Effects of first-generation in utero exposure to diesel engine exhaust on second-generation placental function, fatty acid profiles and foetal metabolism in rabbits: preliminary results.

Atmospheric pollution has major health effects on directly exposed subjects but intergenerational consequences are poorly characterized. We previously reported that diesel engine exhaust (DE) could lead to structural changes in the placenta of in utero exposed rabbits (first generation, F1). The effects of maternal exposure to DE were further studied on second-generation (F2) rabbits. Pregnant F0 females were exposed to filtered, diluted DE (1 mg/m3, median particle diameter: 69 nm) or clean filtered air (controls) for 2 h/day, 5 days/week by nose-only exposure during days 3-27 post-conception (dpc). Adult female offspring (F1) were mated to control males: F1 tissues and F2 foeto-placental units were collected at 28 dpc and placental structure and gene expression (microarray) analysed. Fatty acid profiles were determined in foetal and maternal plasma, maternal liver and placenta. In F1, compared to controls, hepatic neutral lipid contents were increased in exposed animals without change in the blood biochemistry. In F2, the placental lipid contents were higher, with higher monounsaturated fatty acids and reduced pro-inflammatory arachidonic acid (AA), without placental structural changes. Conversely, the proportion of anti-inflammatory n-3 polyunsaturated fatty acids in F2 plasma was increased while that of AA was decreased. Gene set enrichment analyses (GSEA) of F2 placenta transcriptomic data identified that the proteasome complex and ubiquitin pathways genes were over-represented and ion channel function and inflammation pathways genes were under-represented in exposed animals. These preliminary results demonstrate that diesel engine exhaust exposure and in utero indirect exposure should be considered as a programming factor within the context of the DOHaD (Developmental Origins of Health and Disease) with a probable intergenerational transmission.

Effects of dietary arginine supplementation in pregnant mares on maternal metabolism, placental structure and function and foal growth.

Foals born to primiparous mares are lighter and less mature than those born to multiparous dams. Factors driving this difference are not totally understood. Using 7 multiparous and 6 primiparous standardbred mares, we demonstrated that, in late gestation, primiparous mares were less insulin resistant compared to multiparous mares, and that their foals had reduced plasma amino-acid concentrations at birth compared to foals born to multiparous mares. Vascular development, as observed through structure and gene expression, and global DNA methylation were also reduced in primiparous placentas. Another group of 8 primiparous mares was orally supplemented with L-arginine (100 g/day, 210d to term). L-arginine improved pregnancy-induced insulin resistance and increased maternal L-arginine and L-ornithine plasma concentrations but foal plasma amino acid concentrations were not affected at birth. At birth, foal weight and placental biometry, structure, ultra-structure and DNA methylation were not modified. Placental expression of genes involved in glucose and fatty acid transfers was increased. In conclusion, maternal insulin resistance in response to pregnancy and placental function are reduced in primiparous pregnancies. Late-gestation L-arginine supplementation may help primiparous mares to metabolically adapt to pregnancy and improve placental function. More work is needed to confirm these effects and ascertain optimal treatment conditions.

Repeated gestational exposure to diesel engine exhaust affects the fetal olfactory system and alters olfactory-based behavior in rabbit offspring.

BACKGROUND: Airborne pollution, especially from diesel exhaust (DE), is known to have a negative effect on the central nervous system in exposed human populations. However, the consequences of gestational exposure to DE on the fetal brain remain poorly explored, with various effects depending on the conditions of exposure, as well as little information on early developmental stages. We investigated the short-term effects of indirect DE exposure throughout gestation on the developing brain using a rabbit model. We analyzed fetal olfactory tissues at the end of gestation and tested behaviors relevant to pups' survival at birth. Pregnant dams were exposed by nose-only inhalation to either clean air or DE with a content of particles (DEP) adjusted to 1 mg/m3 by diluting engine exhaust, for 2 h/day, 5 days/week, from gestational day 3 (GD3) to day 27 (GD27). At GD28, fetal olfactory mucosa, olfactory bulbs and whole brains were collected for anatomical and neurochemical measurements. At postnatal day 2 (PND2), pups born from another group of exposed or control female were examined for their odor-guided behavior in response to the presentation of the rabbit mammary pheromone 2-methyl-3-butyn-2-ol (2MB2). RESULTS: At GD28, nano-sized particles were observed in cilia and cytoplasm of the olfactory sensory neurons in the olfactory mucosa and in the cytoplasm of periglomerular cells in the olfactory bulbs of exposed fetuses. Moreover, cellular and axonal hypertrophies were observed throughout olfactory tissues. Concomitantly, fetal serotoninergic and dopaminergic systems were affected in the olfactory bulbs. Moreover, the neuromodulatory homeostasis was disturbed in a sex-dependent manner in olfactory tissues. At birth, the olfactory sensitivity to 2MB2 was reduced in exposed PND2 pups. CONCLUSION: Gestational exposure to DE alters olfactory tissues and affects monoaminergic neurotransmission in fetuses' olfactory bulbs, resulting in an alteration of olfactory-based behaviors at birth. Considering the anatomical and functional continuum between the olfactory system and other brain structures, and due to the importance of monoamine neurotransmission in the plasticity of neural circuits, such alterations could participate to disturbances in higher integrative structures, with possible long-term neurobehavioral consequences.

