The effects of partial sleep restriction and altered sleep timing on olfactory performance.

Olfaction can increase the drive to eat and may partially explain the consistent increases in energy intake (EI) following sleep restriction. We investigated the effects of 50% sleep restriction with altered sleep timing on olfactory performance. We also evaluated whether changes (Δ) in olfactory performance were associated with Δ24 h EI. Twelve men and six women (age: 23±4 years; BMI: 23±3 kg/m2) completed three randomized cross-over conditions: habitual sleep duration, 50% sleep restriction with advanced wake-time, and 50% sleep restriction with delayed bedtime. Sleep was measured in-laboratory (polysomnography). Olfactory performance ('sniffin sticks') and 24 h EI (food menu) were evaluated the next day. A trend for a significant condition*sex interaction was noted for threshold-discrimination-identification (TDI) scores (P=0.09); TDI scores were lowest in women and highest in men, following sleep restriction with advanced wake-time. Δolfactory performance were not associated with Δ24 h EI. The impact of sleep restriction on olfactory performance may differ between sexes. Changes in olfactory performance were not associated with changes in 24 h EI. Studies investigating prolonged effects of sleep loss on the relationship between olfactory performance with EI are needed.

Effects of dorsolateral prefrontal cortex lesion on motor habit and performance assessed with manual grasping and control of force in macaque monkeys.

In the context of an autologous adult neural cell ecosystem (ANCE) transplantation study, four intact adult female macaque monkeys underwent a unilateral biopsy of the dorsolateral prefrontal cortex (dlPFC) to provide the cellular material needed to obtain the ANCE. Monkeys were previously trained to perform quantitative motor (manual dexterity) tasks, namely, the "modified-Brinkman board" task and the "reach and grasp drawer" task. The aim of the present study was to extend preliminary data on the role of the prefrontal cortex in motor habit and test the hypothesis that dlPFC contributes to predict the grip force required when a precise level of force to be generated is known beforehand. As expected for a small dlPFC biopsy, neither the motor performance (score) nor the spatiotemporal motor sequences were affected in the "modified-Brinkman board" task, whereas significant changes (mainly decreases) in the maximal grip force (force applied on the drawer knob) were observed in the "reach and grasp drawer" task. The present data in the macaque monkey related to the prediction of grip force are well in line with the previous fMRI data reported for human subjects. Moreover, the ANCE transplantation strategy (in the case of stroke or Parkinson's disease) based on biopsy in dlPFC does not generate unwanted motor consequences, at least as far as motor habit and motor performance are concerned in the context of a sequential grasping a small objects, which does not require the development of significant force levels.

The sacral autonomic outflow is sympathetic.

A kinship between cranial and pelvic visceral nerves of vertebrates has been accepted for a century. Accordingly, sacral preganglionic neurons are considered parasympathetic, as are their targets in the pelvic ganglia that prominently control rectal, bladder, and genital functions. Here, we uncover 15 phenotypic and ontogenetic features that distinguish pre- and postganglionic neurons of the cranial parasympathetic outflow from those of the thoracolumbar sympathetic outflow in mice. By every single one, the sacral outflow is indistinguishable from the thoracolumbar outflow. Thus, the parasympathetic nervous system receives input from cranial nerves exclusively and the sympathetic nervous system from spinal nerves, thoracic to sacral inclusively. This simplified, bipartite architecture offers a new framework to understand pelvic neurophysiology as well as development and evolution of the autonomic nervous system.

Neurodevelopment. Parasympathetic ganglia derive from Schwann cell precursors.

Neural crest cells migrate extensively and give rise to most of the peripheral nervous system, including sympathetic, parasympathetic, enteric, and dorsal root ganglia. We studied how parasympathetic ganglia form close to visceral organs and what their precursors are. We find that many cranial nerve-associated crest cells coexpress the pan-autonomic determinant Paired-like homeodomain 2b (Phox2b) together with markers of Schwann cell precursors. Some give rise to Schwann cells after down-regulation of PHOX2b. Others form parasympathetic ganglia after being guided to the site of ganglion formation by the nerves that carry preganglionic fibers, a parsimonious way of wiring the pathway. Thus, cranial Schwann cell precursors are the source of parasympathetic neurons during normal development.

Cell locations for AQP1, AQP4 and 9 in the non-human primate brain.

