Daphné Capela

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

Effects of pubertal exposure to low doses of di-(2-ethylexyl)phthalate on reproductive behaviors in male mice

Reproductive behaviors are tightly regulated by sex steroid hormones. Interference with these hormones or their neural signaling pathways leads to behavioral alterations. We have previously shown that oral exposure of adult male mice to di(2-ethylhexyl) phthalate (DEHP), an organic environmental endocrine disruptor, altered sexual behavior. In this study, we examined the effects of pubertal exposure to DEHP and analyzed whether pubertal and adult exposures to DEHP trigger long-term effects. For pubertal exposure, male mice were exposed orally to the vehicle or DEHP at 5 or 50 µg/kg/d from postnatal day (PND) 30 to PND60. Exposure was arrested and animals were analyzed on PND120. They exhibited normal olfactory preference but showed modified emission of ultrasonic vocalizations. DEHP exposure also affected partner preference and mating components.

These modifications were associated with normal circulating testosterone levels and weight of androgen-sensitive tissues. In contrast, androgen receptor (AR) protein amount was reduced in the hypothalamic preoptic area in particular for the DEHP-50 group. Pubertal exposure also increased the anxiety-state level without changing circadian activity. When adult male mice were exposed to DEHP at the same doses from PND60 to PND105 and analyzed two months later, no effects of treatment on reproductive and anxiety-related behaviors or hypothalamic AR protein amount were seen.

Our data show that pubertal exposure of male mice to DEHP induces long-term behavioral changes in contrast to the adult exposure. This highlights the sensitivity of the nervous system to low doses of DEHP during the critical period of puberty.

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Introduction

In rodents, male sexual behavior is induced by olfactory cues emitted by sexually receptive females. These pheromonal cues are detected by the main olfactory epithelium and vomeronasal organ of the male and are transmitted, respectively, to the main and accessory olfactory bulbs, then to chemosensory responsive nuclei in the medial amygdala, to the stria terminalis bed nucleus and the hypothalamic medial preoptic area, where they are processed into behavioral responses. These neural structures are tightly regulated by sex steroid hormones, which start acting as early as the perinatal period when testosterone released by the fetal and neonatal testes induces neural masculinization and defeminization [START_REF] Morris | Sexual differentiation of the vertebrate nervous system[END_REF][START_REF] Phoenix | Organizing action of prenatally administered testosterone propionate on the tissues mediating mating behavior in the female guinea pig[END_REF]. Later, in adulthood, testosterone is also required in the activation of these behaviors. The pubertal period is another key time, during which testosterone exerts long-term effects on reproductive behaviors and on underlying neural structures to ensure the maturation of processes initiated during the perinatal period [START_REF] Ahmed | Pubertal hormones modulate the addition of new cells to sexually dimorphic brain regions[END_REF][START_REF] De Lorme | Pubertal testosterone organizes regional volume and neuronal number within the medial amygdala of adult male Syrian hamsters[END_REF][START_REF] Sodersten | Sexual behavior in developing male rats[END_REF][START_REF] Zancan | Remodeling of the number and structure of dendritic spines in the medial amygdala: From prepubertal sexual dimorphism to puberty and effect of sexual experience in male rats[END_REF]. For instance, in hamsters prepubertal castration of males has been shown to decrease sexual behavior with fewer mountings and intromissions and longer times to ejaculation [START_REF] Schulz | Gonadal hormones masculinize and defeminize reproductive behaviors during puberty in the male Syrian hamster[END_REF]. These perinatal, pubertal and adult periods are highly sensitive to hormonal changes, and could therefore also be sensitive to exposure to exogenous substances exhibiting endocrine disrupting activities.

Phthalates are organic pollutants that are detected ubiquitously in the environment, due to their massive use in the manufacture of several everyday products, including those made of polyvinyl chloride plastic. Di(2-ethylhexyl) phthalate (DEHP), the most abundant of this family, exhibits antiandrogenic effects such as the reduction of testosterone production [START_REF] Barakat | Prenatal exposure to DEHP induces premature reproductive senescence in male mice[END_REF][START_REF] Pocar | Exposure to di(2-ethyl-hexyl) phthalate (DEHP) in utero and during lactation causes long-term pituitary-gonadal axis disruption in male and female mouse offspring[END_REF]. Previous studies examined the effects of perinatal DEHP exposure on sexual behavior [START_REF] Andrade | A dose response study following in utero and lactational exposure to di-(2-ethylhexyl) phthalate (DEHP): reproductive effects on adult male offspring rats[END_REF][START_REF] Dalsenter | Phthalate affect the reproductive function and sexual behavior of male Wistar rats[END_REF][START_REF] Moore | Abnormalities of sexual development in male rats with in utero and lactational exposure to the antiandrogenic plasticizer di(2-ethylhexyl) phthalate[END_REF]. In a recent study, we reported that adult exposure to low doses of DEHP reduced the emission of courtship ultrasonic vocalizations (USVs), diminished male attractiveness and delayed the initiation of mating [START_REF] Dombret | Neural mechanisms underlying disruption of male courtship behavior by adult exposure to di-(2-ethylexyl)phthalate in mice[END_REF].

In particular, these effects were observed at doses equivalent to or lower than the tolerable daily intake dose (TDI) of 50 µg/kg body weight/d, which was established and recently updated by the European Food Safety Authority [START_REF] Efsa | Opinion of the Scientific Panel on food additives, flavourings, processing aids and materials in contact with food (AFC) on a request from the Commission related to bis(2-ethylhexyl)phthalate (DEHP) for use in food contact materials[END_REF][START_REF] Efsa | Update of the risk assessment of di-butylphthalate (DBP), butyl-benzyl-phthalate (BBP), bis(2-ethylhexyl)phthalate (DEHP), di-isononylphthalate (DINP) and di-isodecylphthalate (DIDP) for use in food contact materials[END_REF].

It has not been examined whether DEHP exposure during the pubertal period also affects the expression of sexual behavior. Only a single study examined the effects on social behavior of prepubertal exposure of male mice to DEHP (1 to 200 mg/kg/d) from postnatal day (PND) 28 to PND42 [START_REF] Wang | Effects of early pubertal exposure to di-(2ethylhexyl) phthalate on social behavior of mice[END_REF]. This reported reduced play and social investigation in pubertal male mice and inhibited social play and social investigation in adult males.

The effects of pubertal exposure to DEHP have been examined on other reproductive parameters. In male rats, DEHP exposure from PND23 to PND56 or PND98 delayed the onset of puberty, reduced androgen-dependent tissue weights at 300 and 900 mg/kg/d of DEHP, and at the higher dose tested reduced serum testosterone and increased serum luteinizing hormone (LH) levels [START_REF] Noriega | Pubertal administration of DEHP delays puberty, suppresses testosterone production, and inhibits reproductive tract development in male Sprague-Dawley and Long-Evans rats[END_REF]. Exposure to lower doses of DEHP (7.5 and 75 mg/kg/d) from PND22 to PND52 did not alter preputial separation or the weight of androgen-sensitive tissues [START_REF] Venturelli | Effects of exposure to di-(2-ethylhexyl) phthalate (DEHP) during lactation and puberty on sexual maturation and glycemic homeostasis in males rats[END_REF]. In another study with a longer treatment period lasting from PND21 to PND120, DEHP exposure at 0.5 μg/kg/d or above induced effects on sperm count and on Sertoli and Leydig cell numbers [START_REF] Oudir | Male rat exposure to low dose of di(2-ethylhexyl) phthalate during pre-pubertal, pubertal and post-pubertal periods: Impact on sperm count, gonad histology and testosterone secretion[END_REF]. In male mice, pubertal exposure of 4-week-old males to DEHP at 0, 125, 250 or 500 mg/kg/d for 28 consecutive days reduced the sperm count and induced histological abnormalities in the seminiferous epithelium and apoptosis of postmeiotic germ cells [START_REF] Liu | Pubertal exposure to di-(2-ethylhexyl)-phthalate inhibits G9amediated histone methylation during spermatogenesis in mice[END_REF]. Furthermore, testosterone levels were reduced at 125 mg/kg/d of DEHP, and testis weight loss was observed at DEHP doses of 250 and 500 mg/kg/d. In the study of [START_REF] Wang | Effects of early pubertal exposure to di-(2ethylhexyl) phthalate on social behavior of mice[END_REF], exposure to DEHP doses of 50 or 200 mg/kg/d reduced the relative weight of bilateral testis and anogenital distance of males exposed to DEHP from PND28 to PND42. It thus appears that, with the exception of the study of [START_REF] Oudir | Male rat exposure to low dose of di(2-ethylhexyl) phthalate during pre-pubertal, pubertal and post-pubertal periods: Impact on sperm count, gonad histology and testosterone secretion[END_REF], the majority of studies used high doses of DEHP, above the TDI and noobserved adverse effect level (NOAEL) of this molecule (5 mg/kg/d).

The present study was undertaken in order to analyze whether pubertal exposure to the TDI and an environmentally relevant dose of DEHP triggers long-term effects on sexual behavior in male mice. Sexual behavior comprises pre-copulatory and copulatory phases. During the pre-copulatory phase, the courting male displays a chemo-investigating behavior towards receptive females followed by the emission of USVs. The produced USVs play a role in attracting the female partner [START_REF] Pomerantz | Female behavior is affected by male ultrasonic vocalizations in house mice[END_REF]. During the copulatory phase, the male exhibits mounting, thrusting, and intromitting behaviors before reaching ejaculation. Male mice were thus exposed to DEHP from PND30 to PND60 to cover the whole period of puberty and brain sensitivity to gonadal sex steroids.

Two months later, they were analyzed for olfactory preference, USV production, partner preference and copulatory behaviors. General behaviors such as locomotor activity and anxiety-state level were analyzed. Whether or not adult exposure to DEHP can also trigger long-term behavioral effects was also investigated. For this, adult male mice were exposed for 6 weeks, and analyzed two months after the end of exposure. The weight of androgen-sensitive tissues, circulating levels of testosterone, and the protein amount of the androgen (AR) and estrogen receptor α (ERα), the two key signaling pathways involved in the expression of male sexual behavior, were analyzed for both pubertal and adult exposures.

