c-fos induction in the choroid plexus, tanycytes and pars tuberalis is an early indicator of spontaneous arousal from torpor in a deep hibernator.

Hibernation is an extreme state of seasonal energy conservation, reducing metabolic rate to as little as 1% of the active state. During the hibernation season, many species of hibernating mammals cycle repeatedly between the active (aroused) and hibernating (torpid) states (T-A cycling), using brown adipose tissue (BAT) to drive cyclical rewarming. The regulatory mechanisms controlling this process remain undefined but are presumed to involve thermoregulatory centres in the hypothalamus. Here, we used the golden hamster (Mesocricetus auratus), and high-resolution monitoring of BAT, core body temperature and ventilation rate, to sample at precisely defined phases of the T-A cycle. Using c-fos as a marker of cellular activity, we show that although the dorsomedial hypothalamus is active during torpor entry, neither it nor the pre-optic area shows any significant changes during the earliest stages of spontaneous arousal. Contrastingly, in three non-neuronal sites previously linked to control of metabolic physiology over seasonal and daily time scales - the choroid plexus, pars tuberalis and third ventricle tanycytes - peak c-fos expression is seen at arousal initiation. We suggest that through their sensitivity to factors in the blood or cerebrospinal fluid, these sites may mediate metabolic feedback-based initiation of the spontaneous arousal process.

ID factors regulate the ability of Müller glia to become proliferating neurogenic progenitor-like cells.

The purpose of this study was to investigate how ID factors regulate the ability of Müller glia (MG) to reprogram into proliferating MG-derived progenitor cells (MGPCs) in the chick retina. We found that ID1 is transiently expressed by maturing MG (mMG), whereas ID4 is maintained in mMG in embryonic retinas. In mature retinas, ID4 was prominently expressed by resting MG, but following retinal damage ID4 was rapidly upregulated and then downregulated in MGPCs. By contrast, ID1, ID2, and ID3 were low in resting MG and then upregulated in MGPCs. Inhibition of ID factors following retinal damage decreased numbers of proliferating MGPCs. Inhibition of IDs, after MGPC proliferation, significantly increased numbers of progeny that differentiated as neurons. In damaged or undamaged retinas inhibition of IDs increased levels of p21Cip1 in MG. In response to damage or insulin+FGF2 levels of CDKN1A message and p21Cip1 protein were decreased, absent in proliferating MGPCs, and elevated in MG returning to a resting phenotype. Inhibition of notch- or gp130/Jak/Stat-signaling in damaged retinas increased levels of ID4 but not p21Cip1 in MG. Although ID4 is the predominant isoform expressed by MG in the chick retina, id1 and id2a are predominantly expressed by resting MG and downregulated in activated MG and MGPCs in zebrafish retinas. We conclude that ID factors have a significant impact on regulating the responses of MG to retinal damage, controlling the ability of MG to proliferate by regulating levels of p21Cip1, and suppressing the neurogenic potential of MGPCs.

A brainstem-hypothalamus neuronal circuit reduces feeding upon heat exposure.

Empirical evidence suggests that heat exposure reduces food intake. However, the neurocircuit architecture and the signalling mechanisms that form an associative interface between sensory and metabolic modalities remain unknown, despite primary thermoceptive neurons in the pontine parabrachial nucleus becoming well characterized1. Tanycytes are a specialized cell type along the wall of the third ventricle2 that bidirectionally transport hormones and signalling molecules between the brain's parenchyma and ventricular system3-8. Here we show that tanycytes are activated upon acute thermal challenge and are necessary to reduce food intake afterwards. Virus-mediated gene manipulation and circuit mapping showed that thermosensing glutamatergic neurons of the parabrachial nucleus innervate tanycytes either directly or through second-order hypothalamic neurons. Heat-dependent Fos expression in tanycytes suggested their ability to produce signalling molecules, including vascular endothelial growth factor A (VEGFA). Instead of discharging VEGFA into the cerebrospinal fluid for a systemic effect, VEGFA was released along the parenchymal processes of tanycytes in the arcuate nucleus. VEGFA then increased the spike threshold of Flt1-expressing dopamine and agouti-related peptide (Agrp)-containing neurons, thus priming net anorexigenic output. Indeed, both acute heat and the chemogenetic activation of glutamatergic parabrachial neurons at thermoneutrality reduced food intake for hours, in a manner that is sensitive to both Vegfa loss-of-function and blockage of vesicle-associated membrane protein 2 (VAMP2)-dependent exocytosis from tanycytes. Overall, we define a multimodal neurocircuit in which tanycytes link parabrachial sensory relay to the long-term enforcement of a metabolic code.

