Neuroscience of eating: Pace and portion control.

Satiety-promoting neurons of the hindbrain have long been known for their role in meal termination. An innovative new study now reveals how different hindbrain cell types mediate appetite on distinct timescales.

The hindbrain and cortico-reticular pathway in adolescent idiopathic scoliosis.

AIM: To characterise the corticoreticular pathway (CRP) in a case-control cohort of adolescent idiopathic scoliosis (AIS) patients using high-resolution slice-accelerated readout-segmented echo-planar diffusion tensor imaging (DTI) to enhance the discrimination of small brainstem nuclei in comparison to automated whole-brain volumetry and tractography and their clinical correlates. MATERIALS AND METHODS: Thirty-four participants (16 AIS patients, 18 healthy controls) underwent clinical and orthopaedic assessments and brain magnetic resonance imaging (MRI) on a 3 T MRI machine. Automated whole-brain volume-based morphometry, tract-based spatial statistics analysis, and manual CRP tractography by two independent raters were performed. Intra-rater and inter-rater agreement of DTI metrics from CRP tractography were assessed by intraclass correlation coefficient. Normalised structural brain volumes and DTI metrics were compared between groups using Student's t-tests. Linear correlation analysis between imaging parameters and clinical scores was also performed. RESULTS: AIS patients demonstrated a significantly larger pons volume compared to controls (p=0.006). Significant inter-side CRP differences in mean (p=0.02) and axial diffusivity (p=0.01) were found in patients only. Asymmetry in CRP fractional anisotropy significantly correlated with the Cobb angle (p=0.03). CONCLUSION: Relative pontine hypertrophy and asymmetry in CRP DTI metrics suggest central supranuclear inter-hemispheric imbalance in AIS, and support the role of the CRP in axial muscle tone. Longitudinal evaluation of CRP DTI metrics in the prediction of AIS progression may be clinically relevant.

Sea lamprey enlightens the origin of the coupling of retinoic acid signaling to vertebrate hindbrain segmentation.

Retinoic acid (RA) is involved in antero-posterior patterning of the chordate body axis and, in jawed vertebrates, has been shown to play a major role at multiple levels of the gene regulatory network (GRN) regulating hindbrain segmentation. Knowing when and how RA became coupled to the core hindbrain GRN is important for understanding how ancient signaling pathways and patterning genes can evolve and generate diversity. Hence, we investigated the link between RA signaling and hindbrain segmentation in the sea lamprey Petromyzon marinus, an important jawless vertebrate model providing clues to decipher ancestral vertebrate features. Combining genomics, gene expression, and functional analyses of major components involved in RA synthesis (Aldh1as) and degradation (Cyp26s), we demonstrate that RA signaling is coupled to hindbrain segmentation in lamprey. Thus, the link between RA signaling and hindbrain segmentation is a pan vertebrate feature of the hindbrain and likely evolved at the base of vertebrates.

ChOP-CT: quantitative morphometrical analysis of the Hindbrain Choroid Plexus by X-ray micro-computed tomography.

The Hindbrain Choroid Plexus is a complex, cerebrospinal fluid-secreting tissue that projects into the 4th vertebrate brain ventricle. Despite its irreplaceability in the development and homeostasis of the entire central nervous system, the research of Hindbrain Choroid Plexus and other Choroid Plexuses has been neglected by neuroscientists for decades. One of the obstacles is the lack of tools that describe the complex shape of the Hindbrain Choroid Plexus in the context of brain ventricles. Here we introduce an effective tool, termed ChOP-CT, for the noninvasive, X-ray micro-computed tomography-based, three-dimensional visualization and subsequent quantitative spatial morphological analysis of developing mouse Hindbrain Choroid Plexus. ChOP-CT can reliably quantify Hindbrain Choroid Plexus volume, surface area, length, outgrowth angle, the proportion of the ventricular space occupied, asymmetries and general shape alterations in mouse embryos from embryonic day 13.5 onwards. We provide evidence that ChOP-CT is suitable for the unbiased evaluation and detection of the Hindbrain Choroid Plexus alterations within various mutant embryos. We believe, that thanks to its versatility, quantitative nature and the possibility of automation, ChOP-CT will facilitate the analysis of the Hindbrain Choroid Plexus in the mouse models. This will ultimately accelerate the screening of the candidate genes and mechanisms involved in the onset of various Hindbrain Choroid Plexus-related diseases.

