Constitutive cell proliferation and neurogenesis in the organum vasculosum lamina terminalis and subfornical organ of adult rats.

Neurogenesis continues throughout adulthood in the subventricular zone, hippocampal subgranular zone, and the hypothalamic median eminence (ME) and the adjacent medio-basal hypothalamus. The ME is one of the circumventricular organs (CVO), which are specialized brain areas characterized by an incomplete blood-brain barrier and, thus, are involved in mediating communication between the central nervous system and the periphery. Additional CVOs include the organum vasculosum laminae terminalis (OVLT) and the subfornical organs (SFO). Previous studies have demonstrated that the ME contains neural stem cells (NSCs) capable of generating new neurons and glia in the adult brain. However, it remains unclear whether the OVLT and SFO also contain proliferating cells, the identity of these cells, and their ability to differentiate into mature neurons. Here we show that glial and mural subtypes exhibit NSC characteristics, expressing the endogenous mitotic maker Ki67, and incorporating the exogenous mitotic marker BrdU in the OVLT and SFO of adult rats. Glial cells constitutively proliferating in the SFO comprise NG2 glia, while in the OVLT, both NG2 glia and tanycytes appear to constitute the NSC pool. Furthermore, pericytes, which are mural cells associated with capillaries, also contribute to the pool of cells constitutively proliferating in the OVLT and SFO of adult rats. In addition to these glial and mural cells, a fraction of NSCs containing proliferation markers Ki67 and BrdU also expresses the early postmitotic neuronal marker doublecortin, suggesting that these CVOs comprise newborn neurons. Notably, these neurons can differentiate and express the mature neuronal marker NeuN. These findings establish the sensory CVOs OVLT and SFO as additional neurogenic niches, where the generation of new neurons and glia persists in the adult brain.

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.

Imaging and spectroscopic methods to investigate adult neurogenesis in vivo: New models and new avenues.

Adult neurogenesis (AN) can be defined as the birth and development of new neurons in adulthood. Until the 1990s, AN was deemed not to happen after birth. Gradually, several groups demonstrated that specific zones of the brain of various species had a neurogenic potential. AN could be the key to treating a large range of neurodegenerative, neuropsychiatric, and metabolic diseases, with a better understanding of the mechanisms allowing for regeneration of new neurons. Despite this promising prospect, the existence of AN has not been validated in vivo in humans and therefore remains controversial. Moreover, the weight of AN-induced plasticity against other mechanisms of brain plasticity is not known, adding to the controversy. In this review, we would like to show that recent technical advances in brain MR imaging methods combined with improved models can resolve the debate.

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.

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.

Proteomic and functional mapping of cardiac NaV1.5 channel phosphorylation sites.

Phosphorylation of the voltage-gated Na+ (NaV) channel NaV1.5 regulates cardiac excitability, yet the phosphorylation sites regulating its function and the underlying mechanisms remain largely unknown. Using a systematic, quantitative phosphoproteomic approach, we analyzed NaV1.5 channel complexes purified from nonfailing and failing mouse left ventricles, and we identified 42 phosphorylation sites on NaV1.5. Most sites are clustered, and three of these clusters are highly phosphorylated. Analyses of phosphosilent and phosphomimetic NaV1.5 mutants revealed the roles of three phosphosites in regulating NaV1.5 channel expression and gating. The phosphorylated serines S664 and S667 regulate the voltage dependence of channel activation in a cumulative manner, whereas the nearby S671, the phosphorylation of which is increased in failing hearts, regulates cell surface NaV1.5 expression and peak Na+ current. No additional roles could be assigned to the other clusters of phosphosites. Taken together, our results demonstrate that ventricular NaV1.5 is highly phosphorylated and that the phosphorylation-dependent regulation of NaV1.5 channels is highly complex, site specific, and dynamic.