The presence of three water channels (aquaporins, AQP), AQP1, AQP4 and AQP9 were observed in normal brain and several rodent models of brain pathologies. Little is known about AQP distribution in the primate brain and its knowledge will be useful for future testing of drugs aimed at preventing brain edema formation. We studied the expression and cellular distribution of AQP1, 4 and 9 in the non-human primate brain. The distribution of AQP4 in the non-human primate brain was observed in perivascular astrocytes, comparable to the observation made in the rodent brain. In contrast with rodent, primate AQP1 is expressed in the processes and perivascular endfeet of a subtype of astrocytes mainly located in the white matter and the glia limitans, possibly involved in water homeostasis. AQP1 was also observed in neurons innervating the pial blood vessels, suggesting a possible role in cerebral blood flow regulation. As described in rodent, AQP9 mRNA and protein were detected in astrocytes and in catecholaminergic neurons. However additional locations were observed for AQP9 in populations of neurons located in several cortical areas of primate brains. This report describes a detailed study of AQP1, 4 and 9 distributions in the non-human primate brain, which adds to the data already published in rodent brains. This relevant species differences have to be considered carefully to assess potential drugs acting on AQPs non-human primate models before entering human clinical trials.

Optimized time-resolved 3D contrast-enhanced MRA at 3T: appreciating the feasibility of assessing cervical paragangliomas.

OBJECTIVES: To describe an optimized 3D time-resolved contrast-enhanced MR angiography (3D TR-CE-MRA) at 3T in diagnosing head and neck paragangliomas and assessing their morphology and relation to neighboring vessels. METHODS: In a prospective study, eight consecutive patients presenting cranial cervical masses suspected to be 10 paragangliomas were examined with 3D TR-CE-MRA at 3T. Two neuroradiologists evaluated the overall image quality, the presence of a paraganglioma, the maximum diameter, as well as the vessel invasion. RESULTS: In all of the cases, the overall image quality was scored as good. The tumors (n=10) were all visualized and localized. The mean maximum diameter was 32.7mm [range 7-80]. Vessel invasion was assessed as uncertain in one case and improbable in nine cases. CONCLUSION: 3D TR-CE-MRA at 3T associated with conventional sequences facilitates a comprehensive investigation of paragangliomas, thus providing the anatomical and functional information.

Glycogen metabolism as a marker of astrocyte differentiation.

Glycogen is a hallmark of mature astrocytes, but its emergence during astrocytic differentiation is unclear. Differentiation of E14 mouse neurospheres into astrocytes was induced with fetal bovine serum (FBS), Leukemia Inhibitory Factor (LIF), or Ciliary Neurotrophic Factor (CNTF). Cytochemical and enzymatic analyses showed that glycogen is present in FBS- or LIF- but not in CNTF-differentiated astrocytes. Glycogenolysis was induced in FBS- and LIF-differentiated astrocytes but glycogen resynthesis was observed only with FBS. Protein targeting to glycogen mRNA expression appeared with glial fibrillary acidic protein and S100beta in FBS and LIF conditions but not with CNTF. These results show that glycogen metabolism constitutes a useful marker of astrocyte differentiation.

Induction of brain aquaporin 9 (AQP9) in catecholaminergic neurons in diabetic rats.

Aquaporin 9 facilitates the diffusion of water but also glycerol and monocarboxylates, known as brain energy substrates. AQP9 was recently observed in catecholaminergic neurons that are implicated in energy homeostasis and also possibly in neuroendocrine effects of diabetes. Recently it has been observed that the level of AQP9 expression in hepatocytes is sensitive to the blood concentration of insulin. Furthermore, insulin injection in the brain is known to be related to the energy homeostasis. Based on these observations, we investigated if the concentration of insulin affects the level of brain AQP9 expression and if so, in which cell types. This study has been carried out, in a model of the diabetic rat generated by streptozotocin injection and on brainstem slices. In diabetic rats showing a decrease in systemic insulin concentration, AQP9 is only increased in brain areas containing catecholaminergic neurons. In contrast, no significant change is detected in the cerebral cortex and the cerebellum. Using immunocytochemistry, we are able to show that the increase in AQP9 expression is specifically present in catecholaminergic neurons. In brainstem slice cultures, 2 microM insulin induces a significant decrease in AQP9 protein levels 6 h after application, suggesting that brain AQP9 is also regulated by the insulin. These results show that the level of expression of brain AQP9 is affected by variations of the concentration of insulin in a diabetic model and in vitro.