Identification of AgRP cells in the murine hindbrain that drive feeding.

OBJECTIVE: The central melanocortin system is essential for the regulation of food intake and body weight. Agouti-related protein (AgRP) is the sole orexigenic component of the central melanocortin system and is conserved across mammalian species. AgRP is currently known to be expressed exclusively in the mediobasal hypothalamus, and hypothalamic AgRP-expressing neurons are essential for feeding. Here we characterized a previously unknown population of AgRP cells in the mouse hindbrain. METHODS: Expression of AgRP in the hindbrain was investigated using gene expression analysis, single-cell RNA sequencing, immunofluorescent analysis and multiple transgenic mice with reporter expressions. Activation of AgRP neurons was achieved by Designer Receptors Exclusively Activated by Designer Drugs (DREADD) and by transcranial focal photo-stimulation using a step-function opsin with ultra-high light sensitivity (SOUL). RESULTS: AgRP expressing cells were present in the area postrema (AP) and the adjacent subpostrema area (SubP) and commissural nucleus of the solitary tract (cNTS) of the mouse hindbrain (termed AgRPHind herein). AgRPHind cells consisted of locally projecting neurons as well as tanycyte-like cells. Food deprivation stimulated hindbrain Agrp expression as well as neuronal activity of subsets of AgRPHind cells. In adult mice that lacked hypothalamic AgRP neurons, chemogenetic activation of AgRP neurons resulted in hyperphagia and weight gain. In addition, transcranial focal photo-stimulation of hindbrain AgRP cells increased food intake in adult mice with or without hypothalamic AgRP neurons. CONCLUSIONS: Our study indicates that the central melanocortin system in the hindbrain possesses an orexigenic component, and that AgRPHind neurons stimulate feeding independently of hypothalamic AgRP neurons.

Hindbrain boundaries as niches of neural progenitor and stem cells regulated by the extracellular matrix proteoglycan chondroitin sulphate.

The interplay between neural progenitors and stem cells (NPSCs), and their extracellular matrix (ECM) is a crucial regulatory mechanism that determines their behavior. Nonetheless, how the ECM dictates the state of NPSCs remains elusive. The hindbrain is valuable to examine this relationship, as cells in the ventricular surface of hindbrain boundaries (HBs), which arise between any two neighboring rhombomeres, express the NPSC marker Sox2, while being surrounded with the membrane-bound ECM molecule chondroitin sulphate proteoglycan (CSPG), in chick and mouse embryos. CSPG expression was used to isolate HB Sox2+ cells for RNA-sequencing, revealing their distinguished molecular properties as typical NPSCs, which express known and newly identified genes relating to stem cells, cancer, the matrisome and cell cycle. In contrast, the CSPG- non-HB cells, displayed clear neural-differentiation transcriptome. To address whether CSPG is significant for hindbrain development, its expression was manipulated in vivo and in vitro. CSPG manipulations shifted the stem versus differentiation state of HB cells, evident by their behavior and altered gene expression. These results provide further understanding of the uniqueness of hindbrain boundaries as repetitive pools of NPSCs in-between the rapidly growing rhombomeres, which rely on their microenvironment to maintain their undifferentiated state during development.

In Situ Hybridization to Characterize Neurulation and Midbrain-Hindbrain Boundary Formation in Zebrafish.

Whole-mount in situ hybridization is cable to harness the inherent advantages of zebrafish as a model organism for developmental biology, particularly when visualizing the formation of the neural tube, specifically at the level of the midbrain-hindbrain boundary. The size and transparency of developing zebrafish embryos allow for the visualization of neural markers in vivo along the length of the developing zebrafish central nervous system. In practice, this technique is useful for examining defects in neurulation and midbrain-hindbrain boundary formation that may arise following gene manipulation, for example, CRISPR mutagenesis. This method describes the process of embryo collection and preparation, RNA probe transcription, probe hybridization in vivo, as well as the process of probe detection and visualization.

Hindbrain REV-ERB nuclear receptors regulate sensitivity to diet-induced obesity and brown adipose tissue pathophysiology.