Maternal exposure to diluted diesel engine exhaust alters placental function and induces intergenerational effects in rabbits.

BACKGROUND: Airborne pollution is a rising concern in urban areas. Epidemiological studies in humans and animal experiments using rodent models indicate that gestational exposure to airborne pollution, in particular diesel engine exhaust (DE), reduces birth weight, but effects depend on exposure duration, gestational window and nanoparticle (NP) concentration. Our aim was to evaluate the effects of gestational exposure to diluted DE on feto-placental development in a rabbit model. Pregnant females were exposed to diluted (1 mg/m(3)), filtered DE (NP diameter ≈ 69 nm) or clean air (controls) for 2 h/day, 5 days/week by nose-only exposure (total exposure: 20 days in a 31-day gestation). RESULTS: DE exposure induced early signs of growth retardation at mid gestation with decreased head length (p = 0.04) and umbilical pulse (p = 0.018). Near term, fetal head length (p = 0.029) and plasma insulin and IGF1 concentrations (p = 0.05 and p = 0.019) were reduced. Placental function was also affected, with reduced placental efficiency (fetal/placental weight) (p = 0.049), decreased placental blood flow (p = 0.009) and fetal vessel volume (p = 0.002). Non-aggregated and "fingerprint" NP were observed at various locations, in maternal blood space, in trophoblastic cells and in the fetal blood, demonstrating transplacental transfer. Adult female offspring were bred with control males. Although fetoplacental biometry was not affected near term, second generation fetal metabolism was modified by grand-dam exposure with decreased plasma cholesterol (p = 0.008) and increased triglyceride concentrations (p = 0.015). CONCLUSIONS: Repeated daily gestational exposure to DE at levels close to urban pollution can affect feto-placental development in the first and second generation.

Fatty acid binding protein 7 and n-3 poly unsaturated fatty acid supply in early rat brain development.

Fatty acid binding protein 7 (FABP7), abundant in the embryonic brain, binds with the highest affinity to docosahexaenoic acid (DHA) and is expressed in the early stages of embryogenesis. Here, we have examined the consequences of the exposure to different DHA levels and of the in utero depletion of FABP7 on early rat brain development. Neurodevelopment was evaluated through the contents of two proteins, connexin 43 (Cx43) and cyclin-dependent kinase 5 (CDK5), both involved in neuroblast proliferation, differentiation, and migration. The dams were fed with diets presenting different DHA contents, from deficiency to supplementation. DHA brain embryos contents already differed at embryonic day 11.5 and the differences kept increasing with time. Cx43 and CDK5 contents were positively associated with the brain DHA levels. When FABP7 was depleted in vivo by injections of siRNA in the telencephalon, the enhancement of the contents of both proteins was lost in supplemented animals, but FABP7 depletion did not modify phospholipid compositions regardless of the diets. Thus, FABP7 is a necessary mediator of the effect of DHA on these proteins synthesis, but its role in DHA uptake is not critical, although FABP7 is localized in phospholipid-rich areas. Our study shows that high contents of DHA associated with FABP7 are necessary to promote early brain development, which prompted us to recommend DHA supplementation early in pregnancy.

Antigen recovery and preservation using the microwave irradiation of biological samples for transmission electron microscopy analysis.

