2.5D Representations Combining in vivo 3D MRI and ex vivo 2D MSI Approaches to Study the Lipid Distribution in the Whole Sheep Brain

National audienceMass Spectrometry Imaging (MSI) provides easily high spatially resolved masses allowing characterization of endogenous lipids. These latter constitute about 70% of the composition of the white matter of the brain which can be implicated in developmental and/or cognitive troubles. In order to examine the molecular distribution of lipids in whole sheep brain, and especially in white/grey matter, we combined in vivo and ex vivo images, obtained in the same animals, using Magnetic Resonance Imaging (MRI) and MSI, respectively. In order to view the topology of the molecular species within the organ, we propose the construction of a 2.5 D representation where a single section imaged with 2D MSI is localized within the tissue volume obtained by 3D MRI. 3D T1-weighted MPRAGE images were acquired on two anesthetized sheep with a 3 Tesla MRI (Siemens, Verio ®). The parameters of acquisition for the MPRAGE were: TR 2500ms, TE 3.2ms, FA 12, NEX 1, matrix 384×384, FOV 192mm, 288 slices with a thickness of 0.5mm. In order to improve data quality, the 3D MRI volumes have been pre-processed using in-house algorithms using volume fitting and Markov random field methods. T1 3D planes corresponding to MSI planes were reconstructed using Osirix imaging software.Brains were collected after sacrifice and frozen at -80°C. Frontal and sagittal 14 µm brain sections were performed with a cryostat adapted to large sections (CM3050 S, Leica) and mounted onto conductive ITO-coated slides. The spray of α-cyano-4-hydroxycinnamic acid matrix was performed using an Image Prep device (Bruker). Spectra were acquired using an UltrafleXtrem MALDI-TOF instrument (Bruker) in the 200–1200 m/z range with a spatial resolution set at 125 µm. Raw spectra were analyzed with SCiLS Lab software to generate 2D ion density maps and segmentation maps (data partitioning). The tissue sections analyzed by MSI were stained with cresyl violet to manually delimitate neuronal nuclei and areas. This histological map was used to delineate the MRI and MSI 2D views and overlay them regardless the same brain areas used as fiducials. After, a 2.5 D representation was proposed to visualize the lipid distribution within the entire organ.In conclusion, in this study, frontal and sagittal whole sheep brain sections analyzed by MSI showed a clear difference in lipid distribution between different compartments of brain tissues, especially between grey and white matter, until the cerebral envelopment presenting circumvolution. Furthermore, the alignment of 2D MALDI-imaging with T1-weighted images showed that MSI can provide finer details on the structural connectivity of myelinated fiber tracts. Here, the 2.5 D representation combining MRI and MSI was presented as an alternative approach to 3D anatomical and molecular atlas providing a perfect topology of the molecular species within an organ. For the moment, 3D MSI of whole sheep brain is a challenge, while the 2.5 D construction demonstrated to be a capable tool for exploring molecular distributions throughout sample volumes.Nowadays, the reported results may serve as a starting point for further experiments associating MSI and dynamic and functional MRI, especially for the characterization of brain

GPR50 is the mammalian ortholog of Mel1c: Evidence of rapid evolution in mammals

<p>Abstract</p> <p>Background</p> <p>The melatonin receptor subfamily contains three members Mel1a, Mel1b and Mel1c, found in all vertebrates except for Mel1c which is found only in fish, Xenopus species and the chicken. Another receptor, the melatonin related receptor known as GPR50, found exclusively in mammals and later identified as a member of the melatonin receptor subfamily because of its identity to the three melatonin receptors despite its absence of affinity for melatonin. The aim of this study was to describe the evolutionary relationships between GPR50 and the three other members of the melatonin receptor subfamily.</p> <p>Results</p> <p>Using an <it>in silico </it>approach, we demonstrated that GPR50 is the ortholog of the high affinity Mel1c receptor. It was necessary to also study the synteny of this gene to reach this conclusion because classical mathematical models that estimate orthology and build phylogenetic trees were not sufficient. The receptor has been deeply remodelled through evolution by the mutation of numerous amino acids and by the addition of a long C-terminal tail. These alterations have modified its affinity for melatonin and probably affected its interactions with the other two known melatonin receptors MT1 and MT2 that are encoded by Mel1a and Mel1b genes respectively. Evolutionary studies provided evidence that the GPR50 group evolved under different selective pressure as compared to the orthologous groups Me11 a, b, and c.</p> <p>Conclusion</p> <p>This study demonstrated that there are only three members in the melatonin receptor subfamily with one of them (Me11c) undergoing rapid evolution from fishes and birds to mammals. Further studies are necessary to investigate the physiological roles of this receptor.</p

Age-Related Changes in the Daily Rhythm of Photoreceptor Functioning and Circuitry in a Melatonin-Proficient Mouse Strain

Retinal melatonin is involved in the modulation of many important retinal functions. Our previous studies have shown that the viability of photoreceptors and ganglion cells is reduced during aging in mice that lack melatonin receptor type 1. This demonstrates that melatonin signaling is important for the survival of retinal neurons. In the present study, we investigate the effects of aging on photoreceptor physiology and retinal organization in CH3-f+/+ mice, a melatonin proficient mouse strain. Our data indicate that the amplitude of the a and b waves of the scotopic and photopic electroretinogram decreases with age. Moreover, the daily rhythm in the amplitude of the a- and b- waves is lost during the aging process. Similarly, the scotopic threshold response is significantly affected by aging, but only when it is measured during the night. Interestingly, the changes observed in the ERGs are not paralleled by relevant changes in retinal morphological features, and administration of exogenous melatonin does not affect the ERGs in C3H-f+/+ at 12 months of age. This suggests that the responsiveness of the photoreceptors to exogenous melatonin is reduced during aging