Modification of Müller Glial Cell Fate and Proliferation with the Use of Small Molecules.

In recent years, reprogramming Müller glia by overexpressing Ascl1 and other transcription factors has shown promise for the regeneration of postmitotic retinal neurons, primarily bipolar cells, following injury. Müller glial proliferation and efficiency of neuronal differentiation can be modified by the use of small molecules in various systems. The molecules and pathways studied thus far share remarkable consistency with astrocytes. In this mini review, we provide an overview on the modulation of Müller glial proliferation and cell fate using small molecules in injury and reprogramming. We also compare these observations to what has been observed in astrocytes.

Inhibition of Foxp4 Disrupts Cadherin-based Adhesion of Radial Glial Cells, Leading to Abnormal Differentiation and Migration of Cortical Neurons in Mice.

Heterozygous loss-of-function variants of FOXP4 are associated with neurodevelopmental disorders (NDDs) that exhibit delayed speech development, intellectual disability, and congenital abnormalities. The etiology of NDDs is unclear. Here we found that FOXP4 and N-cadherin are expressed in the nuclei and apical end-feet of radial glial cells (RGCs), respectively, in the mouse neocortex during early gestation. Knockdown or dominant-negative inhibition of Foxp4 abolishes the apical condensation of N-cadherin in RGCs and the integrity of neuroepithelium in the ventricular zone (VZ). Inhibition of Foxp4 leads to impeded radial migration of cortical neurons and ectopic neurogenesis from the proliferating VZ. The ectopic differentiation and deficient migration disappear when N-cadherin is over-expressed in RGCs. The data indicate that Foxp4 is essential for N-cadherin-based adherens junctions, the loss of which leads to periventricular heterotopias. We hypothesize that FOXP4 variant-associated NDDs may be caused by disruption of the adherens junctions and malformation of the cerebral cortex.

Activation of enzymatic ethanol oxidation by ATP hydrolysis energy transferred to ADH1A enzyme molecule over Müller cell intermediate filaments.

Presently exciton activation of enzymatic oxidation of ethanol by human alcohol dehydrogenase (ADH) 1A enzyme is reported. The ADH1A enzyme was activated by infrared (IR) excitons transferred over Müller cell (MC) intermediate filaments (IFs). These IR excitons were generated by energy liberated upon enzymatic ATP hydrolysis and transferred to IFs. Also, the emission spectrum was recorded of the electronically excited ADH1A…NAD+…EtOH complexes obtained by energy transfer from IR excitons that traveled along IFs. These results support the hypothesis that ATP hydrolysis energy may be transmitted in vivo in the form of IR excitons, over the network of IFs, both within and between cells.

Metabolic lactate production coordinates vasculature development and progenitor behavior in the developing mouse neocortex.

Proper neural progenitor behavior in conjunction with orderly vasculature formation is fundamental to the development of the neocortex. However, the mechanisms coordinating neural progenitor behavior and vessel growth remain largely elusive. Here we show that robust metabolic production of lactate by radial glial progenitors (RGPs) co-regulates vascular development and RGP division behavior in the developing mouse neocortex. RGPs undergo a highly organized lineage progression program to produce diverse neural progeny. Systematic single-cell metabolic state analysis revealed that RGPs and their progeny exhibit distinct metabolic features associated with specific cell types and lineage progression statuses. Symmetrically dividing, proliferative RGPs preferentially express a cohort of genes that support glucose uptake and anaerobic glycolysis. Consequently, they consume glucose in anaerobic metabolism and produce a high level of lactate, which promotes vessel growth. Moreover, lactate production enhances RGP proliferation by maintaining mitochondrial length. Together, these results suggest that specific metabolic states and metabolites coordinately regulate vasculature formation and progenitor behavior in neocortical development.