Most studies using microwave irradiation (MWI) for the preparation of tissue samples have reported an improvement in structural integrity. However, there have been few studies on the effect of microwave (MW) on antigen preservation during sample preparation prior to immunolocalization. This report documents our experience of specimen preparation using an automatic microwave apparatus to obtain antigen preservation and retrieval. We tested the effects of MW processing vs. conventional procedures on the morphology and antigenicity of two different tissues: the brain and mammary gland, whose chemical composition and anatomical organization are quite different. We chose to locate the transcription factor PPARß/δ using immunocytochemistry on brain tissue sections from hamsters. Antigen retrieval protocols involving MWI were used to restore immunoreactivity. We also studied the efficiency of the ultrastructural immunolocalization of both PPARγ and caveolin-1 following MWI vs. conventional treatment, on mammary gland tissue from mice at 10 days of lactation. Our findings showed that the treatment of tissue samples with MWI, in the context of a process lasting just a few hours from fixation to immunolocalization, enabled similar, or even better, results than conventional protocols. The quantification of immunolabeling for cav-1 indicated an increase in density of up to three-fold in tissues processed in the microwave oven. Furthermore, MW treatment permitted the localization of PPARß/δ in glutaraldehyde-fixed specimens, which was impossible in the absence of MWI. This study thus showed that techniques involving the use of microwaves could largely improve both ultrastructure and immunodetection.

The role of PPARß/δ in the regulation of glutamatergic signaling in the hamster suprachiasmatic nucleus.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily and function as transcription factors that regulate gene expression in numerous biological processes. Although the PPARß/δ subtype is highly expressed in the brain, its physiological roles in neuronal function remain to be elucidated. In this study, we examined the presence of PPARß/δ in the master circadian clock of the Syrian hamster and investigated its putative functional role in this structure. In mammals, the central circadian clock, located in the suprachiasmatic nucleus (SCN), is entrained by the light-dark (LD) cycle via photic6 signals conveyed by a direct pathway whose terminals release glutamate. Using immunocytochemical and qRT-PCR analysis, we demonstrated that the rhythmic expression of PPAR ß/δ within the SCN of hamsters raised under an LD cycle was detectable only at the transcriptional level when the hamsters were maintained under constant darkness (DD). The increase in the number of immunoreactive PPARß/δ cells observed under DD after light stimulation during the early subjective night (CT14), but not during the subjective day (CT06), demonstrated that the expression of PPARß/δ can be up-regulated according to the photosensitive phase of the circadian clock. All of the PPARß/δ-positive cells in the SCN also expressed the glutamate receptor NMDAR1. Moreover, we demonstrated that at the photosensitive point (CT14), the administration of L-16504, a specific agonist of PPARß/δ, amplified the phase delay of the locomotor response induced by a light pulse. Taken together, these data suggest that PPARß/δ activation modulates glutamate release that mediates entrainment of the circadian clock by light.

Leptin and its receptors are present in the rat olfactory mucosa and modulated by the nutritional status.

Leptin is an adipocyte-derived cytokine that regulates body weight mainly via the long form of the leptin receptor (Ob-Rb). Leptin and its receptors are expressed in several tissues, suggesting that leptin might also be effective peripherally. We hypothesized that, as shown in taste cells, leptin and its receptors isoforms (Ob-Rs) could be present in the rat olfactory mucosa (OM). Using RT-PCR, light and electron microscopy immunohistochemistry (ICC), we found that different isoforms of the receptor were expressed in OM and localized in sustentacular cells and in a subpopulation of maturating neurons; in addition, immunoreactivity was also present in differentiated neurons and enriched at the cilia membranes, where the odorants bind to their receptors. Moreover, using RT-PCR, ICC and RIA measurements, we showed that leptin is synthesized locally in the olfactory mucosa. In addition, we demonstrate that fasting causes a significant enhanced transcription of both leptin and Ob-Rs in rat OM by quantitative RT-PCR data. Altogether, these results strongly suggested that leptin, acting as an endocrine or a paracrine factor, could be an important regulator of olfactory function, as a neuromodulator of the olfactory message in cilia of mature olfactory receptors neurons (ORN), but also for the homeostasis of this complex tissue, acting on differentiating neurons and on sustentacular cells.

Quantitative assessment of olfactory receptors activity in immobilized nanosomes: a novel concept for bioelectronic nose.

