NG2-Glia, a New Player in Energy Balance.

There is increasing evidence that glia act not only as neuronal support cells, but that they can also influence physiological outcomes via effects on neural signalling. The role of NG2-glia in this regard is especially enigmatic, as they are known to interact with neural circuits but their precise functions other than as oligodendrocyte progenitor cells remain elusive. Here, we summarise recent evidence suggesting that NG2-glia play a role in the maintenance of energy homeostasis, most notably via the support of leptin-sensing neural circuits. We also discuss the potential clinical implication of these findings specifically in the context of cranial radiation therapy.

Functional organization of an Mbp enhancer exposes striking transcriptional regulatory diversity within myelinating glia.

In mammals, large caliber axons are ensheathed by myelin, a glial specialization supporting axon integrity and conferring accelerated and energy-efficient action potential conduction. Myelin basic protein (MBP) is required for normal myelin elaboration with maximal mbp transcription in oligodendrocytes requiring the upstream M3 enhancer. To further characterize the mechanism regulating mbp transcription, we defined M3 structure/function relationships by evaluating its evolutionary conservation, DNA footprints and the developmental programing conferred in mice by M3 derivatives. Multiple M3 regulatory element combinations were found to drive expression in oligodendrocytes and Schwann cells with a minimal 129 bp sequence conferring expression in oligodendrocytes throughout myelin elaboration, maintenance and repair. Unexpectedly, M3 derivatives conferred markedly different spatial and temporal expression programs thus illuminating striking transcriptional heterogeneity within post-mitotic oligodendrocytes. Finally, one M3 derivative engaged only during primary myelination, not during adult remyelination, demonstrating that transcriptional regulation in the two states is not equivalent.

Extensive regenerative plasticity among adult NG2-glia populations is exclusively based on self-renewal.

NG2-glia are known to proliferate in the adult brain, however the extent of their mitotic and regenerative capacity and particularly their adult origin is uncertain. By employing a paradigm of mitotic blockade in conjunction with genetic fate tracing we demonstrate that intracerebroventricular mitotic blocker infusion leads to wide-spread and complete ablation of NG2-glial cells in the hypothalamus and other periventricular brain regions. However, despite the extensive glia loss, parenchymal NG2-glia coverage is fully restored to pretreatment levels within two weeks. We further reveal that in response to mitotic blocker treatment, NG2-glia bordering the ablated territories start to express the stem cell marker nestin, divide and migrate to replace the lost cells. Importantly, the migration front of repopulating NG2-glia invariably proceeds from the distal parenchyma towards the ventricles, ruling out contributions of the subventricular zone neurogenic niche or the corresponding area of the third ventricle as source of new NG2-glia. NG2-CreER-based fate tracing further substantiates that NG2-glia which have been spared from mitotic blockade are the sole source of regenerating NG2-glia. Collectively, our data reveals that all adult NG2-glia retain the ability to divide and that they are capable of fully restoring parenchymal NG2-glia coverage after wide-spread NG2 cell loss, indicating complete self-sufficiency in maintaining NG2-glia population levels in the adult brain.

The neurons that drive infradian sleep-wake and mania-like behavioral rhythms.

Infradian mood and sleep-wake rhythms with periods of 48 hr and beyond have been observed in bipolar disorder (BD) subjects that even persist in time isolation, indicating an endogenous origin. Here we show that mice exposed to methamphetamine (Meth) in drinking water develop infradian locomotor rhythms with periods of 48 hr and beyond which extend to sleep length and mania-like behaviors in support of a model for cycling in BD. This cycling capacity is abrogated upon genetic disruption of DA production in DA neurons of the ventral tegmental area (VTA) or ablation of nucleus accumbens (NAc) projecting, dopamine (DA) neurons. Chemogenetic activation of NAc-projecting DA neurons leads to locomotor period lengthening in clock deficient mice, while cytosolic calcium in DA processes of the NAc was found fluctuating synchronously with locomotor behavior. Together, our findings argue that BD cycling relies on infradian rhythm generation that depends on NAc-projecting DA neurons.

High-resolution intravital imaging of the murine hypothalamus using GRIN lenses and confocal microscopy.

Intravital studies of cellular morphology in structures such as the hypothalamus are challenging because of their location at the bottom of the brain. Here, we describe an intravital imaging protocol based on gradient refractive index (GRIN) lenses in conjunction with confocal microscopy to inspect fluorescent cells at high resolution in deep-brain areas. The approach relies on implanted guide-tubes for the interchangeable use of GRIN lenses, thereby allowing imaging at different magnifications and increasing the effective field of view. For complete details on the use and execution of this profile, please refer to Butiaeva et al. (2021).

Leptin receptor-expressing pericytes mediate access of hypothalamic feeding centers to circulating leptin.