Transcytosis of tanycytes in the circumventricular organs of adult mouse brain.

Tanycytes are specialized ependymal cells lining the ventricular spaces of the adult brain and thereby provide an interface between the cerebrospinal fluid (CSF) and brain parenchyma. They act as energy homeostasis, neuroendocrine regulation, and CSF-brain barrier; however, their functional significance in CSF-brain communication currently remains unknown. In the present study, we investigated the presence of tanycytic transcytosis using fluorescent tracers; a GM1 ligand, cholera toxin B (CTB), and a mannose-6-phosphate/insulin-like growth factor-Ⅱ receptor ligand, wheat germ agglutinin (WGA). Both CTB and WGA were incorporated by tanycytes and then released into brain parenchyma in the circumventricular organs such as the organum vasculosum laminae terminalis, subfornical organ, and median eminence, arcuate nucleus, and medullary central canal. Incorporated fluorescent CTB and WGA were released from tanycytes to distribute at neuronal somata. These results indicate that tanycytes of all examined brain regions possess the transport capability of macromolecules from CSF to brain neurons.

Temperature dependence of IR exciton emission spectra in Müller cell intermediate filaments.

Temperature dependences of IR exciton properties in Müller cell (MC) intermediate filaments (IFs) isolated from porcine retina were studied. It was found that the widths of the spectral emission bands in the 2500 cm-1 and 5000 cm-1 energy ranges grow with temperature. It was found that temperature effects on the bandwidth may be described by thermal activation of the low-frequency vibrational modes of the IFs. The average activation energies for the two IR bands were estimated. Considering the dynamics of IR emission, its buildup time was independent on the sample temperature, while its decay time decreased with temperature. Thus, the emission decay rate increased exponentially with the sample temperature. The mechanisms explaining the observed temperature effects were proposed and discussed. Taking into account that MC IFs are capable of transmitting ATP hydrolysis energy within and between cells, with these properties being apparently common for all IFs, these IFs may be used by cells for physical energy transport and communications. As presently reported, temperature effects upon IR exciton spectra should not affect these proposed physiological functions to any significant extent. Therefore, the currently reported data are important for improving our understanding of the physical communication mechanisms operating within and between cells.

Effects of pulsed electric fields on exciton propagation efficiency along Müller cell intermediate filaments. Possible separation mechanism of high- and low-contrast images by the eye-brain system.

In the current study, we tested a possible mechanism of low- and high-contrast image component discrimination by the vertebrate eye-brain system. Apparently the eye-brain system has to discriminate between the low-contrast image component formed by light scattered within the retina, due to interaction of photons with cells and their parts, and the high-contrast image component transmitted by excitons via the quantum mechanism. Presently, effects of pulsed electric fields applied to Müller cell (MC) intermediate filaments (IFs) on the efficiency of exciton propagation were explored. The effects of both pulse duration and amplitude were recorded. These experimental results show that the eye-brain system may be using signal modulation to discriminate between high- and low-contrast image components, improving our understanding of high-contrast vision in vertebrates.

Excitatory neurotransmission activates compartmentalized calcium transients in Müller glia without affecting lateral process motility.

Neural activity has been implicated in the motility and outgrowth of glial cell processes throughout the central nervous system. Here, we explore this phenomenon in Müller glia, which are specialized radial astroglia that are the predominant glial type of the vertebrate retina. Müller glia extend fine filopodia-like processes into retinal synaptic layers, in similar fashion to brain astrocytes and radial glia that exhibit perisynaptic processes. Using two-photon volumetric imaging, we found that during the second postnatal week, Müller glial processes were highly dynamic, with rapid extensions and retractions that were mediated by cytoskeletal rearrangements. During this same stage of development, retinal waves led to increases in cytosolic calcium within Müller glial lateral processes and stalks. These regions comprised distinct calcium compartments, distinguished by variable participation in waves, timing, and sensitivity to an M1 muscarinic acetylcholine receptor antagonist. However, we found that motility of lateral processes was unaffected by the presence of pharmacological agents that enhanced or blocked wave-associated calcium transients. Finally, we found that mice lacking normal cholinergic waves in the first postnatal week also exhibited normal Müller glial process morphology. Hence, outgrowth of Müller glial lateral processes into synaptic layers is determined by factors that are independent of neuronal activity.