Adult Neurogenesis: Ultrastructure of a Neurogenic Niche and Neurovascular Relationships

The first-generation precursors producing adult-born neurons in the crayfish (Procambarus clarkii) brain reside in a specialized niche located on the ventral surface of the brain. In the present work, we have explored the organization and ultrastructure of this neurogenic niche, using light-level, confocal and electron microscopic approaches. Our goals were to define characteristics of the niche microenvironment, examine the morphological relationships between the niche and the vasculature and observe specializations at the boundary between the vascular cavity located centrally in the niche. Our results show that the niche is almost fully encapsulated by blood vessels, and that cells in the vasculature come into contact with the niche. This analysis also characterizes the ultrastructure of the cell types in the niche. The Type I niche cells are by far the most numerous, and are the only cell type present superficially in the most ventral cell layers of the niche. More dorsally, Type I cells are intermingled with Types II, III and IV cells, which are observed far less frequently. Type I cells have microvilli on their apical cell surfaces facing the vascular cavity, as well as junctional complexes between adjacent cells, suggesting a role in regulating transport from the blood into the niche cells. These studies demonstrate a close relationship between the neurogenic niche and vascular system in P. clarkii. Furthermore, the specializations of niche cells contacting the vascular cavity are also typical of the interface between the blood/cerebrospinal fluid (CSF)-brain barriers of vertebrates, including cells of the subventricular zone (SVZ) producing new olfactory interneurons in mammals. These data indicate that tissues involved in producing adult-born neurons in the crayfish brain use strategies that may reflect fundamental mechanisms preserved in an evolutionarily broad range of species, as proposed previously. The studies described here extend our understanding of neurovascular relationships in the brain of P. clarkii by characterizing the organization and ultrastructure of the neurogenic niche and associated vascular tissues

Rythmes biologiques et reproduction

Chapitre 15absen

Characterization of the adult hypothalamic neurogenic niche in sheep and influence of an environmental factor: the photoperiod

National audienc

Seasonal regulation of structural plasticity and neurogenesis in the adult mammalian brain: focus on the sheep hypothalamus

To cope with variations in the environment, most mammalian species exhibit seasonal cycles in physiology and behaviour. Seasonal plasticity during the lifetime contributes to seasonal physiology. Over the years, our ideas regarding adult brain plasticity and, more specifically, hypothalamic plasticity have greatly evolved. Along with the two main neurogenic regions, namely the hippocampal subgranular and lateral ventricle subventricular zones, the hypothalamus, which is the central homeostatic regulator of numerous physiological functions that comprise sexual behaviours, feeding and metabolism, also hosts neurogenic niches. Both endogenous and exogenous factors, including the photoperiod, modulate the hypothalamic neurogenic capacities. The present review describes the effects of season on adult morphological plasticity and neurogenesis in seasonal species, for which the photoperiod is a master environmental cue for the successful programming of seasonal functions. In addition, the potential functional significance of adult neurogenesis in the mediation of the seasonal control of reproduction and feeding is discussed

Surgical pinealectomy in ewes

Chapitre 7The pineal gland once regarded as the “seat of the soul” by René Descartes is the structure attached to the caudal part of the roof of the third ventricle. In mammals, the pineal gland ensures the transduction of the photoperiodic information captured by the retina and transmitted to the pineal gland through a transsynaptic network and translated into a hormonal signal through the nocturnal secretion of melatonin. The circadian rhythm of melatonin synthesis (high levels at night, low levels during the day) is triggered by the circadian ‘clock’ located in the suprachiasmatic nuclei of the hypothalamus that projects to the pineal gland via a multi-synaptic pathway. In sheep, the duration of melatonin secretion is proportional to the length of the night and thus providing cues about the time of the year to the animals. Melatonin is involved in several physiological functions including the seasonal control of reproduction in mammals from temperate latitudes. The key role of the pineal gland in this function has been demonstrated by numerous experiments showing that pinealectomy strongly affected the effect of photoperiod on seasonal reproduction. In sheep, the pineal gland is hidden at the bottom of cerebral transverse fissure, in a medial position, close to the cerebral vein. This deep localization makes the surgical excision of the pineal gland very difficult and requires a specific training. In order to avoid pain and side effects both in pre and post surgical care, we designed a protocol of analgesic treatment made of morphine in combination with antiinflammatory compounds. Future improvements of our approach should arise from the use of scanner and magnetic resonance imaging techniques

Thyroid hormone and hypothalamic stem cells in seasonal functions

International audienceSeasonal rhythms are a pervasive feature of most living organisms, which underlie yearly timeliness in breeding, migration, hibernation or weight gain and loss. To achieve this, organisms have developed inner timing devices (circannual clocks) that endow them with the ability to predict then anticipate changes to come, usually using daylength as the proximate cue. In Vertebrates, daylength interpretation involves photoperiodic control of TSH production by the pars tuberalis (PT) of the pituitary, which governs a seasonal switch in thyroid hormone (TH) availability in the neighboring hypothalamus. Tanycytes, specialized glial cells lining the third ventricle (3V), are responsible for this TH output through the opposite, PT-TSH-driven, seasonal control of deiodinases 2/3 (Dio 2/3). Tanycytes comprise a photoperiod-sensitive stem cell niche and TH is known to play major roles in cell proliferation and differentiation, which suggests that seasonal control of tanycyte proliferation may be involved in the photoperiodic synchronization of seasonal rhythms. Here we review our current knowledge of the molecular and neuroendocrine pathway linking photoperiodic information to seasonal changes in physiological functions and discuss the potential implication of tanycytes, TH and cell proliferation in seasonal timing