Elevated genetic risk for multiple sclerosis emerged in steppe pastoralist populations.

Multiple sclerosis (MS) is a neuro-inflammatory and neurodegenerative disease that is most prevalent in Northern Europe. Although it is known that inherited risk for MS is located within or in close proximity to immune-related genes, it is unknown when, where and how this genetic risk originated1. Here, by using a large ancient genome dataset from the Mesolithic period to the Bronze Age2, along with new Medieval and post-Medieval genomes, we show that the genetic risk for MS rose among pastoralists from the Pontic steppe and was brought into Europe by the Yamnaya-related migration approximately 5,000 years ago. We further show that these MS-associated immunogenetic variants underwent positive selection both within the steppe population and later in Europe, probably driven by pathogenic challenges coinciding with changes in diet, lifestyle and population density. This study highlights the critical importance of the Neolithic period and Bronze Age as determinants of modern immune responses and their subsequent effect on the risk of developing MS in a changing environment.

The selection landscape and genetic legacy of ancient Eurasians.

The Holocene (beginning around 12,000 years ago) encompassed some of the most significant changes in human evolution, with far-reaching consequences for the dietary, physical and mental health of present-day populations. Using a dataset of more than 1,600 imputed ancient genomes1, we modelled the selection landscape during the transition from hunting and gathering, to farming and pastoralism across West Eurasia. We identify key selection signals related to metabolism, including that selection at the FADS cluster began earlier than previously reported and that selection near the LCT locus predates the emergence of the lactase persistence allele by thousands of years. We also find strong selection in the HLA region, possibly due to increased exposure to pathogens during the Bronze Age. Using ancient individuals to infer local ancestry tracts in over 400,000 samples from the UK Biobank, we identify widespread differences in the distribution of Mesolithic, Neolithic and Bronze Age ancestries across Eurasia. By calculating ancestry-specific polygenic risk scores, we show that height differences between Northern and Southern Europe are associated with differential Steppe ancestry, rather than selection, and that risk alleles for mood-related phenotypes are enriched for Neolithic farmer ancestry, whereas risk alleles for diabetes and Alzheimer's disease are enriched for Western hunter-gatherer ancestry. Our results indicate that ancient selection and migration were large contributors to the distribution of phenotypic diversity in present-day Europeans.

Ingested non-essential amino acids recruit brain orexin cells to suppress eating in mice.

Ingested nutrients are proposed to control mammalian behavior by modulating the activity of hypothalamic orexin/hypocretin neurons (HONs). Previous in vitro studies showed that nutrients ubiquitous in mammalian diets, such as non-essential amino acids (AAs) and glucose, modulate HONs in distinct ways. Glucose inhibits HONs, whereas non-essential (but not essential) AAs activate HONs. The latter effect is of particular interest because its purpose is unknown. Here, we show that ingestion of a dietary-relevant mix of non-essential AAs activates HONs and shifts behavior from eating to exploration. These effects persisted despite ablation of a key neural gut → brain communication pathway, the cholecystokinin-sensitive vagal afferents. The behavioral shift induced by the ingested non-essential AAs was recapitulated by targeted HON optostimulation and abolished in mice lacking HONs. Furthermore, lick microstructure analysis indicated that intragastric non-essential AAs and HON optostimulation each reduce the size, but not the frequency, of consumption bouts, thus implicating food palatability modulation as a mechanism for the eating suppression. Collectively, these results suggest that a key purpose of HON activation by ingested, non-essential AAs is to suppress eating and re-initiate food seeking. We propose and discuss possible evolutionary advantages of this, such as optimizing the limited stomach capacity for ingestion of essential nutrients.

Convergent inputs from electrically and topographically distinct orexin cells to locus coeruleus and ventral tegmental area.