When it comes to studying the nervous system, neurons often steal the limelight; yet, they can only work properly thanks to an ensemble cast of cell types whose roles are only just emerging. For example, 'glial cells' ­ their name derives from the Greek word for glue ­ were once thought to play only a passive, supporting function in nervous tissues. Now, growing evidence shows that they are, in fact, integrated into neural circuits: their activity is influenced by neurons, and, in turn, they help neurons to function properly. The role of glial cells is becoming clear in the retina, the thin, light-sensitive layer that lines the back of the eye and relays visual information to the brain. There, beautifully intricate Müller glial cells display fine protrusions (or 'processes') that intermingle with synapses, the busy space between neurons where chemical messengers are exchanged. These messengers can act on Müller cells, triggering cascades of molecular events that may influence the structure and function of glia. This is of particular interest during development: as Müller cells mature, they are exposed to chemicals released by more fully formed retinal neurons. Tworig et al. explored how neuronal messengers can influence the way Müller cells grow their processes. To do so, they tracked mouse retinal glial cells 'live' during development, showing that they were growing fine, highly dynamic processes in a region rich in synapses just as neurons and glia increased their communication. However, using drugs to disrupt this messaging for a short period did not seem to impact how the processes grew. Extending the blockade over a longer timeframe also did not change the way Müller cells developed, with the cells still acquiring their characteristic elaborate process networks. Taken together, these results suggest that the structural maturation of Müller glial cells is not impacted by neuronal signaling, giving a more refined understanding of how glia form in the retina and potentially in the brain.

Dietary Supplement Enriched in Antioxidants and Omega-3 Promotes Glutamine Synthesis in Müller Cells: A Key Process against Oxidative Stress in Retina.

To prevent ocular pathologies, new generation of dietary supplements have been commercially available. They consist of nutritional supplement mixing components known to provide antioxidative properties, such as unsaturated fatty acid, resveratrol or flavonoids. However, to date, only one preclinical study has evaluated the impact of a mixture mainly composed of those components (Nutrof Total®) on the retina and demonstrated that in vivo supplementation prevents the retina from structural and functional injuries induced by light. Considering the crucial role played by the glial Müller cells in the retina, particularly to regulate the glutamate cycle to prevent damage in oxidative stress conditions, we questioned the impact of this ocular supplement on the glutamate metabolic cycle. To this end, various molecular aspects associated with the glutamate/glutamine metabolism cycle in Müller cells were investigated on primary Müller cells cultures incubated, or not, with the commercially mix supplement before being subjected, or not, to oxidative conditions. Our results demonstrated that in vitro supplementation provides guidance of the glutamate/glutamine cycle in favor of glutamine synthesis. These results suggest that glutamine synthesis is a crucial cellular process of retinal protection against oxidative damages and could be a key step in the previous in vivo beneficial results provided by the dietary supplementation.

YAP/TAZ maintain the proliferative capacity and structural organization of radial glial cells during brain development.

The Hippo pathway regulates the development and homeostasis of many tissues and in many species. It controls the activity of two paralogous transcriptional coactivators, YAP and TAZ (YAP/TAZ). Although previous studies have established that aberrant YAP/TAZ activation is detrimental to mammalian brain development, whether and how endogenous levels of YAP/TAZ activity regulate brain development remain unclear. Here, we show that during mammalian cortical development, YAP/TAZ are specifically expressed in apical neural progenitor cells known as radial glial cells (RGCs). The subcellular localization of YAP/TAZ undergoes dynamic changes as corticogenesis proceeds. YAP/TAZ are required for maintaining the proliferative potential and structural organization of RGCs, and their ablation during cortical development reduces the numbers of cortical projection neurons and causes the loss of ependymal cells, resulting in hydrocephaly. Transcriptomic analysis using sorted RGCs reveals gene expression changes in YAP/TAZ-depleted cells that correlate with mutant phenotypes. Thus, our study has uncovered essential functions of YAP/TAZ during mammalian brain development and revealed the transcriptional mechanism of their action.