Material and Methods

Animals and treatment

Analyses were performed according to European legal requirements (Decree 2010/63/UE) and were approved by the "Charles Darwin" Ethical committee (project number 01490-01). Animals were obtained from breeding of male and female C57BL/6J mice (Janvier) housed in nest-enriched polysulfone cages maintained at 22°C. Mice were kept under an inverted light schedule, i.e. the dark time began at 1:30 PM with a 12:12 h light-dark cycle. The mice were fed ad libitum a standard diet of food and water.

Oral exposure to DEHP was performed as described previously [START_REF] Capela | Effects of combined exposure of adult male mice to di-(2-ethylexyl)phthalate and nonylphenol on behavioral and neuroendocrine responses[END_REF][START_REF] Dombret | Neural mechanisms underlying disruption of male courtship behavior by adult exposure to di-(2-ethylexyl)phthalate in mice[END_REF]. Male mice were fed ad libitum a standard diet containing the vehicle (control group) or DEHP (Sigma-Aldrich). This compound was dissolved in an ethanol-water mixture and was incorporated into the food, such that exposure was equivalent to 5 µg/kg/d (DEHP-5 group) or 50 µg/kg/d (DEHP-50 group). DEHP doses were calculated for a daily food intake of 3.5 g per animal from 1 month to 1.5 months of age, and of 5 g per animal for later stages. Mice were weighed weekly and DEHP doses adjusted according to changes in their body weight. For pubertal exposure, male mice were treated from PND30 until PND60 and the analyses were started 2 months later on PND120 (Figure 1A). For adult treatment, male mice were exposed from PND60 until PND105 and the analyses started two months later on PND165 (Figure 1B). For a group of adult males, analyses started after the treatment period and DEHP exposure was maintained during the whole period of analyses as explained below and previously described [START_REF] Dombret | Neural mechanisms underlying disruption of male courtship behavior by adult exposure to di-(2-ethylexyl)phthalate in mice[END_REF].

Behavioral tests

Tests, which were conducted under red-light illumination, started 2 h after lights were turned off at 1:30 PM as indicated above, and were videotaped for later analysis as previously described [START_REF] Capela | Adult male mice exposure to nonylphenol alters courtship vocalizations and mating[END_REF][START_REF] Capela | Effects of combined exposure of adult male mice to di-(2-ethylexyl)phthalate and nonylphenol on behavioral and neuroendocrine responses[END_REF][START_REF] Dombret | Neural mechanisms underlying disruption of male courtship behavior by adult exposure to di-(2-ethylexyl)phthalate in mice[END_REF]. Naïve male mice were housed individually for 3 days and then paired with a sexually receptive female for their first sexual experience.

Analyses of recordings were performed by blind observation, males being identified by numbers attributed at weaning without any information concerning their treatment details.

For pubertal exposure, behavioral analyses were conducted on a first cohort including 11 males of the vehicle group, 12 males of the DEHP-5 group and 12 males of the DEHP-50 group. For adult exposure, analyses were performed on a second cohort including 12 males of the vehicle group, 12 males of the DEHP-5 group and 11 males of the DEHP-50 group. The tests were conducted following the order indicated below, starting with olfactory preference, then USV analysis, partner preference, mating and ending with locomotor activity and anxiety-related behavior. A third cohort of adult male mice (n = 11 for the vehicle group; n = 11 for the DEHP-5 group; n = 10 for the DEHP-50 group) was added to test the effects of continuous exposure to DEHP on anxiety-related behavior. These males were exposed to the vehicle or DEHP for the same duration as the second cohort and treatment was maintained during the analyses as previously described [START_REF] Dombret | Neural mechanisms underlying disruption of male courtship behavior by adult exposure to di-(2-ethylexyl)phthalate in mice[END_REF].

Preparation of sexually receptive females

C57BL/6J female mice used as stimuli were ovariectomized under general anesthesia (xylazine/ketamine) and implanted with SILASTIC implants (Dow Corning, Midland, MI) filled with 50 µg of estradiol-benzoate (Sigma-Aldrich) in 30 µl of sesame oil. Four to 5 h before the tests, females were given a subcutaneous injection of 1 mg of progesterone (Sigma-Aldrich) in 100 µl of sesame oil. Female receptivity was verified before the beginning of experiments, using sexually experienced males.

Olfactory preference

Olfactory preference was assessed in an enclosed plexiglass Y-maze. On the day of the test, male mice were offered the choice between an anesthetized sexually receptive female and an anesthetized non-receptive female. Non-receptive females were not administered progesterone before the test.

The number of entries into each arm of the Y-maze and the time spent in chemo-investigation of each stimulus were scored over 10 min.

Ultrasonic vocalization recording

Each male mouse was tested in its home cage, in the presence of a sexually receptive female. After introduction of the female into the cage, vocalizations were recorded for 4 min with a microphone (UltraSoundGate) connected to an ultrasound recording interface plugged into a computer equipped with Avisoft-SASLab Pro 5.2.09 recording software. Then the vocalizations were analyzed using SASLab Pro (Avisoft Bioacoustic). Spectrograms were generated for each call detected (frequency resolution: FFT-length: 512; frame size: 100%; overlap: 50%). The parameters used for the automatic quantification of the ultrasonic vocalizations were: cut-off frequency of 30 kHz, element separation based on an automatic single threshold with a hold time of 15 ms. Syllables of the simple (short, flat, upward, and downward), complex (modulated, complex) and frequency jump (mixed, one-jump, frequency-jump) categories were identified as previously described [START_REF] Capela | Adult male mice exposure to nonylphenol alters courtship vocalizations and mating[END_REF][START_REF] Dombret | Neural mechanisms underlying disruption of male courtship behavior by adult exposure to di-(2-ethylexyl)phthalate in mice[END_REF]. The total number and duration of USVs and the total number and duration of each syllable were analyzed.

Partner preference

A three-chamber test was conducted as previously described [START_REF] Dombret | Neural mechanisms underlying disruption of male courtship behavior by adult exposure to di-(2-ethylexyl)phthalate in mice[END_REF]. Females were allowed to become familiar with the three-chamber arena for 10 min on each of two consecutive days. On the day of the test, each sexually receptive female was placed in the neutral chamber and allowed to freely explore each chamber of the testing arena for 10 min. A DEHP-treated male was placed inside a corral and randomly assigned to the left or right chamber while a vehicle-treated male was placed inside a corral in the other chamber. The time spent in each compartment, the number of entries into each compartment and the time spent by the female sniffing each male, over the 10-min test, were scored.

Mating

Each male mouse was tested in its home cage for 10 h after introduction of the receptive female.

The latency to the first mount, intromission and ejaculation (time from the female introduction into the cage until the behavioral event), the mating duration (time from the first mount until ejaculation), the frequencies of mounts, intromissions and thrusts were scored.

Locomotor activity

Activity was analyzed in a computed circular corridor. Briefly, the male subject was introduced into a circular corridor made up of two concentric cylinders crossed by four diametrically opposite infrared beams (Imetronic). Locomotor activity was counted when animals interrupted two successive beams and had thus traveled a quarter of the circular corridor. Spontaneous activity was recorded for 12 h and was expressed as cumulative activity every 30 min as previously described [START_REF] Dombret | Neural mechanisms underlying disruption of male courtship behavior by adult exposure to di-(2-ethylexyl)phthalate in mice[END_REF][START_REF] Raskin | Conditional inactivation of androgen receptor gene in the nervous system: effects on male behavioral and neuroendocrine responses[END_REF].

Anxiety-related behavior

Male mice were placed in the closed arms of the O-maze and allowed to explore the maze freely for 9 min. The latency time before entry into the open arms, the number of entries and the time spent in the open arms were analyzed. A mouse was considered to be in an open arm once all its 4 paws had entered the arm.

Urogenital tract weight and hormone measurements

At the end of experiments, the weights of body and androgen-sensitive tissues (seminal vesicles, testis and Tyson gland) were measured for the two first cohorts of males analyzed in behavioral tests. The absolute organ weights (mg) and relative weights, expressed as percentage of body weight, were compared between the three-treatment groups. In addition, blood was collected from pubertally exposed males to prepare serum as previously described [START_REF] Capela | Effects of combined exposure of adult male mice to di-(2-ethylexyl)phthalate and nonylphenol on behavioral and neuroendocrine responses[END_REF][START_REF] Raskin | Conditional inactivation of androgen receptor gene in the nervous system: effects on male behavioral and neuroendocrine responses[END_REF]. Circulating levels of testosterone were measured by RIA using 3 H-testosterone at the hormonal assay platform of the laboratory of behavioral and reproductive physiology (UMR INRAE/ CNRS / Université de Tours) as previously reported [START_REF] Capela | Adult male mice exposure to nonylphenol alters courtship vocalizations and mating[END_REF][START_REF] Capela | Effects of combined exposure of adult male mice to di-(2-ethylexyl)phthalate and nonylphenol on behavioral and neuroendocrine responses[END_REF][START_REF] Dombret | Neural mechanisms underlying disruption of male courtship behavior by adult exposure to di-(2-ethylexyl)phthalate in mice[END_REF]. The mean intra-assay variation coefficient was 7%, and the assay sensitivity was 125 pg/ml.

Western blotting

Protein extracts (30 μg) from punches of the medial preoptic area were prepared from 4-6 males per treatment group of the two first cohorts as previously described [START_REF] Capela | Adult male mice exposure to nonylphenol alters courtship vocalizations and mating[END_REF][START_REF] Picot | Neural androgen receptor deletion impairs the temporal processing of objects and hippocampal CA1-dependent mechanisms[END_REF]. Proteins were then separated on a 4-12% polyacrylamide gel (Invitrogen), and transferred onto a nitrocellulose membrane. Blots were probed overnight with polyclonal anti-AR (1:400, Santa-Cruz Biotechnology), anti-ERα (1:400, Santa-Cruz Biotechnology) or anti-glyceraldehyde 3phosphate dehydrogenase gene GAPDH (1:10000, Santa-Cruz Biotechnology) and then with peroxidase-conjugated second antibodies diluted at 1:5000 (Amersham Bioscience). Signals were visualized with the Super Signal detection kit (Thermo Scientific), quantified with ImageJ software (NIH) and normalized with respect to GAPDH.

Statistical analyses

Data were expressed as means ± S.E.M. Normality tests (Kolmogorov-Smirnov and Shapiro-Wilks tests) were performed before using the parametric analyses. Two-way ANOVA was used to analyze the main effects of treatment and stimuli for olfactory preference, and treatment and time for locomotor activity. A paired Student's t test was used to analyze the results of partner preference.