Orexin/hypocretin (orx/hcrt) neurons are thought to ensure that reward-seeking is accompanied by alertness, but the underlying circuit organization is unclear. Reports of differential regulation of lateral versus medial orx/hcrt cells produced a hypothesis of 'efferent dichotomy', in which lateral orx/hcrt cells innervate the ventral tegmental area (VTA) and control reward, while medial orx/hcrt cells innervate locus coeruleus (LC) and control arousal. Two distinct types of orx/hcrt cells also emerged from analysis of intrinsic and input-driven single-cell electrical activity. To examine the projections of these emerging orx/hcrt subtypes to LC and VTA, we injected retrograde tracer into these regions in the mouse brain in vivo, and then examined the properties of tracer-containing orx/hcrt cells in hypothalamic slices. VTA- and LC-projecting orx/hcrt cells were found across the entire orx/hcrt field, including the zona incerta, perifornical area, dorsomedial/anterior and lateral hypothalamus. Within these areas, orx/hcrt cells had similar probabilities of projecting to VTA or LC. Examination of lateral versus medial sections revealed that VTA and LC received inputs from both lateral and medial orx/hcrt cells, but, unexpectedly, lateral orx/hcrt cells were more likely to project to LC than medial orx/hcrt cells. Finally, patch-clamp recordings revealed that VTA and LC received projections from both electrical classes of orx/hcrt cells, which had similar likelihoods of projecting to VTA or LC. Contrary to previous predictions, our data suggest that medial and lateral orx/hcrt cells, and the different electrical and morphological subclasses of orx/hcrt cells identified to date, send projections to both LC and VTA.

Activation of central orexin/hypocretin neurons by dietary amino acids.

Hypothalamic orexin/hypocretin (orx/hcrt) neurons regulate energy balance, wakefulness, and reward; their loss produces narcolepsy and weight gain. Glucose can lower the activity of orx/hcrt cells, but whether other dietary macronutrients have similar effects is unclear. We show that orx/hcrt cells are stimulated by nutritionally relevant mixtures of amino acids (AAs), both in brain slice patch-clamp experiments, and in c-Fos expression assays following central or peripheral administration of AAs to mice in vivo. Physiological mixtures of AAs electrically excited orx/hcrt cells through a dual mechanism involving inhibition of K(ATP) channels and activation of system-A amino acid transporters. Nonessential AAs were more potent in activating orx/hcrt cells than essential AAs. Moreover, the presence of physiological concentrations of AAs suppressed the glucose responses of orx/hcrt cells. These results suggest a new mechanism of hypothalamic integration of macronutrient signals and imply that orx/hcrt cells sense macronutrient balance, rather than net energy value, in extracellular fluid.

Direct and indirect control of orexin/hypocretin neurons by glycine receptors.

Hypothalamic hypocretin/orexin (hcrt/orx) neurons promote arousal and reward seeking, while reduction in their activity has been linked to narcolepsy, obesity and depression. However, the mechanisms influencing the activity of hcrt/orx networks in situ are not fully understood. Here we show that glycine, a neurotransmitter best known for its actions in the brainstem and spinal cord, elicits dose dependent postsynaptic Cl⁻ currents in hcrt/orx cells in acute mouse brain slices. The effect was blocked by the glycine receptor (GLyR) antagonist strychnine and mimicked by the GlyR agonist alanine. Postsynaptic GlyRs on hcrt/orx cells remained functional during both early postnatal and adult periods, and gramicidin-perforated patch-clamp recordings revealed that they progressively switch from excitatory to inhibitory during the first two postnatal weeks. The pharmacological profile of the glycine response suggested that developed hcrt/orx neurons contain α/ß-heteromeric GlyRs that lack α2-subunits, whereas α2-subunits, whereas α2-subunits are present in early postnatal hcrt/orx neurons. All postsynaptic currents (PSCs) in developed hcrt/orx cells were blocked by inhibitors of GABA and glutamate receptors, with no evidence of GlyR-mediated PSCs. However, the frequency but not amplitude of miniature PSCs was reduced by strychnine and increased by glycine in ~50% of hcrt/orx neurons. Together, these results provide the first evidence for functional GlyRs in identified hcrt/orx circuits and suggest that the activity of developed hcrt/orx cells is regulated by two GlyR pools: inhibitory extrasynaptic GlyRs located on all hcrt/orx cells and excitatory GlyRs located on presynaptic terminals contacting some hcrt/orx cells.

Stimulation of orexin/hypocretin neurones by thyrotropin-releasing hormone.