Knowledge of how leptin receptor (LepR) neurons of the mediobasal hypothalamus (MBH) access circulating leptin is still rudimentary. Employing intravital microscopy, we found that almost half of the blood-vessel-enwrapping pericytes in the MBH express LepR. Selective disruption of pericytic LepR led to increased food intake, increased fat mass, and loss of leptin-dependent signaling in nearby LepR neurons. When delivered intravenously, fluorescently tagged leptin accumulated at hypothalamic LepR pericytes, which was attenuated upon pericyte-specific LepR loss. Because a paracellular tracer was also preferentially retained at LepR pericytes, we pharmacologically targeted regulators of inter-endothelial junction tightness and found that they affect LepR neuronal signaling and food intake. Optical imaging in MBH slices revealed a long-lasting, tonic calcium increase in LepR pericytes in response to leptin, suggesting pericytic contraction and vessel constriction. Together, our data indicate that LepR pericytes facilitate localized, paracellular blood-brain barrier leaks, enabling MBH LepR neurons to access circulating leptin.

TLR4 links innate immunity and fatty acid-induced insulin resistance.

TLR4 is the receptor for LPS and plays a critical role in innate immunity. Stimulation of TLR4 activates proinflammatory pathways and induces cytokine expression in a variety of cell types. Inflammatory pathways are activated in tissues of obese animals and humans and play an important role in obesity-associated insulin resistance. Here we show that nutritional fatty acids, whose circulating levels are often increased in obesity, activate TLR4 signaling in adipocytes and macrophages and that the capacity of fatty acids to induce inflammatory signaling in adipose cells or tissue and macrophages is blunted in the absence of TLR4. Moreover, mice lacking TLR4 are substantially protected from the ability of systemic lipid infusion to (a) suppress insulin signaling in muscle and (b) reduce insulin-mediated changes in systemic glucose metabolism. Finally, female C57BL/6 mice lacking TLR4 have increased obesity but are partially protected against high fat diet-induced insulin resistance, possibly due to reduced inflammatory gene expression in liver and fat. Taken together, these data suggest that TLR4 is a molecular link among nutrition, lipids, and inflammation and that the innate immune system participates in the regulation of energy balance and insulin resistance in response to changes in the nutritional environment.

Evidence for constitutive neural cell proliferation in the adult murine hypothalamus.

Compelling evidence suggests that the mammalian brain is capable of generating new neurons throughout adult life. While neurogenesis can be induced at various brain sites by exogenous cues, constitutive birth of new neurons has been unambiguously demonstrated within the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus. The lack of strong evidence that constitutive neurogenesis occurs elsewhere in the adult brain could be due to its exclusive restriction to the SVZ and SGZ or, for instance, to the inadequacy of the methods used to reveal new-born neurons at other brain sites. By using intracerebroventricular (icv) delivery of the mitotic marker bromodeoxyuridine (BrdU) we demonstrate that new cells are born continuously and in substantial numbers in the adult murine hypothalamus and that many of these cells appear to differentiate into neurons as assessed by the expression of doublecortin (Dcx) and other neuronal fate markers. As compared to intraperitoneal (ip) BrdU injections, central BrdU infusion also uncovers a higher-fold induction of hypothalamic cell proliferation by ciliary neurotrophic factor (CNTF). It appears that new cells are born throughout the hypothalamic parenchyma without an apparent restriction to a specific neurogenic layer, as seen in the SVZ. Thus, we provide evidence that the adult hypothalamus is constitutively neurogenic and that hypothalamic cell proliferation is highly responsive to mitogen action.

Adult NG2-Glia Are Required for Median Eminence-Mediated Leptin Sensing and Body Weight Control.

While leptin is a well-known regulator of body fat mass, it remains unclear how circulating leptin is sensed centrally to maintain energy homeostasis. Here we show that genetic and pharmacological ablation of adult NG2-glia (also known as oligodendrocyte precursors), but not microglia, leads to primary leptin resistance and obesity in mice. We reveal that NG2-glia contact the dendritic processes of arcuate nucleus leptin receptor (LepR) neurons in the median eminence (ME) and that these processes degenerate upon NG2-glia elimination, which explains the consequential attenuation of these neurons' molecular and electrical responses to leptin. Our data therefore indicate that LepR dendrites in the ME represent the principal conduits of leptin's anorexigenic action and that NG2-glia are essential for their maintenance. Given that ME-directed X-irradiation confirmed the pharmacological and genetically mediated ablation effects on body weight, our findings provide a rationale for the known obesity risk associated with cranial radiation therapy.

A highly tunable dopaminergic oscillator generates ultradian rhythms of behavioral arousal.

Ultradian (~4 hr) rhythms in locomotor activity that do not depend on the master circadian pacemaker in the suprachiasmatic nucleus have been observed across mammalian species, however, the underlying mechanisms driving these rhythms are unknown. We show that disruption of the dopamine transporter gene lengthens the period of ultradian locomotor rhythms in mice. Period lengthening also results from chemogenetic activation of midbrain dopamine neurons and psychostimulant treatment, while the antipsychotic haloperidol has the opposite effect. We further reveal that striatal dopamine levels fluctuate in synchrony with ultradian activity cycles and that dopaminergic tone strongly predicts ultradian period. Our data indicate that an arousal regulating, dopaminergic ultradian oscillator (DUO) operates in the mammalian brain, which normally cycles in harmony with the circadian clock, but can desynchronize when dopamine tone is elevated, thereby producing aberrant patterns of arousal which are strikingly similar to perturbed sleep-wake cycles comorbid with psychopathology.