One-way ANOVA was used to analyze the effects of treatment on the remaining data; except for testosterone levels, which were analyzed by non parametric Kruskal-Wallis test. Post-hoc tests were used to determine group differences. P values of less than 0.05 were considered to be significant.

Results

Effects of pubertal exposure to DEHP on olfactory preference and USV production

Male mice were exposed to the vehicle or DEHP at 5 or 50 µg/kg/d from PND30 to PND60, with exposure covering the whole period of puberty and brain sensitivity to gonadal sex steroids (Figure 1A). Exposure was arrested, and 2 months later, adult males were sexually experienced before starting the behavioral analyses.

In the olfactory preference test, male mice were given the choice between non-receptive and receptive females placed in opposite arms of the Y maze. The females were both anesthetized in order to avoid any social interaction. Two-way ANOVA showed no effect of stimulus (F(1, 32) = 0.13, p = 0.72) or treatment on the number of entries into the stimulus arms (F(2, 32) = 1.2, p = 0.31) (Figure 2A). The total time spent investigating the two stimuli was also comparable between the three groups (p = 0.4; Figure 2B). In contrast, there was an effect of the stimulus (F(1, 32) = 12.32, p = 0.001) but not of treatment (F(2, 32) = 0.93, p = 0.41) on the time spent in investigation (Figure 2C). Males were more interested in the receptive female than in the non-receptive one, regardless of their treatment status.

Male mice were also analyzed for the emission of USVs in the presence of a receptive female. Oneway ANOVA showed no effect of treatment on the total number of emitted USVs (p = 0.81; Figure 3A). A detailed analysis of the number of each syllable category (Figure 3B-D) shows a significant effect of treatment on short syllables (p < 0.05), with a lower number (-52%) in males of the DEHP-50 group by comparison to the DEHP-5 group (Figure 3B). There were no effects on the other syllables (flat: p = 0.70; upward: p = 0.49; downward: p = 0.16; mixed: p = 0.39; frequency-jump: p = 0.60; one-jump: p = 0.11; modulated: p = 0.64; complex: p = 0.63). The analysis of the total duration of emitted syllables shows an effect of treatment (p = 0.05; Figure 3E). Further analyses of each category show treatment effects on modulated (p = 0.03), mixed (p = 0.03) and one-jump syllables (p = 0.03) (Figure 3F-H). No treatment effects were observed on the other syllables (short: p = 0.10; flat: p = 0.77; upward: p = 0.46; downward: p = 0.17; frequency-jump: p = 0.55; complex: p = 0.7). Altogether, these data show that pubertal exposure to DEHP alters the number of short syllables, and the total duration of syllables, with a significant effect on modulated, mixed and onejump syllables. All these effects seemed to target the DEHP-5 group.

Effects of pubertal exposure to DEHP on partner preference and mating

Partner preference was investigated using the three-chamber paradigm containing a compartment with a box containing a vehicle-treated male and a compartment with a box containing a DEHPtreated male at either side of the middle chamber. Receptive female mice were given the choice between the two stimuli. Figure 4A shows that females spent a comparable total time investigating the male compartments. The number of entries in the vehicle-versus DEHP-treated compartments was also similar (Figure 4B). In contrast, females spent less time investigating males of the DEHP-50 group (-23%) in comparison to vehicle-exposed males (p = 0.007; Figure 4C).

Analyses of mating tests in the presence of a receptive female show no effect of treatment on the latency to mount (p = 0.48) or intromit (p = 0.37), but an effect on the latency to reach ejaculation (p = 0.0053) (Figure 4D). Posthoc analyses show that males of the DEHP-50 group reached ejaculation later than the vehicle group (+50%, p < 0.01). There was also an effect of treatment on mating duration (p = 0.011; Figure 4E), this being longer for males of the DEHP-50 group compared to the vehicle group (+51%, p < 0.05). Furthermore, analyses of the number of behavioral events (Figure 4F-H) show a significant effect of treatment on the number of intromissions (p = 0.04), and thrusts (p = 0.019).

Effects of pubertal exposure to DEHP on general behavior

Locomotor activity was monitored for 12 h during the dark phase (Figure 5A). There was an effect of time (F(23, 759) = 16.57, p < 0.0001), but not of treatment (F(2, 33) = 0.62, p = 0.55). Males exhibited a maximal activity during the first five hours, regardless of their treatment; then activity decreased progressively. In the elevated O-maze paradigm, an effect of treatment was observed on the latency to the first entry into the open arms (p = 0.015), with a significantly higher latency for DEHP-5 treated males than the vehicle group (+61%; Figure 5B). The number of entries into the open arms was not statistically significant, although a tendency to decrease was observed for the two DEHPtreated groups (Figure 5C). Moreover, there was an effect on the time spent in the open arms (p = 0.04) as shown in Figure 5D. Altogether, these data show that pubertal DEHP exposure did not affect locomotor activity but increased the anxiety state level of DEHP-treated mice.

Urogenital tract, testosterone levels and hypothalamic AR and ERα protein amounts

DEHP has been widely described as an anti-androgenic compound that affects testosterone production. We thus compared the weight of androgen-sensitive tissues between the three treatment groups and measured circulating levels of testosterone at the end of the behavioral analyses. Table 1 shows an effect of DEHP exposure on body weight (p = 0.02). Males of the DEHP-50 group had a lower weight (-4%) than males from the vehicle group (p < 0.05). There were no effects of DEHP exposure on the absolute or relative weights of the seminal vesicle, testis or Tyson gland.

Measurement of circulating levels of testosterone showed no effects of DEHP treatment (p = 0.37), with comparable levels between the three treatment groups (vehicle: 1.96 ± 0.30 ng/ml; DEHP-5: 2.01 ± 0.50 ng/ml; DEHP-50: 1.73 ± 0.50 ng/ml). These data show that pubertal exposure to DEHP reduced body weight in the DEHP-50 group, but did not affect testosterone levels or the weight of androgen-sensitive tissues in either of the DEHP-exposed groups.

Endocrine disruption of male sexual behavior by phthalates can also occur through modifications of the expression of the two key AR and ERα mediating testosterone-induced regulation of this behavior [START_REF] Naulé | Revisiting the neural role of estrogen receptor beta in male sexual behavior by conditional mutagenesis[END_REF][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-alpha gene disruption in male mice[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] Wersinger | Masculine sexual behavior is disrupted in male and female mice lacking a functional estrogen receptor a gene[END_REF].

Indeed, in the male nervous system, testosterone can act either directly through the activation of the AR or be metabolized by the neural aromatase cytochrome P450 into estradiol, which stimulates ER. We thus quantified the protein amounts of AR and ERα proteins by Western blotting in the medial preoptic area, the key hypothalamic region where chemosignals are processed in behavioral responses. Figure 4I shows an effect of pubertal exposure to DEHP on the amount of AR protein (p = 0.04), with a significant decrease (-47%) in the DEHP-50 group compared to males exposed to the vehicle. No effect of phthalate treatment was observed on the protein amount of ERα (p = 0.62; Figure 4J).

Effects of DEHP exposure arrest during adulthood

In previous research, we showed that chronic exposure of adult male mice to DEHP at 5 or 50 µg/kg/d reduced USV emission and attractiveness, delayed the time to reach ejaculation without affecting olfactory preference or locomotor activity, and down-regulated the expression of the neural AR [START_REF] Dombret | Neural mechanisms underlying disruption of male courtship behavior by adult exposure to di-(2-ethylexyl)phthalate in mice[END_REF]. In this previous study, males were exposed to DEHP for 6 weeks and exposure was maintained during the whole period of behavioral analyses. Thus, we wanted to know whether exposure to DEHP during adulthood triggers long-term modifications similarly to pubertal exposure, in particular of the behaviors and parameters affected by continuous exposure.

For this purpose, adult male mice were exposed to DEHP from PND60 for 6 weeks before exposure cessation for 2 months (Figure 1B). The mice were then analyzed for reproductive behaviors that were impacted by continuous DEHP treatment [START_REF] Dombret | Neural mechanisms underlying disruption of male courtship behavior by adult exposure to di-(2-ethylexyl)phthalate in mice[END_REF]. One-way ANOVA showed no significant effect of DEHP treatment on the total number (p = 0.27) or total duration (p = 0.53) of emitted USVs in the presence of a sexually receptive female (Figure 6A-B). Analysis of partner preference in the three-chamber paradigm shows that receptive females displayed a comparable total time and number of entries in the stimuli compartments (Figure 6C-D), and time investigating the vehicle-and DEHP-treated males (Figure 6E). In mating tests, there was no treatment effect on the latency to the first mount (p = 0.85), the first intromission (p = 0.51) or time to reach ejaculation (p = 0.62) (Figure 6F). Similarly, no effect of treatment on mating duration was observed (p = 0.61).

Anxiety state level was also compared between adult males exposed to DEHP, with or without an exposure arrest (Figure 7). In adult males where exposure was maintained throughout the behavioral analyses, there was an effect of adult DEHP exposure (p < 0.05) on the number of entries and the time spent in the open arms of the O-maze (Figure 7A-B). In particular, males of the DEHP-50 group exhibited a lower number of entries (-40% compared the DEHP-5 group) and time spent in the open arms (-30% versus the vehicle and DEHP-5 groups). In contrast, when adult male mice were exposed to DEHP and then analyzed after a two-months exposure arrest, there was no effect of DEHP exposure on the number of entries or time spent in the open arms (Figure 7C-D).

At the end of the behavioral analyses, male mice were analyzed for their body weight and for the weight of androgen-sensitive tissues. Data presented in Table 1 show that DEHP treatment had no effect on body weight (p = 0.37), or weights of the seminal vesicles (p = 0.99), testis (p = 0.37) or Tyson gland (p = 0.77). Quantification by Western blotting of the protein amounts of AR and ERα was also performed in the medial preoptic area of male mice. Figure 6H-I shows no changes in the AR (p = 0.76) and ERα protein amounts (p = 0.69) between the treatment groups.

Discussion

The present study shows that pubertal exposure of male mice to the TDI dose of DEHP (50 µg/kg/d) and to a 10-fold lower dose, which is close to the estimated environmental exposure, triggered long-term alteration of sexual behavior and anxiety-state level, which were seen two months after exposure cessation. In contrast, adult exposure to similar DEHP doses did not trigger such alterations, since no treatment effects were observed after a comparable duration of exposure arrest.

