Multiparametric MR Evaluation of the Photoperiodic Regulation of Hypothalamic Structures in Sheep.

Most organisms on earth, humans included, have developed strategies to cope with environmental day-night and seasonal cycles to survive. For most of them, their physiological and behavioral functions, including the reproductive function, are synchronized with the annual changes of day length, to ensure winter survival and subsequent reproductive success in the following spring. Sheep are sensitive to photoperiod, which also regulates natural adult neurogenesis in their hypothalamus. We postulate that the ovine model represents a good alternative to study the functional and metabolic changes occurring in response to photoperiodic changes in hypothalamic structures of the brain. Here, the impact of the photoperiod on the neurovascular coupling and the metabolism of the hypothalamic structures was investigated at 3T using BOLD fMRI, perfusion-MRI and proton magnetic resonance spectroscopy (1H-MRS). A longitudinal study involving 8 ewes was conducted during long days (LD) and short days (SD) revealing significant BOLD, rCBV and metabolic changes in hypothalamic structures of the ewe brain between LD and SD. More specifically, the transition between LD and SD revealed negative BOLD responses to hypercapnia at the beginning of SD period followed by significant increases in BOLD, rCBV, Glx and tNAA concentrations towards the end of the SD period. These observations suggest longitudinal mechanisms promoting the proliferation and differentiation of neural stem cells within the hypothalamic niche of breeding ewes. We conclude that multiparametric MRI studies including 1H-MRS could be promising non-invasive translational techniques to investigate the existence of natural adult neurogenesis in-vivo in gyrencephalic brains.

Seasonal remodeling of the progenitor pool and its distribution in the ewe mediobasal hypothalamus.

Recent studies have reported the presence of adult neurogenesis in the arcuate nucleus periventricular space (pvARH) and in the median eminence (ME), two structures involved in reproductive function. In sheep, a seasonal mammal, decreasing daylight in autumn induces a higher neurogenic activity in these two structures. However, the different types of neural stem and progenitor cells (NSCs/NPCs) that populate the arcuate nucleus and median eminence, as well as their location, have not been evaluated. Here, using semi-automatic image analyzing processes, we identified and quantified the different populations of NSCs/NPCs, showing that, during short days, higher densities of [SOX2 +] cells are found in pvARH and ME. In the pvARH, higher densities of astrocytic and oligodendrocitic progenitors mainly contribute to these variations. The different populations of NSCs/NPCs were mapped according to their position relative to the third ventricle and their proximity to the vasculature. We showed that [SOX2 +] cells extended deeper into the hypothalamic parenchyma during short days. Similarly, [SOX2 +] cells were found further from the vasculature in the pvARH and the ME, at this time of year, indicating the existence of migratory signals. The expression levels of neuregulin transcripts (NRGs), whose proteins are known to stimulate proliferation and adult neurogenesis and to regulate progenitor migration, as well as the expression levels of ERBB mRNAs, cognate receptors for NRGs, were assessed. We showed that mRNA expression changed seasonally in pvARH and ME, suggesting that the ErbB-NRG system is potentially involved in the photoperiodic regulation of neurogenesis in seasonal adult mammals.

Seasonal vascular plasticity in the mediobasal hypothalamus of the adult ewe.

Sheep, like most seasonal mammals, exhibit a cyclic adaptive reproductive physiology that allows ewes to give birth to their progeny during the spring when environmental conditions are favorable to their survival. This process relies on the detection of day length (or photoperiod) and is associated with profound changes in cellular plasticity and gene expression in the hypothalamic-pituitary-gonadal axis, mechanisms that are suggested to participate in the seasonal adaptation of neuroendocrine circuits. Recently, pituitary vascular growth has been proposed as a seasonally regulated process in which the vascular endothelial growth factor A (VEGFA), a well-known angiogenic cytokine, is suspected to play a crucial role. However, whether this mechanism is restricted to the pituitary gland or also occurs in the mediobasal hypothalamus (MBH), a crucial contributor to the control of the reproductive function, remains unexplored. Using newly developed image analysis tools, we showed that the arcuate nucleus (ARH) of the MBH exhibits an enhanced vascular density during the long photoperiod or non-breeding season, associated with higher expression of VEGFA. In the median eminence (ME), a structure connecting the MBH to the pituitary gland, higher VEGFA, kinase insert domain receptor (KDR/VEGFR2) and plasmalemma vesicle-associated protein (PLVAP) gene expressions were detected during the long photoperiod. We also found that VEGFA and its receptor, VEGFR2, are expressed by neurons and tanycytes in both the ARH and ME. Altogether, these data show variations in the MBH vasculature according to seasons potentially through a VEGFA-dependent pathway, paving the way for future studies aiming to decipher the role of these changes in the hypothalamic control of seasonal reproduction.

Negative regulation of melatonin secretion by melatonin receptors in ovine pinealocytes.

Melatonin (MLT) is a biological modulator of circadian and seasonal rhythms and reproduction. The photoperiodic information is detected by retinal photoreceptors and transmitted through nerve transmissions to the pineal gland, where MLT is synthesized and secreted at night into the blood. MLT interacts with two G protein-coupled receptors, MT1 and MT2. The aim of our work was to provide evidence for the presence of MLT receptors in the ovine pineal gland and define their involvement on melatonin secretion. For the first time, we identified the expression of MLT receptors with the specific 2-[125I]-MLT agonistic radioligand in ovin pinealocytes. The values of Kd and Bmax are 2.24 ± 1.1 nM and 20 ± 6.8 fmol/mg. MLT receptors are functional and inhibit cAMP production and activate ERK1/2 through pertussis toxin-sensitive Gi/o proteins. The MLT receptor antagonist/ inverse agonist luzindole increased cAMP production (189 ± 30%) and MLT secretion (866 ± 13%). The effect of luzindole on MLT secretion was additive with the effect of well-described activators of this pathway such as the ß-adrenergic agonist isoproterenol and the α-adrenergic agonist phenylephrine. Co-incubation of all three compounds increased MLT secretion by 1236 ± 199%. These results suggest that MLT receptors are involved in the negative regulation of the synthesis of its own ligand in pinealocytes. While adrenergic receptors promote MLT secretion, MLT receptors mitigate this effect to limit the quantity of MLT secreted by the pineal gland.