Mutations of the RET gene in isolated and syndromic Hirschsprung's disease in human disclose major and modifier alleles at a single locus.

BACKGROUND: In Hirschsprung's disease (HSCR), a hypomorphic allele of a major gene, RET, accounts for most isolated (non-syndromic) cases, along with other autosomal susceptibility loci under a multiplicative model. However, some syndromic forms of HSCR are monogenic entities, for which the disease causing gene is known. OBJECTIVE: To determine whether RET could be considered a modifier gene for the enteric phenotype on the background of a monogenic trait. METHODS: The syndromic HSCR entities studied were congenital central hypoventilation (CCHS) and Mowat-Wilson syndrome (MWS), caused by PHOX2B and ZFHX1B gene mutations, respectively. The RET locus was genotyped in 143 CCHS patients, among whom 44 had HSCR, and in 30 MWS patients, among whom 20 had HSCR. The distribution of alleles, genotypes, and haplotypes was compared within the different groups. To test the interaction in vivo, heterozygous mice were bred for a null allele of Phox2b and Ret genes. RESULTS: RET was shown to act as a modifier gene for the HSCR phenotype in patients with CCHS but not with MWS. The intestine of double heterozygote mice was indistinguishable from their littermates. A loss of over 50% of each gene function seemed necessary in the mouse model for an enteric phenotype to occur. CONCLUSIONS: In CCHS patients, the weak predisposing haplotype of the RET gene can be regarded as a quantitative trait, being a risk factor for the HSCR phenotype, while in MWS, for which the HSCR penetrance is high, the role of the RET predisposing haplotype is not significant. It seems likely that there are both RET dependent and RET independent HSCR cases.

Distribution of Aquaporin 9 in the adult rat brain: preferential expression in catecholaminergic neurons and in glial cells.

Aquaporin 9 (AQP9) is a recently cloned water channel that is permeable to monocarboxylate, glycerol and urea. In rat, AQP9 has been found in testis and liver as well as in brain where its expression has been initially shown in glial cells in forebrain. However, the expression of AQP9 has not been investigated in the brainstem. The purpose of this study is to describe the distribution of AQP9-immunoreactive cells throughout the adult rat brain using reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry. We performed immunolabeling on brain from animals perfused with fixative and we show that AQP9 is expressed (i) in astrocytes in the glia limitans, in the white matter and in glial cells of the cerebellum, (ii) in the endothelial cells of pial vessels, and (iii) in specific groups of neurons. The neuronal AQP9 expression was almost exclusively observed in catecholaminergic cells including the adrenergic, noradrenergic and dopaminergic groups, but not in other monoaminergic neurons such as serotonergic or histaminergic cells. A slight labeling was also observed in non-catecholaminergic neurons localized in the paraventricular nucleus of the hypothalamus. These results indicate that AQP9 has a unique brain distribution with a preferential localization in catecholaminergic nuclei known to be involved in many cerebral functions. While the presence of AQP9 in glia limitans and in endothelial cells of the pial vessels could be related to water transport through the blood-brain barrier, its expression in neuronal cells, not directly involved in the osmoregulation, suggests that brain AQP9 could also be used as a metabolite channel since lactate and glycerol can be energy substrates for neurons.

Early acquisition of typical metabolic features upon differentiation of mouse neural stem cells into astrocytes.

Specific metabolic features, such as glutamate reuptake, have been associated with normal functions of mature astrocytes. In this study, we examined whether these characteristics are acquired together with classical phenotypic markers of differentiated astrocytes. Differentiation of E14 mouse neurospheres into astrocytes was induced by the addition of fetal bovine serum (FBS). Degree of differentiation was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence for both GFAP and nestin. Neural stem cells expressed nestin but not GFAP, while differentiated astrocytes were immunopositive for GFAP but displayed low levels of nestin expression. A strong increase in the expression of the glutamate transporter GLAST and the monocarboxylate transporter MCT1 accompanied phenotypic changes. In addition, active glutamate transport appeared in differentiated astrocytes, as well as their capacity to increase aerobic glycolysis in response to glutamate. Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor, but not interleukin-6, triggered the expression of phenotypic and morphological characteristics of astrocytes. In addition, exposure to LIF led to the appearance of metabolic features typically associated with astrocytes. Altogether, our results show that acquisition of some specific metabolic features by astrocytes occurs early in their differentiation process and that LIF represents a candidate signal to induce their expression.