Exposure of male mice to DEHP during the whole period covering puberty and brain sensitivity to sex steroids triggered behavioral modifications. The assessment of sexual behavior of adult males shows that both the precopultaory and copulatory phases were affected. For the precopulatory phase, there was a treatment effect on the total number of short syllables produced and on the total duration of emitted ultrasonic vocalizations, in particular for syllables of the modulated, mixed and one-jump category. Compared to the vehicle group, DEHP-exposed males were also less able to attract females, in particular for the DEHP-50 group. During the copulatory phase, effects of DEHP treatment were seen on the latency to reach ejaculation, with a longer duration of mating for the DEHP-50 group. DEHP-exposed mice also exhibited more intromissions and thrusts than the vehicle group. Therefore, it seems that DEHP-exposed male mice had more difficulties than the vehicle group in attracting females, and initiating and terminating mating.

Behavioral changes were not due to an altered olfactory preference, since DEHP-exposed males were able to discriminate between non-receptive and receptive females, and showed more interest in the latter females. This suggests that the modifications induced by pubertal DEHP exposure are located downstream from the olfactory system, in the chemosensory areas such as the medial amygdala, the bed nucleus of stria terminalis and the medial preoptic area where signals are processed in behavioral responses.

Analyses of general behavior showed that locomotor activity during the dark phase, the period during which mating tests were performed, was unaffected by DEHP treatment. In contrast, both DEHP-treatment groups displayed an increased anxiety-related behavior as assessed by the O-maze test. Therefore, the altered sexual behavior of male mice exposed to DEHP during the pubertal period could be explained by changes in the neural circuitry underlying sexual behavior together with an increased anxiety-state level, which could interfere with the expression of reproductive behaviors.

The behavioral modifications induced by pubertal exposure to DEHP were observed two months after exposure arrest. This could be due either to long-term effects of DEHP or to the persistence of DEHP metabolites in the body of animals tested. Phthalates such as DEHP are rapidly degraded and their metabolites extensively excreted within a few days, mainly via urine and feces. Indeed, no accumulation in the plasma or testes of rats and marmosets was reported following oral administration of DEHP [START_REF] Kurata | Metabolism of di(2-ethylhexyl) phthalate (DEHP): comparative study in juvenile and fetal marmosets and rats[END_REF][START_REF] Pollack | Effects of route of administration and repetitive dosing on the disposition kinetics of di(2-ethylhexyl) phthalate and its mono-deesterified metabolite in rats[END_REF][START_REF] Teirlynck | Disposition of orally administered di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate in the rat[END_REF]. However, the retention of DEHP and its active metabolite MEHP (mono-2-ethylhexyl phthalate) was reported in vivo in the fat tissues of mice [START_REF] Lindgren | A whole body autoradiographic study on the distribution of 14C-labelled di-(2-ethylhexyl)phthalate in mice[END_REF] and monkeys [START_REF] Jacobson | Effects of a plasticizer leached from polyvinyl chloride on the subhuman primate: a consequence of chronic transfusion therapy[END_REF], and in vitro in fat cells where MEHP impacted cellular metabolism [START_REF] Chiang | Mono(2ethylhexyl)phthalate accumulation disturbs energy metabolism of fat cells[END_REF]. In the present study, the body weight of male mice exposed during the pubertal period did not increase, in contrast to what has previously been reported for the perinatal exposure [START_REF] Hao | Perinatal exposure to diethyl-hexyl-phthalate induces obesity in mice[END_REF]. We observed unchanged or reduced body weight of male mice exposed during the pubertal period to DEHP at 5 or 50 µg/kg/d, respectively. Furthermore, in the same experimental conditions, male mice exposed during adulthood exhibited normal behavior after a similar duration of exposure arrest. Together, these observations led us to suggest that the behavioral effects induced by pubertal exposure to DEHP may be mainly due to long-term effects.

The behavioral alterations induced by pubertal exposure were associated with unchanged circulating levels of testosterone and weight of the androgen-sensitive tissues (seminal vesicles, testis and Tyson gland). In agreement with these results, no alteration of the androgenic status of rats was observed following peripubertal exposure to DEHP at 7.7 and 75 mg/kg/d [START_REF] Venturelli | Effects of exposure to di-(2-ethylhexyl) phthalate (DEHP) during lactation and puberty on sexual maturation and glycemic homeostasis in males rats[END_REF]. In contrast, pubertal exposure to higher doses of DEHP ranging from 125 to 6000 mg/kg/d resulted in reduced androgen levels and androgen-dependent tissue weights in rats and mice [START_REF] David | Reversibility of the chronic effects of di(2-ethylhexyl)phthalate[END_REF][START_REF] Liu | Pubertal exposure to di-(2-ethylhexyl)-phthalate inhibits G9amediated histone methylation during spermatogenesis in mice[END_REF][START_REF] Noriega | Pubertal administration of DEHP delays puberty, suppresses testosterone production, and inhibits reproductive tract development in male Sprague-Dawley and Long-Evans rats[END_REF][START_REF] Wang | Effects of early pubertal exposure to di-(2ethylhexyl) phthalate on social behavior of mice[END_REF]. Therefore, it seems from these observations that pubertal exposure to low DEHP doses does not affect testosterone levels and consequently androgen-sensitive tissues of the urogenital tract.

Quantification of the protein amounts of the two key receptors involved in the activation of male sexual behavior showed an effect of DEHP treatment on AR protein amount in the medial preoptic area. In particular, a significantly reduced protein amount was observed at 50 µg/kg/d of DEHP, but a tendency of this amount to be decreased was also noticed for the DEHP-5 group. No treatment effects were observed on ERα protein amount in the medial preoptic area between the three treatment groups. In our previous work on adult exposure with continuous DEHP treatment, the same DEHP doses as in the present study were found to alter sexual behavior and triggered selective modifications in the expression levels of the neural AR, without affecting the integrity of the hypothalamus-pituitary-gonad axis or hormonal levels [START_REF] Dombret | Neural mechanisms underlying disruption of male courtship behavior by adult exposure to di-(2-ethylexyl)phthalate in mice[END_REF]. Altogether, these data indicate first that pubertal and adult exposures to low DEHP doses target preferentially the AR signaling pathway in the neural circuitry underlying sexual behavior. Second, the structures underlying sexual behavior are more sensitive than the urogenital tract to pubertal and adult exposure to DEHP at low doses (5 and 50 µg/kg/d). Whether such differences in tissue sensitivity to DEHP also occur for perinatal exposure needs further studies at equally low doses. A previous work reported that gestational and lactational exposure to 50 µg/kg/d or 5 mg/kg/d of DEHP reduced testis and seminal vesicle weights in male mice [START_REF] Pocar | Exposure to di(2-ethyl-hexyl) phthalate (DEHP) in utero and during lactation causes long-term pituitary-gonadal axis disruption in male and female mouse offspring[END_REF]. This suggests a high sensitivity of the urogenital tract to DEHP exposure during this developmental period.

The TDI dose for DEHP is equivalent to 50 µg/kg/d in Europe. The recent revision by the EFSA of 5 phthalates including DEHP maintains the TDI dose at 50 µg/kg/d on the basis of their ability to reduce fetal testosterone production [START_REF] Efsa | Update of the risk assessment of di-butylphthalate (DBP), butyl-benzyl-phthalate (BBP), bis(2-ethylhexyl)phthalate (DEHP), di-isononylphthalate (DINP) and di-isodecylphthalate (DIDP) for use in food contact materials[END_REF]. The present behavioral study, together with increasing evidence in the literature of in vivo effects of DEHP on several biological systems at low doses in comparison to the previously used high doses in the majority of experimental studies addressing phthalate exposure, should lead to revision of the reference values established for this molecule.

We have previously reported that chronic exposure of adult male mice to DEHP at 5 or 50 µg/kg/d reduced the emission of ultrasonic vocalizations, the interest of female partners towards exposed males, and increased the latency to reach ejaculation [START_REF] Dombret | Neural mechanisms underlying disruption of male courtship behavior by adult exposure to di-(2-ethylexyl)phthalate in mice[END_REF]. These behavioral changes were associated with a selective down-regulation of AR expression in the neural circuitry underlying male sexual behavior. The present study shows that when mice were exposed to the same DEHP doses and analyzed two months after exposure arrest, there were no significant behavioral effects of DEHP treatment. Indeed, the production USVs as well as their ability to attract a receptive female and initiate and terminate mating were comparable between the vehicle and DEHP-treated groups of mice. Moreover, the protein amount of AR in the medial preoptic area was also unchanged between the three treatment groups. This reversibility was not specific to reproductive behaviors and underlying mechanisms. Indeed, in adult males where exposure was maintained throughout the behavioral analyses there was an increased anxiety-state level. In contrast, adult male mice analyzed after a DEHP exposure arrest of two months exhibited an anxiety-state level comparable to that of the vehicle group. These data indicate that the effects induced by adult exposure to low doses of DEHP are reversible after arrest of exposure in male mice, thereby highlighting the plasticity of neural structures underlying reproductive and anxietyrelated behaviors. In agreement with these observations, a previous rat study showed that cessation of chronic adult exposure improved the effects induced by high doses of DEHP on the kidney, liver and testes [START_REF] David | Reversibility of the chronic effects of di(2-ethylhexyl)phthalate[END_REF]. Such reversibility can be explained by the rapid degradation of DEHP and excretion of its metabolites as discussed above. However, further investigation as to whether the nervous system or peripheral tissues are able to adapt following repetitive environmental exposure to endocrine disruptors is needed.

Conclusion

The data presented here and summarized in Table 2 show that pubertal exposure to low doses of DEHP induced long-term effects in male mice. Indeed, these animals showed altered sexual and anxiety-related behaviors during adulthood. At the DEHP doses used in this study, there were no effects on circulating levels of testosterone or weight of the androgen-sensitive tissues, but a significant decrease of AR protein amount was observed in the medial preoptic area, a key hypothalamic region involved in male sexual behavior. This suggests a higher sensitivity of neural structures to pubertal exposure to low doses of DEHP. A lower body weight was noticed, suggesting that other peripheral and/or neural effects must be induced by such pubertal exposure. In contrast to pubertal exposure, an exposure arrest of two months after adult exposure of male mice to DEHP did not trigger long-term effects as evidenced by the unchanged sexual behavior and anxiety-related behavior, as well as comparable AR protein amounts in the medial preoptic area between the treatment groups (Table 2). This indicates that exposure arrest completely reversed the modifications observed in behavior and hypothalamic AR expression when DEHP treatment was maintained during the period of analyses (Table 2). Altogether, these observations point out the high sensitivity of neural structures to low DEHP doses, in particular during critical period such as puberty. The weight of body and androgen-sensitive tissues (seminal vesicles, testis, Tyson gland) in adult male mice exposed to DEHP at 5 or 50 µg/kg/d during the pubertal period, or during adulthood.