We describe how mammalian olfactory receptors (ORs) could be used as sensing elements of highly specific and sensitive bioelectronic noses. An OR and an appropriate G(alpha) protein were co-expressed in Saccharomyces cerevisiae cells from which membrane nanosomes were prepared, and immobilized on a sensor chip. By Surface Plasmon Resonance, we were able to quantitatively evaluate OR stimulation by an odorant, and G protein activation. We demonstrate that ORs in nanosomes discriminate between odorant ligands and unrelated odorants, as in whole cells. This assay also provides the possibility for quantitative assessment of the coupling efficiency of the OR with different G(alpha) subunits, without the interference of the cellular transduction pathway. Our findings will be useful to develop a new generation of electronic noses for detection and discrimination of volatile compounds, particularly amenable to micro- and nano-sensor formats.

Anatomical evidence for glutamatergic transmission in primary sensory neurons and onto postganglionic neurons controlling penile erection in rats: an ultrastructural study with neuronal tracing and immunocytochemistry.

In male rats, the dorsal penile nerve (DPN) conveys sensory information from the genitals to the lumbosacral spinal segments of the spinal cord. DPN is the afferent limb of a reflex loop that supports reflexive erections, and that includes a network of spinal interneurons and autonomic and somatic motoneurons to the penis and perineal striated muscles. Autonomic efferent pathways to the penis relay in the major pelvic ganglion (MPG). Glutamate (Glu) is a likely candidate as a neurotransmitter of reflexive erections. Both AMPA and NMDA glutamatergic receptor subunits are present in the lumbosacral spinal cord, and AMPA and NMDA receptor antagonists block reflexive erections. In the present study, we used tract-tracing experiments combined with immunohistochemical and immunocytochemical techniques to ascertain the presence of Glu at two different levels of the network controlling reflexive erections. DPN afferents were localized in the dorsal horn of the lumbosacral cord and displayed the characteristics of either C-fibers or Adelta fibers. DPN terminals (some of them glutamatergic) were mainly distributed in the medial edge of the dorsal horn in the L6 spinal segment. GluR1 subunits were present in some DPN afferents, suggesting that they could be autoreceptors. DPN fibers were also present in the MPG, as were Glu terminals and GluR4 subunits. The results reveal the presence of Glu in DPN fibers and terminals and suggest that both the spinal cord and the MPG use glutamatergic transmission to control reflexive erections.

Orexin A modulates mitral cell activity in the rat olfactory bulb: patch-clamp study on slices and immunocytochemical localization of orexin receptors.

Orexin A and B are involved in feeding behaviors, and recently fibers containing these peptides were found in the rat olfactory bulb. These fibers, which originate from the lateral and posterior hypothalamus and the perifornical area, are distributed in the glomerular, mitral cell, and granule cell layers. Orexin receptors are mainly expressed by mitral cells. In the present study, RT-PCR experiments were done to determine orexin receptor expression during the early postnatal life of rats, and immunocytochemical experiments were performed to further clarify the structural and ultrastructural localization of orexin receptors in the olfactory bulb. Furthermore, a functional electrophysiological approach examined the action of orexin A on mitral cell excitability and spontaneous activity using in vitro patch-clamp techniques. RT-PCR results show that mRNA of the two type receptors, type 1 orexin receptors and type 2 orexin receptors, are expressed in the olfactory bulb of rat from 10 d to the adult stage. At the same ages, immunocytochemical data show that orexin 1 receptors are localized in the cell bodies of periglomerular, mitral/tufted, and granule cells. Immunoreactivity was also demonstrated in mitral/tufted cell dendrites arborizing in the glomerulus and mitral/tufted and granule cell processes running in the external plexiform layer. Functionally, orexin A produced either a direct, tetrodotoxin-insensitive depolarization in one group of mitral cells (7%), or, in another group (30%), an indirect, tetrodotoxin-sensitive hyperpolarization. Both actions were mediated by type 1 orexin receptors because the response was antagonized by SB-334867-A, a selective antagonist. Mitral cell recordings performed under bicuculline [gamma-aminobutyric acid (GABA)A receptor antagonist], indicate that the orexin-induced indirect hyperpolarization was partly mediated through GABA(A) receptors. Because granule cells and periglomerular cells express orexin receptors and are GABAergic cells, they could be both involved in this hyperpolarization. Other mechanisms, which could support an indirect hyperpolarization of mitral cells through dopamine interneuron solicitation, are proposed. Our results provide data that should allow us to better understand neural communication and regulation mechanisms between the hypothalamic feeding centers and the olfactory bulb.