Therapeutically viable generation of neurons with antisense oligonucleotide suppression of PTB.

Methods to enhance adult neurogenesis by reprogramming glial cells into neurons enable production of new neurons in the adult nervous system. Development of therapeutically viable approaches to induce new neurons is now required to bring this concept to clinical application. Here, we successfully generate new neurons in the cortex and dentate gyrus of the aged adult mouse brain by transiently suppressing polypyrimidine tract binding protein 1 using an antisense oligonucleotide delivered by a single injection into cerebral spinal fluid. Radial glial-like cells and other GFAP-expressing cells convert into new neurons that, over a 2-month period, acquire mature neuronal character in a process mimicking normal neuronal maturation. The new neurons functionally integrate into endogenous circuits and modify mouse behavior. Thus, generation of new neurons in the dentate gyrus of the aging brain can be achieved with a therapeutically feasible approach, thereby opening prospects for production of neurons to replace those lost to neurodegenerative disease.

Biphasic Roles of Hedgehog Signaling in the Production and Self-Renewal of Outer Radial Glia in the Ferret Cerebral Cortex.

The neocortex, the center for higher brain function, emerged in mammals and expanded in the course of evolution. The expansion of outer radial glia (oRGs) and intermediate progenitor cells (IPCs) plays key roles in the expansion and consequential folding of the neocortex. Therefore, understanding the mechanisms of oRG and IPC expansion is important for understanding neocortical development and evolution. By using mice and human cerebral organoids, we previously revealed that hedgehog (HH) signaling expands oRGs and IPCs. Nevertheless, it remained to be determined whether HH signaling expanded oRGs and IPCs in vivo in gyrencephalic species, in which oRGs and IPCs are naturally expanded. Here, we show that HH signaling is necessary and sufficient to expand oRGs and IPCs in ferrets, a gyrencephalic species, through conserved cellular mechanisms. HH signaling increases oRG-producing division modes of ventricular radial glia (vRGs), oRG self-renewal, and IPC proliferation. Notably, HH signaling affects vRG division modes only in an early restricted phase before superficial-layer neuron production peaks. Beyond this restricted phase, HH signaling promotes oRG self-renewal. Thus, HH signaling expands oRGs and IPCs in two distinct but continuous phases during cortical development.

Hypothalamic Rax+ tanycytes contribute to tissue repair and tumorigenesis upon oncogene activation in mice.

Hypothalamic tanycytes in median eminence (ME) are emerging as a crucial cell population that regulates endocrine output, energy balance and the diffusion of blood-born molecules. Tanycytes have recently been considered as potential somatic stem cells in the adult mammalian brain, but their regenerative and tumorigenic capacities are largely unknown. Here we found that Rax+ tanycytes in ME of mice are largely quiescent but quickly enter the cell cycle upon neural injury for self-renewal and regeneration. Mechanistically, Igf1r signaling in tanycytes is required for tissue repair under injury conditions. Furthermore, Braf oncogenic activation is sufficient to transform Rax+ tanycytes into actively dividing tumor cells that eventually develop into a papillary craniopharyngioma-like tumor. Together, these findings uncover the regenerative and tumorigenic potential of tanycytes. Our study offers insights into the properties of tanycytes, which may help to manipulate tanycyte biology for regulating hypothalamic function and investigate the pathogenesis of clinically relevant tumors.

Photoreceptor ablation following ATP induced injury triggers Müller glia driven regeneration in zebrafish.