Figure 1 .

 1 Figure 1. Paradigms of pubertal versus adult exposure to DEHP. A. For pubertal DEHP

Figure 2 .

 2 Figure 2. Effects of pubertal exposure to DEHP on olfactory preference of adult male mice. A.

Figure 3 .

 3 Figure 3. Effects of pubertal exposure to DEHP on the emission of ultrasonic vocalizations by

Figure 4 .

 4 Figure 4. Effects of pubertal exposure to DEHP on partner preference, mating and

Figure 5 .

 5 Figure 5. Effects of pubertal exposure on locomotor activity and anxiety-related behavior in

  Absolute (mg) and relative organ weights (% of body weight (bw)) are expressed as means ± S.E.M of 11-12 males per treatment group for pubertal exposure, and 11-12 males per treatment group for adult exposure. * p < 0.05 versus the vehicle group.

Acknowledgements

This work was supported by the "Agence Nationale de Sécurité Sanitaire de l'Alimentation, de l'Environnement et du Travail" (Anses; Project n° 2012-2-077). We thank the INRAE hormonal assay platform for testosterone assays and Sorbonne University platforms for taking care of the animals.

SU

https://dropsu.sorbonne-universite.fr/

Table 2

Summary of the behavioral and neuroendocrine effects induced by pubertal or adult exposure to DEHP at 5 or 50 µg/kg/d in male mice. AR: androgen receptor, ERα: estrogen receptor α, ND: not determined.


 We examine immobility in Rio de Janeiro using the 2002/2003 Household Travel Survey  Almost half of inhabitants made no trips at all during the day  Income has only a slight effect on the probability of being immobile  Unemployed workers and homemakers have the greatest number of immobile  Strong geographic dimension of immobility is due to demographic structure effects

Immobility in Rio de Janeiro, beyond poverty

Introduction

Average levels of individual mobility in Brazilian cities are low, in terms of both kilometers traveled and number of trips made. An initial review of the 2002/2003 Rio de Janeiro Metropolitan Area (RJMA) Household Travel Survey (HTS) (CENTRAL 2005) suggests very high levels of immobility. While immobility is sometimes mentioned in studies on Latin American cities [START_REF] Vasconcellos | Transport metabolism, social diversity and equity: The case of São Paulo, Brazil[END_REF][START_REF] Ureta | To Move or Not to Move? Social Exclusion, Accessibility and Daily Mobility among the Low-income Population in Santiago, Chile[END_REF]), immobility has never been studied as a subject in itself, despite the fact that it is a major characteristic of these cities. We have undertaken such a study, and results are presented in this article.

According to the 2002/2003 HTS 1 nearly half of RJMA residents (46%) did not travel at all during the day. This degree of immobility is much higher than that observed in European countries, where it is between 10 and 26 percent, depending on the country, sources, and population studied [START_REF] Hubert | Immobility and Mobility Seen Through Trip-Based Versus Time-Use Surveys[END_REF]. The figure for another major Brazilian metropolitan area, São Paulo, is similar: [START_REF] Vasconcellos | Transport metabolism, social diversity and equity: The case of São Paulo, Brazil[END_REF] observes that the level of immobility there varies from 24 to 55 percent depending on individuals' income level.

Poverty is often cited as an explanation for these high levels of immobility in Brazilian cities (CENTRAL 2005;[START_REF] Vasconcellos | Transport metabolism, social diversity and equity: The case of São Paulo, Brazil[END_REF], without specifying exactly which characteristics of poverty or impoverished populations 2 lead to immobility. There are very few examples of research on the subject of immobility, and those that do exist concentrate on measuring degrees of immobility rather than studying its characteristics and causes [START_REF] Madre | Immobility in travel diary surveys[END_REF][START_REF] Hubert | Immobility and Mobility Seen Through Trip-Based Versus Time-Use Surveys[END_REF].

Examining the literature on the connections between poverty and mobility, one notable review of which is given by [START_REF] Fol | La mobilité des pauvres[END_REF], we can identify several potential explanations for immobility. First, low incomes lead people to limit or avoid certain trips that are too costly. Next, income tends to affect the number of trips for certain purposes, notably those for purchases and health services, which are less numerous in poorer populations. Finally, impoverished populations are less mobile overall. They are affected by spatial mismatch [START_REF] Kain | Housing segregation, negro employment, and metropolitan decentralization[END_REF]) which limits their access to employment, as well as their social capital [START_REF] Putnam | Bowling alone: America's declining social capital[END_REF][START_REF] Putnam | Bowling alone: The collapse and revival of American community[END_REF], and by diminished motility [START_REF] Kaufmann | Motility: mobility as capital[END_REF] in the neighborhoods where they live.

In the city of Rio de Janeiro, which is strongly segregated, the poorer part of the population is concentrated in the North and the richer part in the South. This segregation could potentially 1 The Rio de Janeiro HTS does take into account all trips, associated with a purpose, unless walking trips of less than 300 meters.

2

The category "poor" is questionable, and subject to criticism. Not only does this category say nothing about the processes that lead to social inequality and poverty, but it is also very vague and depends on geographic and historical context (Paugam and Duvoux, 2008). Impoverished individuals experience a partial lack of social representation or are denigrated as dependent on social programs (Farge et al., 2002). Nonetheless, we use the term "poor" (and its variants) in this article, both for simplicity and because it can today be connected with a statistical definition of poverty that allows comparative analysis. manifest itself as a strong geographic dimension to immobility, which would be very present in the North while the South would be situated at the level of large European cities.

Our results partially contradict this hypothesis, as we find that, all other things equal, individuals in the northern zone of Rio de Janeiro are not more immobile than those in the South. For example, an unemployed man around thirty years of age with a high school level of education has the same probability of being immobile no matter where he lives. Nevertheless, immobility remains much higher in the North, because of the socio-demographic and activity structure of the populations who live there. Indeed, an analysis of the Rio de Janeiro HTS shows that more so than income, employment status is the principal factor behind immobility. The northern zone is home to more homemakers and unemployed workers, and almost 80 percent of people in these two categories are immobile.

Less mobility among the poor

Mobility is unequally distributed

Over the course of recent decades, mobility has experienced rapid growth, particularly in North America and Europe, driven by growing vehicle ownership [START_REF] Orfeuil | L'évolution de la mobilité quotidienne:(comprendre les dynamiques, éclairer les controverses)[END_REF]. The United States is much more mobile than other countries, because motorization took place earlier and was more intense [START_REF] Fol | La mobilité des pauvres[END_REF]. The distances traveled per capita there are almost twice those observed in France [START_REF] Orfeuil | L'évolution de la mobilité quotidienne:(comprendre les dynamiques, éclairer les controverses)[END_REF]. In Brazil, a low level of car ownership (almost eight times lower than in the US, with 110 versus 830 cars per 1000 inhabitants) [START_REF] Lopes | Elaboração de Modelos Matemáticos para Análise[END_REF]) leads to lower overall mobility. The number of trips per person per day is 1.77 in Rio de Janeiro in 2002[START_REF] Barnes | Determinants of individual neighborhood ties and social resources in poor urban neighborhoods[END_REF](CENTRAL 2005), as compared to an average of 4 in the United States [START_REF] Pucher | Socioeconomics of urban travel: evidence from the 2001 NHTS[END_REF] and 3.5 in the Paris region (called Ile-de-France) [START_REF] Dreif | Les déplacements des franciliens en 2001-2002[END_REF]. This corresponds to an average travel distance of 14.8 kilometers per day per inhabitant in São Paulo, as opposed to 17.5 kilometers for an inhabitant of Ile-de-France. The number of trips by car is particularly low in Brazilian cities: 0.3 trips per day per person in Rio de Janeiro and 0.6 in São Paulo, compared to 1.5 in Paris.

The different forms of mobility and in particular automobile mobility [START_REF] Urry | Social Inequalities in Network Capital[END_REF]) require economic resources, and this is a source of inequality between individuals. Indeed, many studies show that access to mobility varies from one social group to another, particularly because access to an automobile is dependent upon income [START_REF] Hivert | Le parc automobile des ménages, étude en fin d'année 1999 à partir de la source" Parc Auto[END_REF][START_REF] Pucher | Socioeconomics of urban travel: evidence from the 2001 NHTS[END_REF][START_REF] Vasconcellos | Transport metabolism, social diversity and equity: The case of São Paulo, Brazil[END_REF]. The poorest households (with incomes below 250 reals 3 per month) have low levels of automobile ownership, with less than one in ten owning a car, as opposed to one in two for the richest households. They use the car for only 12.1% of trips as opposed to 66.1% for the richest group (with incomes above 3600 reals per month) [START_REF] Vasconcellos | Transport metabolism, social diversity and equity: The case of São Paulo, Brazil[END_REF]. This also leads to strong differences in travel distance. Among the poorest individuals in Rio de Janeiro, even public transit use is limited, and almost 60 percent of trips are made on foot (CENTRAL 2005). Such clear income-based differences in mobility are not found in French cities [START_REF] Olvera | Daily mobility and inequality: the situation of the poor[END_REF][START_REF] Paulo | Inégalités de mobilités: disparité des revenus, hétérogénéité des effets[END_REF]. While trip distance is lower for poorer populations, in general they still have access to automobiles. The average number of trips for poor and rich households is comparable, with the exception of work-related trips. In large Brazilian cities like São Paulo and Rio de Janeiro, the poorest populations have an average number of trips almost half that of the rich (CENTRAL 2005;[START_REF] Vasconcellos | Transport metabolism, social diversity and equity: The case of São Paulo, Brazil[END_REF]). These strong differences can be explained by the greater income differences between the rich and the poor, but also by their higher levels of unemployment and differences in employment status that lead to a lower number of work-related trips.