Central orexin/hypocretin neurones are critical for sustaining consciousness: their firing stimulates wakefulness and their destruction causes narcolepsy. We explored whether the activity of orexin cells is modulated by thyrotropin-releasing hormone (TRH), an endogenous stimulant of wakefulness and locomotor activity whose mechanism of action is not fully understood. Living orexin neurones were identified by targeted expression of green fluorescent protein (GFP) in acute brain slices of transgenic mice. Using whole-cell patch-clamp recordings, we found that TRH robustly increased the action potential firing rate of these neurones. TRH-induced excitation persisted under conditions of synaptic isolation, and involved a Na(+)-dependent depolarization and activation of a mixed cation current in the orexin cell membrane. By double-label immunohistochemistry, we found close appositions between TRH-immunoreactive nerve terminals and orexin-A-immunoreactive cell bodies. These results identify a new physiological modulator of orexin cell firing, and suggest that orexin cell excitation may contribute to the arousal-enhancing actions of TRH.

Adaptive sugar sensors in hypothalamic feeding circuits.

Brain glucose sensing is critical for healthy energy balance, but how appropriate neurocircuits encode both small changes and large background values of glucose levels is unknown. Here, we report several features of hypothalamic orexin neurons, cells essential for normal wakefulness and feeding: (i) A distinct group of orexin neurons exhibits only transient inhibitory responses to sustained rises in sugar levels; (ii) this sensing strategy involves time-dependent recovery from inhibition via adaptive closure of leak-like K(+) channels; (iii) combining transient and sustained glucosensing allows orexin cell firing to maintain sensitivity to small fluctuations in glucose levels while simultaneously encoding a large range of baseline glucose concentrations. These data provide insights into how vital behavioral orchestrators sense key features of the internal environment while sustaining a basic activity tone required for the stability of consciousness.

Control of hypothalamic orexin neurons by acid and CO2.

Hypothalamic orexin/hypocretin neurons recently emerged as key orchestrators of brain states and adaptive behaviors. They are critical for normal stimulation of wakefulness and breathing: Orexin loss causes narcolepsy and compromises vital ventilatory adaptations. However, it is unclear how orexin neurons generate appropriate adjustments in their activity during changes in physiological circumstances. Extracellular levels of acid and CO2 are fundamental physicochemical signals controlling wakefulness and breathing, but their effects on the firing of orexin neurons are unknown. Here we show that the spontaneous firing rate of identified orexin neurons is profoundly affected by physiological fluctuations in ambient levels of H+ and CO2. These responses resemble those of known chemosensory neurons both qualitatively (acidification is excitatory, alkalinization is inhibitory) and quantitatively (approximately 100% change in firing rate per 0.1 unit change in pHe). Evoked firing of orexin cells is similarly modified by physiologically relevant changes in pHe: Acidification increases intrinsic excitability, whereas alkalinization depresses it. The effects of pHe involve acid-induced closure of leak-like K+ channels in the orexin cell membrane. These results suggest a new mechanism of how orexin/hypocretin networks generate homeostatically appropriate firing patterns.

The value of animal models for drug development in multiple sclerosis.

The rodent model for multiple sclerosis, experimental allergic (autoimmune) encephalomyelitis (EAE), has been used to dissect molecular mechanisms of the autoimmune inflammatory response, and hence to devise and test new therapies for multiple sclerosis. Clearly, artificial immunization against myelin may not necessarily reproduce all the pathogenetic mechanisms operating in the human disease, but most therapies tested in multiple sclerosis patients are nevertheless based on concepts derived from studies in EAE. Unfortunately, several treatments, though successful in pre-clinical EAE trials, were either less effective in patients, worsened disease or caused unexpected, severe adverse events, as we review here. These discrepancies must, at least in part, be due to genetic and environmental differences, but the precise underlying reasons are not yet clear. Our understanding of EAE pathogenesis is still incomplete and so, therefore, are any implications for drug development in these models. Here, we suggest some potential explanations based on new thinking about key pathogenic concepts and differences that may limit extrapolation from EAE to multiple sclerosis. To try to circumvent these rodent-human dissimilarities more systematically, we propose that pre-clinical trials should be started in humanized mouse models.