OBJECTIVE: The dorsal vagal complex (DVC) of the hindbrain is a major point of integration for central and peripheral signals that regulate a wide variety of metabolic functions to maintain energy balance. The REV-ERB nuclear receptors are important modulators of molecular metabolism, but their role in the DVC has yet to be established. METHODS: Male REV-ERBα/ß floxed mice received stereotaxic injections of a Cre expressing virus to the DVC to create the DVC REV-ERBα/ß double knockout (DVC RDKO). Control littermates received stereotaxic injections to the DVC of a green fluorescent protein expressing virus. Animals were maintained on a normal chow diet or a 60% high-fat diet to observe the metabolic phenotype arising from DVC RDKO under healthy and metabolically stressed conditions. RESULTS: DVC RDKO animals on high-fat diet exhibited increased weight gain compared to control animals maintained on the same diet. Increased weight gain in DVC RDKO animals was associated with decreased basal metabolic rate and dampened signature of brown adipose tissue activity. RDKO decreased gene expression of calcitonin receptor in the DVC and tyrosine hydroxylase in the brown adipose tissue. CONCLUSIONS: These results suggest a previously unappreciated role of REV-ERB nuclear receptors in the DVC for maintaining energy balance and metabolic rate potentially through indirect sympathetic outflow to the brown adipose tissue.

The molecular cytoarchitecture of the adult mouse brain.

The function of the mammalian brain relies upon the specification and spatial positioning of diversely specialized cell types. Yet, the molecular identities of the cell types and their positions within individual anatomical structures remain incompletely known. To construct a comprehensive atlas of cell types in each brain structure, we paired high-throughput single-nucleus RNA sequencing with Slide-seq1,2-a recently developed spatial transcriptomics method with near-cellular resolution-across the entire mouse brain. Integration of these datasets revealed the cell type composition of each neuroanatomical structure. Cell type diversity was found to be remarkably high in the midbrain, hindbrain and hypothalamus, with most clusters requiring a combination of at least three discrete gene expression markers to uniquely define them. Using these data, we developed a framework for genetically accessing each cell type, comprehensively characterized neuropeptide and neurotransmitter signalling, elucidated region-specific specializations in activity-regulated gene expression and ascertained the heritability enrichment of neurological and psychiatric phenotypes. These data, available as an online resource ( www.BrainCellData.org ), should find diverse applications across neuroscience, including the construction of new genetic tools and the prioritization of specific cell types and circuits in the study of brain diseases.

Brain-wide correspondence of neuronal epigenomics and distant projections.

Single-cell analyses parse the brain's billions of neurons into thousands of 'cell-type' clusters residing in different brain structures1. Many cell types mediate their functions through targeted long-distance projections allowing interactions between specific cell types. Here we used epi-retro-seq2 to link single-cell epigenomes and cell types to long-distance projections for 33,034 neurons dissected from 32 different regions projecting to 24 different targets (225 source-to-target combinations) across the whole mouse brain. We highlight uses of these data for interrogating principles relating projection types to transcriptomics and epigenomics, and for addressing hypotheses about cell types and connections related to genetics. We provide an overall synthesis with 926 statistical comparisons of discriminability of neurons projecting to each target for every source. We integrate this dataset into the larger BRAIN Initiative Cell Census Network atlas, composed of millions of neurons, to link projection cell types to consensus clusters. Integration with spatial transcriptomics further assigns projection-enriched clusters to smaller source regions than the original dissections. We exemplify this by presenting in-depth analyses of projection neurons from the hypothalamus, thalamus, hindbrain, amygdala and midbrain to provide insights into properties of those cell types, including differentially expressed genes, their associated cis-regulatory elements and transcription-factor-binding motifs, and neurotransmitter use.

Chiari III malformation in a neonate.

Chiari malformation (CM) is a group of complex deformities of the posterior fossa and hindbrain, of which CMIII is the rarest. We report a term neonate, with an antenatal diagnosis of occipital encephalocele, who underwent resection of the encephalocele and ligation of vessels, with repair of a large scalp defect and dural reconstruction on day 4 of life. The parents of the child had been counselled for a guarded and poor prognosis on initial diagnosis. The child has had a good postoperative course without complications but suffers from cortical visual impairment and global developmental delay.

scMultiome analysis identifies embryonic hindbrain progenitors with mixed rhombomere identities.