One of the most extreme expressions of income-related mobility disparity is the very high proportion of immobile individuals, i.e. those that make no trips at all: 46.6% of the inhabitants of Rio de Janeiro according to 2004/2005 HTS data. The level of immobility in Brazilian cities is particularly high in comparison with those observed in European countries, which are generally situated between the values of 17.4% in France and 22% in the United Kingdom [START_REF] Madre | Immobility in travel diary surveys[END_REF][START_REF] Hubert | Immobility and Mobility Seen Through Trip-Based Versus Time-Use Surveys[END_REF]. For [START_REF] Vasconcellos | Transport metabolism, social diversity and equity: The case of São Paulo, Brazil[END_REF], the reason that values are so high in Brazilian cities is probably to be found in the very low income of a large part of the population, as well as unemployment and underemployment. In Rio de Janeiro, the percentage of immobile individuals reaches 54.3% among the poor, as compared to 32.9% among households earning more than 20 times the minimum wage (CENTRAL 2005). In the São Paulo HTS, we observe the same kind of differences between the richest and poorest households. Among the poorest individuals, immobility reaches 55% while it is only 24% in the richest category of individuals (Vasconcellos 2005).

Spatial mismatch, neighborhood effects, and social capital

Besides the income and underemployment of poorer populations, very high levels of immobility could be explained by the spatial mismatch hypothesis and the intense concentration of poverty in certain neighborhoods and areas. The spatial mismatch hypothesis [START_REF] Kain | Housing segregation, negro employment, and metropolitan decentralization[END_REF]) has long been used in North American research to address the absence or low level of mobility among poor households, as well as the living conditions and social trajectory of these households. These households' weak or nonexistent mobility is considered particularly problematic there [START_REF] Bacqué | Inequality Regarding Mobility: Observations and Policies[END_REF][START_REF] Fol | La mobilité des pauvres[END_REF]. It is considered to be a consequence of segregation processes combined with the decentralization of employment, which together increase the distance between poorer populations and employment opportunities, reducing both their chances of finding a job [START_REF] Holzer | Work, search, and travel among white and black youth[END_REF] and their ability to travel.

In the municipality of Rio de Janeiro, the principal employment centers are in the south area and downtown (figure 2), whereas the poor population is concentrated in the northern neighborhoods [START_REF] Marques | A comparison of social structures in Rio de Janeiro and Sao Paulo[END_REF] where unemployment is particularly high. We would thus expect to observe high levels of immobility there. However, the spatial mismatch theories are controversial. In the end, spatial mismatch is only a secondary factor in the higher risk of unemployment for individuals who are members of ethnic minorities, for whom the principal barriers to employment are education, qualifications, and child care [START_REF] Blumenberg | Beyond the spatial mismatch: welfare recipients and transportation policy[END_REF]. Thus, spatial mismatch might have but a very limited impact on general mobility, and particularly on immobility [START_REF] Fol | Transport policy and the car divide in the UK, the US and France: Beyond the environmental debate[END_REF].

Looking beyond spatial mismatch, the concentration of poor populations in certain neighborhoods tends to create a "social isolation" similar to that observed in inner cities in the United States, as well as certain neighborhoods of French cities [START_REF] Wilson | The truly disadvantaged: The inner city, the underclass, and public policy[END_REF][START_REF] Breton | Bouger pour s'en sortir. Mobilité quotidienne et intégration sociale[END_REF]. Within the Rio de Janeiro municipality, the hilly landscape contributes to the separation of the northern neighborhoods (that are home to a large number of people living in extreme poverty) from the richer southern neighborhoods [START_REF] Marques | A comparison of social structures in Rio de Janeiro and Sao Paulo[END_REF]. According to [START_REF] Putnam | Bowling alone: The collapse and revival of American community[END_REF], the poor neighborhoods and the people who live there possess little social capital4 because spatial and social isolation deprive them of contact with people from other social groups. Openness to other social groups allows access to employment and social success. Thus, the concentration of impoverished households in the northern zone of Rio de Janeiro could make an even greater number of them immobile, perhaps through a "contagion" as postulated by [START_REF] Crane | The epidemic theory of ghettos and neighborhood effects on dropping out and teenage pregnancy[END_REF] or low social capital [START_REF] Putnam | Bowling alone: America's declining social capital[END_REF], Coleman 2007). However, these ideas are contested (Bordreuil 1998[START_REF] Imbroscio | United and actuated by some common impulse of passion: challenging the dispersal consensus in american housing policy research[END_REF]) and analyses give mitigated and sometimes contradictory results [START_REF] Marpsat | Le chômage des jeunes est-il aggravé par l'appartenance à un quartier en difficulté ?[END_REF]. In the end, existing work does not conclusively link lower mobility levels with neighborhood effects, beyond a simple income-related lack of access to automobiles. Here again we might discover that belonging to a certain neighborhood has a limited effect on the number of trips made by the populations who live there, and on their immobility levels.

The explanation for these uncertain results is probably to be found in the fact that the spatial mismatch hypothesis and Putnam's social capital approach [START_REF] Putnam | Bowling alone: The collapse and revival of American community[END_REF] ignore or underestimate the degree of local solidarity and the strength of social networks in impoverished neighborhoods. Many studies show that these networks and other forms of solidarity play an essential role in the daily life of inhabitants [START_REF] Barnes | Determinants of individual neighborhood ties and social resources in poor urban neighborhoods[END_REF], notably in issues concerning mobility [START_REF] Coutard | Suburban poverty: local dependance or automobile dependance?: Spaces, directions for use[END_REF], Fol et al. 2007), including the search for employment [START_REF] Chapple | Time to work: Job search strategies and commute time for women on welfare in San Francisco[END_REF]. Additionally, distance to the workplace is not necessarily a barrier to employment in poor neighborhoods, considering that job markets for the least qualified people are primarily local [START_REF] Shen | A spatial analysis of job openings and access in a US metropolitan area[END_REF].

Mobility has grown in our societies not only because it has become technically easier but also because it has social value [START_REF] Rémy | Mobilités et ancrages: vers une autre définition de la ville[END_REF]. But the "injunction to be mobile conflicts partially with the experience of poor households and the concrete strategies they apply to confront poverty" [START_REF] Bacqué | Inequality Regarding Mobility: Observations and Policies[END_REF]. These strategies do not necessarily require the transformation of potential mobility into actual mobility, as shown by studies on the subject [START_REF] Kaufmann | Motility: mobility as capital[END_REF], and this leads to a reduced number of trips among certain categories of the population, specifically poor households.

For certain authors defending a new paradigm of mobility (Hannan et al., 2006;[START_REF] Urry | Social Inequalities in Network Capital[END_REF]Adey, 2010;Cresswell, 2010), mobility and immobility are "neither a dualism nor an opposition rather a relational continuum" (Pellegrino 2011). Mobility has a respiration-like rhythm (Creswell 2010) that manifests through periods of relative immobility during which the individual remains active. For [START_REF] Urry | Social Inequalities in Network Capital[END_REF], mobility requires these "mooring" phases to remain fluid over time and space. Depending on the scale of analysis, locality or proximity can be mistaken for immobility.

Individuals belonging to poor households tend to connect mobility and proximity in a particular way, relying more heavily on proximity [START_REF] Bacqué | Inequality Regarding Mobility: Observations and Policies[END_REF][START_REF] Fol | La mobilité des pauvres[END_REF]. This necessarily leads to reductions in travel distance, which are clearly observed among the poorer population of several countries, including Brazil. But it may also result in a lower observed number of trips and even apparent immobility when the means for measuring mobility (the Household Travel Surveys) are not designed to record short-range travel on foot. We shall examine this problem more closely and describe the data used in this study below.

Studying immobility using 2002/2003 Household Travel Survey (HTS) data

The RJMA, a territory with 11 million inhabitants

The Rio de Janeiro household travel survey took place between October 2002 and December 2003, with a sample of 34,000 households participating in face-to-face interviews. These households are distributed throughout the entire RJMA, which had an estimated population of 11.3 million in 2003. The RJMA includes 20 municipalities, and stretches over 120 kilometers from east to west, and 50 km in the north-south direction. The municipality of Rio de Janeiro is the largest and most populous one in the metropolitan area, with around 53.8 percent of the population and roughly the same proportion of jobs.

The geography of human settlement in the RJMA is characterized first and foremost by its coastal location facing the Atlantic Ocean, surrounding the Guanabara Bay. Next, population distribution can be understood in connection with the very hilly terrain throughout the RJMA. The steepest hills have quite low population or none at all, creating very low-density areas in the heart of the city, only a few hundred meters from the center.

Almost 100,000 people interviewed

Out of the 99,300 individuals interviewed for the Rio de Janeiro HTS we analyzed 97,536, excluding records with incomplete information and those where incoherency was found in the household characteristics. Women make up 52.9% of the sample. There are more women in higher age groups, which can be explained by shorter life expectancy for men and a sharp peak in their mortality between 15 and 25 years of age, due largely to violence.

The RJMA population has several notable characteristics. First, employment is low (52,3%) among individuals between 18 and 60 years of age. In Ile-de-France this figure is 73.4%. Low employment is explained by the number of unemployed people and homemakers among the 18-60 year old population in the RJMA. These two categories make up 15.2 and 16.6 percent of this age class, as compared to 9.1 and 4.1 percent in Ile-de-France. Next, the population of the RJMA is younger overall than that of Ile-de-France, but the difference is rather small. Thus, we find a somewhat smaller percentage of the population is aged 60 or over (14% of the total RJMA population as opposed to 16.2% in IDF) and the proportion of children under 15 years old is 20.5% as opposed to the 19.8% observed in Ile-de-France.

Low levels of mobility revealed by the Rio de Janeiro Household Travel Survey

The HTS reveals that the inhabitants of the RJMA have a low level of mobility: only 19 million trips are carried out daily, or 1.77 per capita, and for more than 46% of survey participants no trips were reported at all. For the sake of comparison, a mobile employed person in RJMA carries out on average 2.4 trips per day, while this figure is over 3.7 for an inhabitant of the Paris region. The least mobile inhabitants of the RJMA are those over 60 years of age and the lowest-income category, with averages of 1.2 and 1.5 trips per day, respectively.