Retinal regeneration research offers hope to people affected by visual impairment due to disease and injury. Ongoing research has explored many avenues towards retinal regeneration, including those that utilizes implantation of devices, cells or targeted viral-mediated gene therapy. These results have so far been limited, as gene therapy only has applications for rare single-gene mutations and implantations are invasive and in the case of cell transplantation donor cells often fail to integrate with adult neurons. An alternative mode of retinal regeneration utilizes a stem cell population unique to vertebrate retina - Müller glia (MG). Endogenous MG can readily regenerate lost neurons spontaneously in zebrafish and to a very limited extent in mammalian retina. The use of adenosine triphosphate (ATP) has been shown to induce retinal degeneration and activation of the MG in mammals, but whether this is conserved to other vertebrate species including those with higher regenerative capacity remains unknown. In our study, we injected a single dose of ATP intravitreal in zebrafish to characterize the cell death and MG induced regeneration. We used TUNEL labelling on retinal sections to show that ATP caused localised death of photoreceptors and ganglion cells within 24 h. Histology of GFP-transgenic zebrafish and BrdU injected fish demonstrated that MG proliferation peaked at days 3 and 4 post-ATP injection. Using BrdU labelling and photoreceptor markers (Zpr1) we observed regeneration of lost rod photoreceptors at day 14. This study has been undertaken to allow for comparative studies between mammals and zebrafish that use the same specific induction method of injury, i.e. ATP induced injury to allow for direct comparison of across species to narrow down resulting differences that might reflect the differing regenerative capacity. The ultimate aim of this work is to recapitulate pro-neurogenesis Müller glia signaling in mammals to produce new neurons that integrate with the existing retinal circuit to restore vision.

VEGF-Independent Activation of Müller Cells by the Vitreous from Proliferative Diabetic Retinopathy Patients.

Proliferative diabetic retinopathy (PDR), a major complication of diabetes mellitus, results from an inflammation-sustained interplay among endothelial cells, neurons, and glia. Even though anti-vascular endothelial growth factor (VEGF) interventions represent the therapeutic option for PDR, they are only partially efficacious. In PDR, Müller cells undergo reactive gliosis, produce inflammatory cytokines/chemokines, and contribute to scar formation and retinal neovascularization. However, the impact of anti-VEGF interventions on Müller cell activation has not been fully elucidated. Here, we show that treatment of MIO-M1 Müller cells with vitreous obtained from PDR patients stimulates cell proliferation and motility, and activates various intracellular signaling pathways. This leads to cytokine/chemokine upregulation, a response that was not mimicked by treatment with recombinant VEGF nor inhibited by the anti-VEGF drug ranibizumab. In contrast, fibroblast growth factor-2 (FGF2) induced a significant overexpression of various cytokines/chemokines in MIO-M1 cells. In addition, the FGF receptor tyrosine kinase inhibitor BGJ398, the pan-FGF trap NSC12, the heparin-binding protein antagonist N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe Boc2, and the anti-inflammatory hydrocortisone all inhibited Müller cell activation mediated by PDR vitreous. These findings point to a role for various modulators beside VEGF in Müller cell activation and pave the way to the search for novel therapeutic strategies in PDR.

Potential roles of astrocytes and Müller cells in the pathogenesis of glaucoma.

Glaucoma, a progressive optic neuropathy and the leading cause of blindness, is characterized by impairment or degeneration of retinal ganglion cells (RGCs), which transmit visual information to the brain. Currently, 70 million people worldwide are affected by glaucoma. Elevated intraocular pressure (IOP), a major risk factor of glaucoma, directly damages RGCs. However, a substantial proportion of glaucoma patients have a normal IOP level. In particular, over 90% of Japanese glaucoma patients are reported to have normal IOP levels. Thus, a new focus for glaucoma pathology has emerged. Glial cells contribute to tissue homeostasis. Under pathological conditions, glial cells become reactive, lose their homeostatic functions, and gain neurotoxic functions, which trigger neurodegeneration in several diseases including glaucoma. Reactive glial cells have been identified in the eyes of glaucoma patients. In a glaucoma animal model, reactive glial cells are observed at early stages of the disease when RGCs are intact, indicating the possible role of glial cells in the pathogenesis of glaucoma. In this review, we introduce potential roles of glial cells in the pathogenesis of glaucoma. We focus on the roles of the ocular macroglial cells such as astrocytes and Müller cells, and discuss their roles in the pathogenesis of glaucoma.