Role of Phox2b and Mash1 in the generation of the vestibular efferent nucleus.

The inner ear (vestibular and cochlear) efferent neurons are a group of atypical motor-like hindbrain neurons which innervate inner ear hair cells and their sensory afferents. They are born in the fourth rhombomere, in close association with facial branchial motor neurons, from which they subsequently part through a specific migration route. Here, we demonstrate that the inner ear efferents depend on Phox2b for their differentiation, behaving in that respect like hindbrain visceral and branchial motor neurons. We also show that the vestibular efferent nucleus is no longer present at its usual site in mice inactivated for the bHLH transcription factor Mash 1. The concomitant appearance of an ectopic branchial-like nucleus at the location where both inner ear efferents and facial branchial motor neurons are born suggests that Mash1 is required for the migration of a subpopulation of rhombomere 4-derived efferents.

Aquaporin 1 and aquaporin 4 expression in human brain after subarachnoid hemorrhage and in peritumoral tissue.

Aquaporins (AQPs) are a protein family of water channels which facilitate the water flux through the plasmatic membranes. The expression of AQPs has been described in rat brain by several studies. Despite recent reports that have shown an over-expression of AQP1 and 4 in human tumoral cells, little is known about AQP expression in human brain. The purpose of this study was to investigate the expression of AQP1 and AQP4 in human brain after subarachnoid hemorrhage (SAH) and in peritumoral tissue by western blot and immunohistochemistry. The results showed a marked increase of the expression of AQP1 and AQP4. This over-expression occurred on the astrocytic processes and polarization on astrocytic end-feet was lost. No expression was observed on neuronal cells. This study is the first demonstration of the induction of AQP1 and AQP4 on reactive astrocytes in an acute brain injury, such as SAH. These results reinforce the hypothesis that AQPs may be involved in the dynamics of brain edema formation or resolution. Further studies are needed to understand their functional role.

Isolation of multipotent neural precursors residing in the cortex of the adult human brain.

Multipotent precursors able to generate neurons, astrocytes, and oligodendrocytes have previously been isolated from human brain embryos and recently from neurogenic regions of the adult human brains. The isolation of multipotent neural precursors from adult human should open new perspectives to study adult neurogenesis and for brain repair. The present study describes the in vitro isolation from adult human brains of a progenitor responsive to both epidermal and basic fibroblast growth factors that forms spheres as it proliferates. Single spheres derived from various regions of the brain generate in vitro neurons, astrocytes, and oligodendrocytes. The clonal origin of the spheres was revealed by genomic viral insertion using lentiviral vector. Interestingly, this vector appears to be a potent tool for gene transfer into human neural progeny. Ninety-six percent of the spheres investigated were multipotent. Multipotent precursors were isolated from all brain regions studied, including the temporal and the frontal cortex, the amygdala, the hippocampus, and the ventricular zone. This study is the first evidence that primitive precursors such as multipotent precursors exist in the adult human cortex and can reside far from the ventricles. Neurogenesis derived from adult human progenitors differ to murine neurogenesis by the requirement of laminin for oligodendrocyte generation and by the action of basic-fibroblast growth factor and platelet derived growth factor that prevented the formation of oligodendrocytes and neurons. Moreover, the differentiation of human adult precursors seems to differ from fetal ones: adult precursors do not necessitate the removal of mitogen for differentiation. These results indicate that the study of adult multipotent precursors is a new platform to study adult human neurogenesis, potentially generate neural cells for transplantation, and design protocols for in vivo stimulation.

The Phox2b transcription factor coordinately regulates neuronal cell cycle exit and identity.