Another striking characteristic of the cariocas'5 mobility is the modal split. Only 15 percent of trips are carried out by car, as compared to 44% in Ile-de-France. The automobile's small mode share stands in contrast with that of public transport, which carries 46.4% of trips as compared to 19.4% in Ile-de-France. Finally, we note that the share of walking trips is practically equal in the two cities, at around 34% of trips.

High levels of immobility: a result of survey methods?

The immobility figures for Rio de Janeiro and São Paulo are both over 45%, considering the whole of the population, and are much higher than those observed in European cities, which vary from 16.9 to 22.6 percent of individuals [START_REF] Hubert | Immobility and Mobility Seen Through Trip-Based Versus Time-Use Surveys[END_REF]. The extreme poverty which affects a large part of the Brazilian population would seem to be a major explanation for these differences. However, two principal differences in survey methods also exist between European travel surveys and those in Rio de Janeiro.

The first difference is in the interview samples. The Rio de Janeiro HTS concerns the entire population, including the youngest children, whereas European surveys do not take everyone into account and use different age cutoffs. [START_REF] Hubert | Immobility and Mobility Seen Through Trip-Based Versus Time-Use Surveys[END_REF] take into account all individuals over 15 years of age in their comparison of three European countries. Applying this threshold to the RJMA, the percentage of immobile individuals remains the same, at 47% of the population. Thus, the inclusion of very young individuals in the survey does not contribute at all to the high level of immobility observed in the RJMA.

The second difference in methods is less obvious, and while its effects are not directly measurable they certainly exist. The Rio de Janeiro HTS does not count walking trips of less than 300 meters. The Paris HTS reveals that 5.3% of the population there did not make any trip of over 300 meters. However, the absence of complementary quantitative or qualitative surveys specifically dealing with the RJMA prevents us from evaluating the effect of this methodological choice on the measurement of immobility. We will see below that the categories of people most likely to be considered immobile are homemakers and the elderly, two categories for whom we also observe many short-range trips on foot near the home. Despite these methodological differences, the magnitude of the difference in immobility between Brazilian and European cities is still surprising. We recall that the HTS is but a snapshot of individual mobility on a particular day, and does not take into account life rhythms and forms of organization that occur over periods of several days [START_REF] Axhausen | Observing the rhythms of daily life: A six-week travel diary[END_REF][START_REF] Axhausen | Fatigue in long-duration travel diaries[END_REF]) which can vary greatly from one place or country to another. For example, an individual who is immobile on one day might be mobile every other day of the week. HTS that observe individual mobility over several days find levels of mobility of only 5% to 8% of the population [START_REF] Chlond | Auswertung zum Deutschen Mobilitäts-Panel 1998/1999: Haushaltsbefragung zur Alltagsmobilität in verschiedenen Raumtypen[END_REF][START_REF] Axhausen | Observing the rhythms of daily life: A six-week travel diary[END_REF][START_REF] Axhausen | Fatigue in long-duration travel diaries[END_REF]) and very similar values in the various places studied [START_REF] Madre | Immobility in travel diary surveys[END_REF]).

Two contrasting territories in Rio de Janeiro

To study the connection between poverty and immobility in more detail, we have compared two areas of the Rio de Janeiro municipality. These two zones are on one hand a vast area containing 1,288,000 inhabitants in the northern, poorer part of the city, and on the other hand an area containing 872,000 inhabitants in the south, the richest part of the city. As the individual income variable in the Rio de Janeiro HTS is of insufficient quality, this comparison is a means to evaluate the effects of poverty on immobility.

The outlines of the two zones were defined based on the "traffic zone" divisions (CENTRAL 2005) specific to the Rio de Janeiro HTS, which are larger than census tracts. This is why the outlines we have chosen seem to deviate slightly from the geography of income in the Rio de Janeiro municipality. We also note that the outlines we use are chosen to assure that the two study zones include a sufficient number of HTS data points to allow a detailed comparison. The "northern zone" includes some of the poorest neighborhoods in the city, where average incomes are below 700 reals per month, but also areas with intermediate incomes, between 700 and 1600 reals. The "southern zone" includes a few areas with intermediate average incomes, but is essentially composed of zones whose average revenue is high (>1600 reals). The principal favelas present in the south of the city are not included in the "southern zone" as we have drawn it.

Structural immobility

Immobility in Rio de Janeiro is strongly correlated with the poverty that affects a large part of the population. In households with a minimum wage income, 54.3% of individuals are immobile. As opposed to only 32.9% for those individuals in households earning twenty times the minimum wage. But the income levels themselves do not seem to be a major explicative factor of the probability of being immobile in our analysis. Immobility depends more on employment status and the demographic structure of impoverished populations.

Employment status highly correlated with immobility level

Immobility takes on very different values depending on individuals' employment status. It varies from 80% for the least mobile individuals -homemakers and unemployed workers -to a little over 20% for the most mobile people, employed workers and students. This is fairly intuitive: we note a strong opposition between the individuals who are most likely to have an activity outside the home (students and employed workers) and those who are least likely to have obligatory trips away from the home (homemakers, unemployed workers, the retired, and young children).

Figure 3: percentage of immobile individuals by employment status and location of residence

A clear contrast can be observed between the northern and southern zones of the Rio de Janeiro municipality, with the level of immobility reaching 50% in the North as opposed to 32% in the South. This distinction between the richer and poorer parts of Rio de Janeiro can be observed for every individual employment status. It is only among employed workers that the difference is very small.

The principal factors behind individual immobility are individual characteristics

The variables that contribute the most to the probability that an individual is immobile are all individual characteristics, rather than ones related to a household or a place of residence.

Among individual characteristics, employment status plays a major role in determining immobility. On one hand, we find students and employed workers whose level of immobility is low, and on the other hand unemployed or inactive workers, homemakers, and retirees or pensioners who have very high levels of immobility. We note that unemployment has a very strong effect on immobility among workers. Among those who do have a job, being employed in the formal economy greatly minimizes the probability of being immobile relative to employment in the informal sector or non-contractual work.

The probability of being immobile varies with age and education level. The more education a person has, the lower the probability of being immobile, while inversely mobility decreases as age increases.

The position within the family has a very strong effect on the probability of being immobile. Spouses (other than the "reference person" or "head of household") and children have a much higher probability of being immobile. This probability is even higher in the case of family members (other than parents and children) living with the household.

Household characteristics have only a limited effect on the probability of being immobile

Among the many variables that characterize households, few of them have significant connections with the dependent variable (immobility). For example, household automobile ownership is not significant, though for those individuals who did make at least one trip in the day, the distance and number of trips depends on the number of cars in the household. The only household variable having a strong, significant link with the probability of immobility is the number of children (or the total number of individuals in the household), which is positively correlated with the probability of immobility.

Another household characteristic that appears to explain the level of immobility is income. However, interviewees often declined to answer this question, and there seems to be a tendency to underdeclare among the richest households. Because of this fact, this variable was not retained for our model.

Connections between territories and immobility are limited

Living in a low-density area, in a single-family house, and particularly in the peripheral areas of the RJMA tends to increase the probability of being immobile. These effects are slight but an overall trend does emerge: immobility is more frequent where density is lower.

Finally, since the household income data are of questionable quality, we have employed another variable that provides information on the average income in the household's place of residence, derived from the census. It indicates that individuals residing in areas with upper-middle incomes have a lesser tendency to be immobile. Nonetheless, this variable has low explicative power.

A comparison of the northern and southern zones: structural effects of population on the level of immobility

The influence of income on mobility is central to our initial question, but this variable appears only very marginally in the results obtained above. It seems that poverty, when expressed in terms of household income level, is only weakly connected to individual immobility. However, our comparison of the northern and southern parts of the city leaves no doubt that immobility is much more common in the northern, poorer area. Applying the model for the entire RJMA to a sample including only the population of these two areas reveals that, all other things being equal, living in one or the other of these areas has no significant connection with individual immobility (appendix 1).

Thus, we hypothesize that the differences in immobility observed between these two territories can be explained by differences in socio-demographic composition. The connection between poverty and immobility would be only indirectly connected to differences in income, and more directly connected to the very different characteristics of poorer and richer populations. The demographics of the "northern zone" are notable for a greater number of individuals from categories with high levels of immobility, such as homemakers and unemployed workers. The "northern zone" is also characterized by a lower level of education, a higher number of children per household, and the predominance of single-family homes. All of these factors are linked with high levels of immobility. On the other hand, the socio-demographic composition of the "southern zone" tends toward lower levels of immobility.

Table 3: Distribution of individuals by employment status (columns are in percent)

The employment status of individuals, which is a particularly important variable affecting immobility, reveals differences between the northern and southern zones. On one hand, in the north of the city there is a greater number of homemakers and inactive workers (table 3). These are the two categories of people who have the highest levels of immobility. On the other hand, in the south it is employed workers and students who are more numerous, and these are the two groups with the lowest percentage of immobility.

Table 4: Distribution of individuals according to number of children in the household (columns are percent) Table 5: Distribution of individuals by education level (columns are percent)

Employment status is not the only characteristic unequally distributed between the north and the south that contributes to a higher level of immobility in the northern, poorer part of the city. The "northern zone" also has a greater number of large families having two or more children (table 4), and its population generally has a low level of education. Almost 90 percent of the population has only primary or secondary education (table 5). Inversely, in the "southern zone" people are generally more educated, and 50.3% of people have finished secondary or at least some postsecondary education. Level of education is thus a major difference between the poorer northern neighborhoods and the richer south.

Table 6: Distribution of employed workers by employment type (columns are percent) Table 7: Distribution of individuals by age (columns are percent)

Some other individual characteristics that are unequally distributed between the north and the south tend on the other hand to moderate the immobility differences between the two study zones. First and foremost for employed workers, this is the employment type (table 6). Though workers employed in the informal sector have high levels of immobility and are more present in the "northern zone", the category "other" which includes heads of businesses and independent professionals has even higher levels of immobility and is found predominantly in the southern zone. Finally, the population of the northern zone is younger than that of the southern zone (table 7), and immobility increases with age.

Discussion, Conclusion

In Rio de Janeiro, nearly half of inhabitants interviewed were found to carry out no travel during the day. This value is much higher than those observed in major European cities, where it does not exceed one in five inhabitants. We were curious about the causes of this very high level of immobility, as well as its individual and social consequences.