Blood oxygen level dependent fMRI and perfusion MRI in the sheep brain.

The ovine model could be an effective translational model but remains underexplored. Here, Blood Oxygen Level dependent functional MRI during visual stimulation and resting-state perfusion MRI were explored. We aimed at investigating the impact of isoflurane anesthesia during visual stimulation and evaluate resting cerebral blood flow and cerebral blood volume parameters in the lamb and adult sheep brain. BOLD fMRI and perfusion MRI after a bolus of DOTAREM were conducted in 4 lambs and 6 adult ewes at 3 T. A visual stimulation paradigm was delivered during fMRI at increasing isoflurane doses (1-3%). Robust but weak BOLD responses (0.21 ± 0.08%) were found in the lateral geniculate nucleus (LGN) up to 3% isoflurane anaesthesia. No significant differences were found beween BOLD responses in the range 1 to 3% ISO (p > 0.05). However, LGN cluster size decreased and functional localization became less reliable at high ISO doses (2.5-3% ISO). BOLD responses were weaker in adult sheep than in lambs (4.6 ± 1.5 versus 13.6 ± 8.5; p = 0.08). Relative cerebral blood volumes (rCBV) and relative cerebral blood flows (rCBF) were significantly higher (p < 0.0001) in lambs than in adult sheep for both gray and white matter. The impact of volatile anesthesia was explored for the first time on BOLD responses demonstrating increased reliability of functional localization of brain activity at low doses. Perfusion MRI was conducted for the first time in both lambs and adult ewes. Assessment of baseline cerebrovascular values are of interest for future studies of brain diseases allowing an improved interpretation of BOLD responses.

Chemerin impairs food intake and body weight in chicken: Focus on hypothalamic neuropeptides gene expression and AMPK signaling pathway.

Unlike mammals, the role of adipokines and more particularly of chemerin in the regulation of food intake is totally unknown in avian species. Here we investigated the effect of chemerin on the food and water consumption and on the body weight in chicken. We studied the effects on the plasma glucose and insulin concentrations and the hypothalamic neuropeptides and AMPK signaling pathway. Female broiler chickens were intraperitoneally injected, daily for 13 days with either vehicle (saline; n = 25) or chemerin (8 µg/kg; n = 25 and 16 µg/kg; n = 25). Food and water intakes were recorded 24 h after each administration. Overnight fasted animals were sacrificed at day 13 (D13), 24 h after the last injection and hypothalamus and left cerebral hemispheres were collected. Chemerin and its receptors protein levels were determined by western-blot. Gene expression of neuropeptide Y (Npy), agouti-related peptide (Agrp), corticotrophin releasing hormone (Crh), pro-opiomelanocortin (Pomc), cocaine and amphetamine-regulated transcript (Cart) and Taste 1 Receptor Member 1 (Tas1r1) were evaluated by RT-qPCR. In chicken, we found that the protein amount of chemerin, CCRL2 and GPR1 was similar in left cerebral hemisphere and hypothalamus whereas CMKLR1 was higher in hypothalamus. Chemerin administration (8 and 16 µg/kg) decreased both food intake and body weight compared to vehicle without affecting water intake and the size or volume of different brain subdivisions as determined by magnetic resonance imaging. It also increased plasma insulin levels whereas glucose levels were decreased. These data were associated with an increase in Npy and Agrp expressions and a decrease in Crh, Tas1r1 mRNA expression within the hypothalamus. Furthermore, chemerin decreased hypothalamic CMKLR1 protein expression and AMPK activation. Taken together, these results support that chemerin could be a peripheral appetite-regulating signal through modulation of hypothalamic peptides expression in chicken.

A high level of male sexual activity is necessary for the activation of the medial preoptic area and the arcuate nucleus during the 'male effect' in anestrous goats.

In small ungulates such as sheep or goats, the introduction of a male among a group of anovulatory females during the anestrus season leads to the reactivation of the gonadotrope axis and ovulation, a phenomenon known as the 'male effect'. In goats, our previous studies have demonstrated the importance of male sexual activity for an efficient reactivation of the gonadotrope axis assessed through ovulation and blood LH pulsatility. In the present experiment, we assessed whether the level of male sexual activity would also induce differential activation of two brain regions of key importance for the reactivation of GnRH activity, namely the medial preoptic area and the hypothalamic arcuate nucleus. In both structures, we observed a differential activation of Fos in females, depending on the level of buck sexual activity. Indeed, goats unexposed to males showed low levels of expression of Fos while those exposed to sexually inactive bucks showed an intermediate level of Fos expression. Finally, the highest level of Fos expression was found in females exposed to sexually active males. However, and contrary to our initial hypothesis, we were not able to find any specific activation of kisspeptin cells in the arcuate nucleus following the introduction of highly sexually active males. As a whole, these results demonstrate that the level of male sexual activity is a key factor to stimulate brain regions involved in the control of the gonadotrope axis in the context of the male effect in goats.