In the vertebrate neural tube, cell cycle exit of neuronal progenitors is accompanied by the expression of transcription factors that define their generic and sub-type specific properties, but how the regulation of cell cycle withdrawal intersects with that of cell fate determination is poorly understood. Here we show by both loss- and gain-of-function experiments that the neuronal-subtype-specific homeodomain transcription factor Phox2b drives progenitor cells to become post-mitotic. In the absence of Phox2b, post-mitotic neuronal precursors are not generated in proper numbers. Conversely, forced expression of Phox2b in the embryonic chick spinal cord drives ventricular zone progenitors to become post-mitotic neurons and to relocate to the mantle layer. In the neurons thus generated, ectopic expression of Phox2b is sufficient to initiate a programme of motor neuronal differentiation characterised by expression of Islet1 and of the cholinergic transmitter phenotype, in line with our previous results showing that Phox2b is an essential determinant of cranial motor neurons. These results suggest that Phox2b coordinates quantitative and qualitative aspects of neurogenesis, thus ensuring that neurons of the correct phenotype are generated in proper numbers at the appropriate times and locations.

The homeodomain protein CePHOX2/CEH-17 controls antero-posterior axonal growth in C. elegans.

An essential aspect of a neuron's identity is the pattern of its axonal projections. In C. elegans, axons extend either longitudinally or circumferentially in response to distinct molecular cues, some of which have been identified. It is currently unclear, however, how the differential capacity to respond to these cues is transcriptionally implemented in distinct neuronal subtypes. Here, we characterise a C. elegans paired-like homeobox gene, CePhox2/ceh-17, expressed in five head neurons, ALA and the 4 SIAs, all of which project axons towards the tail along the lateral and sublateral cords. Abrogation of ceh-17 function, while leaving intact many phenotypic traits of these neurons, disrupts their antero-posterior axonal elongation beyond the mid-body region. Conversely, ectopic expression of ceh-17 in the mechanoreceptors, several of which are known to pioneer their tract, leads to exaggerated longitudinal axonal outgrowth. Thus, ceh-17 is a novel gene involved in fasciculation-independent longitudinal axonal navigation.

Role of the target in the pathfinding of facial visceral motor axons.

Axon navigation depends, in part, on guidance cues emanating from the target. We have investigated the possible role of the target in the pathfinding of visceral motor axons to cranial parasympathetic ganglia. Mice homozygous for a tau-LacZ transgene targeted in the Phox2a locus lack the sphenopalatine ganglion, which is the normal target of visceral motor axons of the facial nerve. We found that in these mutants, facial visceral motor axon pathfinding was disrupted, and some axons were misrouted to an alternative parasympathetic ganglion. Moreover, the absence of correct facial visceral motor pathways was concomitant with defects in the pathfinding of rostrally-projecting sympathetic axons.

Specification of the central noradrenergic phenotype by the homeobox gene Phox2b.

The closely related homeobox genes Phox2a and Phox2b are expressed in all central and peripheral noradrenergic neurons. Our previous results have shown that Phox2a controls the differentiation of the main noradrenergic center of the brain, the locus coeruleus, but leaves unaffected the other noradrenergic centers. Here, we report that Phox2b has a wider and overlapping role, in that it is required for the differentiation of all noradrenergic centers in the brain, including the locus coeruleus. Together with the previously reported lack of dopamine-b-hydroxylase and tyrosine hydroxylase expression in the peripheral nervous system of Phox2b knock-out embryos, our present findings make Phox2b a master regulator of all central and peripheral noradrenergic differentiation. We discuss the nonredundancy of Phox2 genes and their complex partnership with the bHLH transcription factor Mash1, which is also required for the differentiation of most noradrenergic cell types.

Control of hindbrain motor neuron differentiation by the homeobox gene Phox2b.

Motor neurons are a widely studied model of vertebrate neurogenesis. They can be subdivided in somatic, branchial and visceral motor neurons. Recent studies on the dorsoventral patterning of the rhombencephalon have implicated the homeobox genes Pax6 and Nkx2.2 in the early divergence of the transcriptional programme of hindbrain somatic and visceral motor neuronal differentiation. We provide genetic evidence that the paired-like homeodomain protein Phox2b is required for the formation of all branchial and visceral, but not somatic, motor neurons in the hindbrain. In mice lacking Phox2b, both the generic and subtype-specific programs of motoneuronal differentiation are disrupted at an early stage. Most motor neuron precursors die inside the neuroepithelium while those that emigrate to the mantle layer fail to switch on early postmitotic markers and to downregulate neuroepithelial markers. Thus, the loss of function of Phox2b in hindbrain motor neurons exemplifies a novel control point in the generation of CNS neurons.