The principal factors explaining high levels of immobility in Rio de Janeiro

Immobility is explained primarily by characteristics of individuals and their households (rather than those of their place of residence). Employment status is the strongest determining factor. The greatest difference is observed between employed workers and students, only 20 percent of whom were immobile, and homemakers and inactive workers, 80 percent of whom were immobile. The figure is around 70 percent for retirees and pensioners. A low level of education, advanced age, and large families are also factors that substantially increase the probability that an individual is immobile. On the other hand, all other things being equal, household income has only a very weak effect on the probability of being immobile. Poverty as expressed by income is not a factor capable of directly explaining high levels of immobility. Nonetheless, we find employment type and level of education to be among the principal determinants of immobility. These are also factors behind poverty.

For this reason, there is a clear difference between the northern zone of Rio de Janeiro, where lowincome populations are located, and the southern zone, where the richest inhabitants live. The former is characterized by a high level of immobility (nearly 50% of the population made no trips) while this figure is a little over 32% in the latter. This geographic dimension to immobility is connected to a particular population structure. The northern zone has a large number of homemakers and unemployed workers, and an overall low level of education. But the high levels of immobility in the northern zone are not at all a consequence of neighborhood effects. These effects, which are contested by numerous authors (Bordreuil 1998[START_REF] Imbroscio | United and actuated by some common impulse of passion: challenging the dispersal consensus in american housing policy research[END_REF] are not found to influence the probability of being immobile, once the effects of individual and household characteristics have been accounted for.

The southern zone still shows levels of immobility above those observed in major European cities. Here again, immobility is explained by the presence of homemakers and unemployed or inactive workers, who are less numerous than in the northern zone. This zone also has a large proportion of retirees and pensioners. We also observe that the employment structure of the southern zone of Rio de Janeiro is different from that in Ile-de-France in that the percentage of employed workers is 10 points lower (48.1% as opposed to 57.5% among the population over 14 years old). This could also be one of the factors explaining the higher degree of immobility in Rio de Janeiro. Several authors have pointed out the percentage of active workers and professional trips as factors capable of explaining differences in mobility level, especially when they vary with income [START_REF] Olvera | Daily mobility and inequality: the situation of the poor[END_REF][START_REF] Paulo | Inégalités de mobilités: disparité des revenus, hétérogénéité des effets[END_REF].

Questions surrounding measurement techniques and travel characteristics may also help explain high levels of reported immobility. The Rio de Janeiro HTS does not count trips of less than 300 meters made on foot. This restrictive definition of a "trip" is characteristic of an HTS intended to measure phenomena relevant to transport engineering, rather than social phenomena. A certain number of trips are thus excluded from observation, and it is impossible to quantify them as there is no point of comparison: urban travel is measured in the same way throughout Brazil. In Paris, where the HTS records all trips over one minute long, we find that 5.3% of people made only walking trips of less than 300 meters during the day. In Rio de Janeiro, this percentage could be even greater because overall travel distances are lower than those in Paris, and more trips should therefore be less than 300 meters. In addition, the proportion of active workers is lower in Rio de Janeiro, and home-work commuting trips are among the longest ones, so the number of individuals who may have made only trips less than 300 meters in length should be higher. Finally, the hilliness and narrow streets of certain neighborhoods, such as the hillside favelas, probably result in a large number of short-range trips on foot, as the use of motorized travel modes is difficult there. However, the northern neighborhoods that we concentrated on do not contain such steep slopes. Measurement methods must also play a role in the level of immobility observed in Rio de Janeiro, but this effect is difficult to quantify.

The individual and social consequences of immobility

In a society that values mobility [START_REF] Rémy | Mobilités et ancrages: vers une autre définition de la ville[END_REF], being immobile means finding oneself excluded, on the fringes of society. Additionally, a large percentage of immobile people could be a sign that a society is functioning poorly, with fewer connections existing between individuals. From this perspective, the levels of immobility seen in Rio de Janeiro seem particularly troubling. However, the literature tends to question these kinds of conclusions, countering them with examples of the concrete strategies used by impoverished households. These strategies do not necessarily include the transformation of potential mobility into actual mobility [START_REF] Kaufmann | Motility: mobility as capital[END_REF]), but tend to combine mobility and locality in a particular fashion, taking advantage of resources in close proximity to the home [START_REF] Bacqué | Inequality Regarding Mobility: Observations and Policies[END_REF]Fol 2007, Fol 2009). Thus, the Rio de Janeiro HTS, which does not count walking trips of less than 300 meters, does not allow us to truly measure the individual and social consequences of immobility, which could only be evaluated with the help of a complementary survey.

Nonetheless, it is possible based on our results to envisage political responses to what might be considered excessive levels of immobility. Some reactions might concern the transport and urban planning sectors, though it is probable that they would have little effect. These measures could include better public transport service for the neighborhoods where immobile populations are concentrated, the construction of streets that better connect certain neighbourhoods to employment centres, or the creation of over-or underpasses across the major infrastructures that can truly function as barriers for the local population [START_REF] Turner | The enclave society: towards a sociology of immobility[END_REF]. Finally, income-based subsidies for transport passes and system-wide integrated ticketing are possible responses to low levels of mobility. But responses targeting the transportation sector would likely have little effect. The low level of access to automobiles does not appear to be a major factor behind immobility, and neither do financial or temporal difficulties in accessing public transport. Much immobility is observed in dense areas that are well-served by public transport, and income levels have only a limited effect on the probability of being immobile.

Appropriate political responses to excessive immobility would instead aim for changes in the labor market and the level of education. A drop in unemployment, an increase in economic activity, and an increase in employment among women would all doubtlessly have a considerable effect on immobility. Since 2003, decreases in unemployment, an increase in employment in the formal sector have most likely led to a significant decrease in immobility, and an increase in education level, particularly in poorer neighborhoods, would allow additional change in this direction.

3

  The minimum wage was 240 reals per month in April 2003. It increased to 545 reals in March 2011. The value of the Brazilian real has fluctuated greatly since 2003. The exchange rate was $1=3,53 reals on January 1, 2003, and $1=1,7 reals in March 2011.

Figure 1 :

 1 Figure 1: Population density of municipalities in the Rio de Janeiro Metropolitan AreaSource: Cartography by the authors from the Rio deJaneiro HTS 2002[START_REF] Barnes | Determinants of individual neighborhood ties and social resources in poor urban neighborhoods[END_REF]

Figure 2 :

 2 Figure 2: Average household income in the municipality of Rio de Janeiro Source: Cartography by the authors from the Rio de Janeiro HTS 2002-2003

Table 1 :

 1 Distribution of "Traffic Zones" in the municipality of Rio de Janeiro by average household income level

Table 2 :

 2 Logistic model: immobile individuals in the Rio de Janeiro HTS

Table 1 :

 1 Distribution of "Traffic Zones" in the municipality of Rio de Janeiro by average household income level

	Income per household	North	South	All RJMA
	Low (<700 reals)	56,7	0,0	55,2
	Medium	43,3	13,1	29,8
	High (>1 600 reals)	0,0	86,9	15,0
	All	100,0	100,0	100,0
	Source: Calculated by the authors from the National Population Census 2000 and the Rio de Janeiro HTS 2002-
		2003

Table 2 :

 2 Logistic model: immobile individuals in the Rio de Janeiro HTSSource: Calculated by the authors from the Rio deJaneiro HTS 2002[START_REF] Barnes | Determinants of individual neighborhood ties and social resources in poor urban neighborhoods[END_REF]

		Standard	Wald
	Estimate	Error	Chi2	Pr>Chi2

Table 3 :

 3 Distribution of individuals by employment status (columns are in percent)Source: Calculated by the authors from the Rio deJaneiro HTS 2002[START_REF] Barnes | Determinants of individual neighborhood ties and social resources in poor urban neighborhoods[END_REF]

		North -
	Employment status	Poor	South -Rich All RJMA
	Student	9,8	12,2	10,8
	Employed	38,5	48,1	42,2
	Unemployed	14,4	5,8	12,5
	Homemaker	16,2	9,2	15,0
	Retiree or
	pensioner	19,3	23,6	17,9
	Other	1,8	1,2	1,5
	All	100,0	100,0	100,0

Table 4 :

 4 Distribution of individuals according to number of children in the household (columns are percent)Source: Calculated by the authors from the Rio deJaneiro HTS 2002[START_REF] Barnes | Determinants of individual neighborhood ties and social resources in poor urban neighborhoods[END_REF]

		North -
	Children	Poor	South -Rich All RJMA
	None	25,6	38,7	26,1
	1 child	29,8	28,5	28,8
	2 children and
	more	44,6	32,7	45,1
	All	100,0	100,0	100,0

Table 5 :

 5 Distribution of individuals by education level (columns are percent)Source: Calculated by the authors from the Rio deJaneiro HTS 2002[START_REF] Barnes | Determinants of individual neighborhood ties and social resources in poor urban neighborhoods[END_REF]

		North -	South -
	Education level	Poor	Rich	All RJMA
	Illiterate	3,1	1,2	3,3
	Primary	47,7	19,8	46,7
	High school	38,2	28,4	33,9
	Some university +	10,9	50,3	15,8
	Other	0,2	0,3	0,3
	All	100,0	100,0	100,0

Table 6 :

 6 Distribution of employed workers by employment type (columns are percent)Source: Calculated by the authors from the Rio deJaneiro HTS 2002[START_REF] Barnes | Determinants of individual neighborhood ties and social resources in poor urban neighborhoods[END_REF]

		North -	South -
	Employment type	Poor	Rich	All RJMA
	Formal Employment	61,8	58,1	57,7
	Informal
	employment	14,1	8,6	17,0
	Other	24,1	33,3	25,3
	All	100,0	100,0	100,0

Table 7 :

 7 Distribution of individuals by age (columns are percent)Source: Calculated by the authors from the Rio deJaneiro HTS 2002[START_REF] Barnes | Determinants of individual neighborhood ties and social resources in poor urban neighborhoods[END_REF] Appendix 1: Logistic model: immobile individuals in Rio de Janeiro north and south areas Source: Calculated by the authors from the Rio deJaneiro HTS 2002[START_REF] Barnes | Determinants of individual neighborhood ties and social resources in poor urban neighborhoods[END_REF]

		North -
	Age	Poor	South -Rich All RJMA

Social capital is defined as a set of ties and norms facilitating coordination and cooperation within a group or a society(Putnam 2000, after Bacqué and Fol 2007).

Cariocas are inhabitants of the municipality of Rio de Janeiro.