfunction), "apathy" (arousal system dysfunction), and "emotional dysregulation" (cognitive control dysfunction) [START_REF] Alexopoulos | Mechanisms and treatment of late-life depression[END_REF]. More generally, as psychotherapy has been reported as efficient as pharmacotherapy, it should not be neglected [START_REF] Pinquart | Treatments for Later-Life Depressive Conditions: A Meta-Analytic Comparison of Pharmacotherapy and Psychotherapy[END_REF]. Augmentation strategy of antidepressants with either lithium, aripiprazole, methylphenidate has also been found effective in LLD unresponsive to an antidepressant and can be considered [START_REF] Alexopoulos | Mechanisms and treatment of late-life depression[END_REF][START_REF] Knöchel | Treatment-resistant Late-life Depression: Challenges and Perspectives[END_REF][START_REF] Lenze | Efficacy, safety, and tolerability of augmentation pharmacotherapy with aripiprazole for treatment-resistant depression in late life: a randomised, double-blind, placebo-controlled trial[END_REF].

Based on the hypothesis that the treatment of LLD requires stimulation intensities that can overcome prefrontal atrophy, a recent trial reported evidence for the efficacy and tolerability of high-dose deep repetitive transcranial magnetic stimulation for LLD [START_REF] Kaster | Efficacy, tolerability, and cognitive effects of deep transcranial magnetic stimulation for late-life depression: a prospective randomized controlled trial[END_REF]. Other therapeutic pathways targeting inflammatory or cerebral hemodynamic systems have also been explored, with a need of confirmation studies [START_REF] Alexopoulos | Mechanisms and treatment of late-life depression[END_REF]. Therefore treatment recommendations for elderly depressive patients favor a multimodal approach and optimization in psychotherapy, pharmacotherapy, biophysical therapy with an appropriate treatment of comorbidities is essential [START_REF] Knöchel | Treatment-resistant Late-life Depression: Challenges and Perspectives[END_REF].

It is of importance to note that response rate to ECT, even in VRF context, is higher than medication [START_REF] Sheline | Support for the Vascular Depression Hypothesis in Late-Life Depression: Results of a 2-Site, Prospective, Antidepressant Treatment Trial[END_REF][START_REF] Spaans | Vascular risk factors in older patients with depression: outcome of electroconvulsive therapy versus medication: VRF in depression: ECT versus medication[END_REF]. ECT is generally well-tolerated and can avoid severe medication adverse effects such as serotonin syndrome, associated to multiple lines of antidepressant, in case of treatment-resistant depression [START_REF] Jurek | The serotonin syndrome: An updated literature review[END_REF].

CONCLUSION

In conclusion, although ECT is an effective and well tolerated treatment for LLD, this study discloses that VRF can impede response to ECT in elderly patients with moderate to severe major depressive disorder. Framingham scores were negatively correlated to MADRS changes across ECT course and could be a useful tool in clinical practice to estimate response to ECT. Further studies are needed to confirm our results and to better understand the impact of VRF on ECT outcomes but also its physiopathology. Nevertheless, primary and secondary prevention of VRF is essential to prevent vascular depression and to improve ECT outcome.

Figure 1 .Figure 2 .

 12 Figure 1. Number of responders to electroconvulsive therapy in the two groups: with and without vascular risk factors. * = p<0.05 / VRF: group with Vascular Risk Factor; Non-VRF : group without Vascular Risk Factor

TABLE :

 :

	Characteristics	No-VRF group N = 32	VFR-group N = 20	p-value 1
	Age, mean years (SD)	70 (9)	67 (7)	0.15
	Sex (%)			0.14
	Female	66%	45%
	Male	34%	55%
	Duration of the ilness, mean (SD)	20 (18)	20 (13)	0.97
	Depression Type, n (%)
	Unipolar Bipolar	75% 25%	55% 45%	0.13
	Treatment
	First generation Antipsychotic (%)	16%	25%	0.48
	Second generation Antipsychotic (%)	53%	45%	0.57
	Antidepressant (%)	78%	80%	>0.99
	Tricyclic (%)	31%	30%	0.92
	Mood Stabilizer (%)	16%	45%	0.020
	Benzodiazepine (%)	41%	35%	0.69
	Comorbidities
	Alcohol Use Disorder (%)	13%	32%	0.15
	Obstructive Sleep Apnea (%)	6.7%	15%	0.38
	Baseline evaluation
	BDI at baseline, mean (SD)	21 (10)	14 (7)	0.070
	MADRS at baseline, mean (SD)	38 (7)	35 (10)	0.13
	MOCA at baseline, mean (SD)	21 (4)	23 (4)	0.15
	MAUDSLEY at baseline, mean (SD)	10 (2)	9 (2)	0.85
	White matter hyperintensities (%)	16 (59%)	7 (44%)	0.32
	ECT characteristics
	n of ECT sessions, mean (SD)	14 (4)	14 (4)	0.84
	EEG seizure duration (sec), mean (SD)	36 (11)	39 (16)	0.39
	Switch from bilateral to unilateral (%)	28%	25%	0.80
	Unipolar stimulation ECT (mC), mean (SD)	423 (223)	299 (132)	0.28
	Bipolar stimulation ECT (mC), mean (SD)	397 (171)	408 (235)	0.84
	Anesthetic agent (%)
	Etomidate Propofol	47% 53%	40% 60%	0.63

Table 1 . Characteristics of studied population.* =

 1 p<0.05 SD: Standard Deviation, VRF: Vascular Risk Factor, MADRS: Montgomery-A ̊sberg Depression Rating Scale, MOCA: Montreal Cognitive Assessment, BDI: Beck Depression Inventory, n: number, ECT: Electroconvulsive Therapy , EEG: Electro Encephalography

		Non-VRF group (n=32) VRF group (n=20)
	Tobacco smoking, n (%)	1 (3.1%)	8 (40%)
	Diabetes mellitus, n (%)	3 (9.4%)	9 (45%)
	Hypertension, n (%)	14 (44%)	16 (80%)
	Hypercholesterolemia, n (%)	3 (12%)	8 (44%)
	History of cardio-vascular disease, n (%)	2 (6.2%)	2 (10%)

Table 2 . Descriptive of vascular risk factors in both groups.

 2

	Characteristic	OR 1 95% CI 1 p-value
	Depression Type
	Unipolar	--
	Bipolar	5.47 0.84, 55.1 0.10
	Age	0.96 0.85, 1.07 0.5
	Sex
	Women	--
	Men	0.37 0.05, 2.00 0.3
	Vascular Risk Factor group
	No	--
	Yes	0.06 0.01, 0.34 0.004
	1 OR = Odds Ratio, CI = Confidence Interval

Table 3 . Logistic regression analysis for response to ECT.

 3