Evidence for NG2-glia derived, adult-born functional neurons in the hypothalamus.

Accumulating evidence suggests that the adult murine hypothalamus, a control site of several fundamental homeostatic processes, has neurogenic capacity. Correspondingly, the adult hypothalamus exhibits considerable cell proliferation that is ongoing even in the absence of external stimuli, and some of the newborn cells have been shown to mature into cells that express neuronal fate markers. However, the identity and characteristics of proliferating cells within the hypothalamic parenchyma have yet to be thoroughly investigated. Here we show that a subset of NG2-glia distributed throughout the mediobasal hypothalamus are proliferative and express the stem cell marker Sox2. We tracked the constitutive differentiation of hypothalamic NG2-glia by employing genetic fate mapping based on inducible Cre recombinase expression under the control of the NG2 promoter, demonstrating that adult hypothalamic NG2-glia give rise to substantial numbers of APC+ oligodendrocytes and a smaller population of HuC/D+ or NeuN+ neurons. Labelling with the cell proliferation marker BrdU confirmed that some NG2-derived neurons have proliferated shortly before differentiation. Furthermore, patch-clamp electrophysiology revealed that some NG2-derived cells display an immature neuronal phenotype and appear to receive synaptic input indicative of their electrical integration in local hypothalamic circuits. Together, our studies show that hypothalamic NG2-glia are able to take on neuronal fates and mature into functional neurons, indicating that NG2-glia contribute to the neurogenic capacity of the adult hypothalamus.

Remodeling of the arcuate nucleus energy-balance circuit is inhibited in obese mice.

In the CNS, the hypothalamic arcuate nucleus (ARN) energy-balance circuit plays a key role in regulating body weight. Recent studies have shown that neurogenesis occurs in the adult hypothalamus, revealing that the ARN energy-balance circuit is more plastic than originally believed. Changes in diet result in altered gene expression and neuronal activity in the ARN, some of which may reflect hypothalamic plasticity. To explore this possibility, we examined the turnover of hypothalamic neurons in mice with obesity secondary to either high-fat diet (HFD) consumption or leptin deficiency. We found substantial turnover of neurons in the ARN that resulted in ongoing cellular remodeling. Feeding mice HFD suppressed neurogenesis, as demonstrated by the observation that these mice both generated fewer new neurons and retained more old neurons. This suppression of neuronal turnover was associated with increased apoptosis of newborn neurons. Leptin-deficient mice also generated fewer new neurons, an observation that was explained in part by a loss of hypothalamic neural stem cells. These data demonstrate that there is substantial postnatal turnover of the arcuate neuronal circuitry in the mouse and reveal the unexpected capacity of diet and leptin deficiency to inhibit this neuronal remodeling. This insight has important implications for our understanding of nutritional regulation of energy balance and brain function.

Neurogenesis in the hypothalamus of adult mice: potential role in energy balance.

Ciliary neurotrophic factor (CNTF) induces weight loss in obese rodents and humans, and for reasons that are not understood, its effects persist after the cessation of treatment. Here we demonstrate that centrally administered CNTF induces cell proliferation in feeding centers of the murine hypothalamus. Many of the newborn cells express neuronal markers and show functional phenotypes relevant for energy-balance control, including a capacity for leptin-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3). Coadministration of the mitotic blocker cytosine-beta-d-arabinofuranoside (Ara-C) eliminates the proliferation of neural cells and abrogates the long-term, but not the short-term, effect of CNTF on body weight. These findings link the sustained effect of CNTF on energy balance to hypothalamic neurogenesis and suggest that regulated hypothalamic neurogenesis in adult mice may play a previously unappreciated role in physiology and disease.

A novel mode of sensory transduction in archaea: binding protein-mediated chemotaxis towards osmoprotectants and amino acids.

Directly upstream of the Halobacterium salinarum transducer genes basT and htpIV we identified two open reading frames (orfs) with significant homologies to genes encoding binding proteins for amino acids and compatible solutes, respectively. Behavioral testing of deletion mutants indicates that halobacterial chemotaxis towards branched-chain amino acids as well as compatible osmolytes of the betaine family requires both a binding and a transducer protein. We therefore named the binding/transducer proteins BasB/BasT for branched-chain and sulfur-containing amino acids and CosB/CosT for compatible solutes. Our data support a signaling mechanism with the binding proteins functioning as lipid-anchored receptors interacting with the extracellular domain of their cognate transducers. Inspection of the halobacterial genome suggests that BasB and CosB exclusively mediate chemotaxis responses without any additional role in transport, which is in contrast to bacterial binding proteins, which are always part of ABC transport systems. The CosB/CosT system is the first instance of a chemotaxis signaling pathway for organic osmolytes in the living world and natural abundance 13C-NMR analysis of cytoplasmic extracts suggests that H.salinarum utilizes these solutes for osmotic adaptation.