Immobilization of native membrane-bound rhodopsin on biosensor surfaces.

In this paper, we evaluated the grafting of G-protein-coupled receptors (GPCRs) onto functionalized surfaces, which is a primary requirement to elaborate receptor-based biosensors, or to develop novel GPCR assays. Bovine rhodopsin, a prototypical GPCR, was used in the form of receptor-enriched membrane fraction. Quantitative immobilization of the membrane-bound rhodopsin either non-specifically on a carboxylated dextran surface grafted with long alkyl groups, or specifically on a surface coated with anti-rhodopsin antibody was demonstrated by surface plasmon resonance. In addition, a new substrate based on mixed self-assembled multilayer that anchors specific anti-receptor antibodies was developed. Electrochemical impedance spectroscopy performed upon deposition of membrane-bound rhodopsin of increasing concentration exhibited a significant change, until a saturation level was reached, indicating optimum receptor immobilization on the substrate. The structures obtained with this new immobilization procedure of the rhodopsin in its native membrane environment are stable, with a controlled density of specific anchoring sites. Therefore, such receptor immobilization method is attractive for a range of applications, especially in the field of GPCR biosensors.

Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening.

The functional expression of olfactory receptors (ORs) is a primary requirement to examine the molecular mechanisms of odorant perception and coding. Functional expression of the rat I7 OR and its trafficking to the plasma membrane was achieved under optimized experimental conditions in the budding yeast Saccharomyces cerevisiae. The membrane expression of the receptor was shown by Western blotting and immunolocalization methods. Moreover, we took advantage of the functional similarities between signal transduction cascades of G protein-coupled receptor in mammalian cells and the pheromone response pathway in yeast to develop a novel biosensor for odorant screening using luciferase as a functional reporter. Yeasts were engineered to coexpress I7 OR and mammalian G(alpha) subunit, to compensate for the lack of endogenous Gpa1 subunit, so that stimulation of the receptor by its ligands activates a MAP kinase signaling pathway and induces luciferase synthesis. The sensitivity of the bioassay was significantly enhanced using mammalian G(olf) compared to the G(alpha15) subunit, resulting in dose-dependent responses of the system. The biosensor was probed with an array of odorants to demonstrate that the yeast-borne I7 OR retains its specificity and selectivity towards ligands. The results are confirmed by functional expression and bioluminescence response of human OR17-40 to its specific ligand, helional. Based on these findings, the bioassay using the luciferase reporter should be amenable to simple, rapid and inexpensive odorant screening of hundreds of ORs to provide insight into olfactory coding mechanisms.

Localization of orexins and their receptors in the rat olfactory system: possible modulation of olfactory perception by a neuropeptide synthetized centrally or locally.

Orexin-A and -B, also known as hypocretins, are two neuropeptides acting on feeding and sleep. They are specific ligands for two different receptors belonging to the G-protein coupled receptors family. Orexin fibers and orexin receptor neurons have been previously described in the forebrain olfactory system. Using immunocytochemistry, we showed that both orexin-A and -B as well as their receptors were present at different levels of the olfactory system, from the nasal mucosa to nuclei of the amygdala. A punctuated staining for orexins and their receptors was detected at the apical part of the olfactory epithelium; in the lamina propria of the mucosa, the staining was localized around olfactory nerves. At the ultrastructural level, olfactory neurons and supporting cells were found immunoreactive for orexins and their receptors. The labeling was localized in dendritic knobs and cilia of neurons, in the apical part and microvilli of supporting cells. The finding of immunolabeled cisternae of reticulum strongly suggests a local synthesis of both peptides and receptors, confirmed by RT-PCR experiments. In forebrain and amygdala regions, we detected numerous orexin fibers. Orexin receptors were present in mitral-tufted cells of the bulb and in many neuronal perikarya in the anterior olfactory nuclei, piriform cortex and amygdala nuclei. Altogether, these results show that orexins and their receptors are present at all levels of the olfactory system, from cilia where odors bind to their receptors to central regions where integration of olfactory signals occurs. They suggest a possible modulation of olfactory perception by these neuropeptides.