Rhombomeres serve to position neural progenitors in the embryonic hindbrain, thereby ensuring appropriate neural circuit formation, but the molecular identities of individual rhombomeres and the mechanism whereby they form has not been fully established. Here, we apply scMultiome analysis in zebrafish to molecularly resolve all rhombomeres for the first time. We find that rhombomeres become molecularly distinct between 10hpf (end of gastrulation) and 13hpf (early segmentation). While the embryonic hindbrain transiently contains alternating odd- versus even-type rhombomeres, our scMultiome analyses do not detect extensive odd versus even molecular characteristics in the early hindbrain. Instead, we find that each rhombomere displays a unique gene expression and chromatin profile. Prior to the appearance of distinct rhombomeres, we detect three hindbrain progenitor clusters (PHPDs) that correlate with the earliest visually observed segments in the hindbrain primordium that represent prospective rhombomere r2/r3 (possibly including r1), r4, and r5/r6, respectively. We further find that the PHPDs form in response to Fgf and RA morphogens and that individual PHPD cells co-express markers of multiple mature rhombomeres. We propose that the PHPDs contain mixed-identity progenitors and that their subdivision into individual rhombomeres requires the resolution of mixed transcription and chromatin states.

Hindbrain Adrenergic/Noradrenergic Control of Integrated Endocrine and Autonomic Stress Responses.

Hindbrain adrenergic/noradrenergic nuclei facilitate endocrine and autonomic responses to physical and psychological challenges. Neurons that synthesize adrenaline and noradrenaline target hypothalamic structures to modulate endocrine responses while descending spinal projections regulate sympathetic function. Furthermore, these neurons respond to diverse stress-related metabolic, autonomic, and psychosocial challenges. Accordingly, adrenergic and noradrenergic nuclei are integrative hubs that promote physiological adaptation to maintain homeostasis. However, the precise mechanisms through which adrenaline- and noradrenaline-synthesizing neurons sense interoceptive and exteroceptive cues to coordinate physiological responses have yet to be fully elucidated. Additionally, the regulatory role of these cells in the context of chronic stress has received limited attention. This mini-review consolidates reports from preclinical rodent studies on the organization and function of brainstem adrenaline and noradrenaline cells to provide a framework for how these nuclei coordinate endocrine and autonomic physiology. This includes identification of hindbrain adrenaline- and noradrenaline-producing cell groups and their role in stress responding through neurosecretory and autonomic engagement. Although temporally and mechanistically distinct, the endocrine and autonomic stress axes are complementary and interconnected. Therefore, the interplay between brainstem adrenergic/noradrenergic nuclei and peripheral physiological systems is necessary for integrated stress responses and organismal survival.

Deficiency of the Heterogeneous Nuclear Ribonucleoprotein U locus leads to delayed hindbrain neurogenesis.

Genetic variants affecting Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU) have been identified in several neurodevelopmental disorders (NDDs). HNRNPU is widely expressed in the human brain and shows the highest postnatal expression in the cerebellum. Recent studies have investigated the role of HNRNPU in cerebral cortical development, but the effects of HNRNPU deficiency on cerebellar development remain unknown. Here, we describe the molecular and cellular outcomes of HNRNPU locus deficiency during in vitro neural differentiation of patient-derived and isogenic neuroepithelial stem cells with a hindbrain profile. We demonstrate that HNRNPU deficiency leads to chromatin remodeling of A/B compartments, and transcriptional rewiring, partly by impacting exon inclusion during mRNA processing. Genomic regions affected by the chromatin restructuring and host genes of exon usage differences show a strong enrichment for genes implicated in epilepsies, intellectual disability, and autism. Lastly, we show that at the cellular level HNRNPU downregulation leads to an increased fraction of neural progenitors in the maturing neuronal population. We conclude that the HNRNPU locus is involved in delayed commitment of neural progenitors to differentiate in cell types with hindbrain profile.

Dimensionality reduction reveals separate translation and rotation populations in the zebrafish hindbrain.

In many brain areas, neuronal activity is associated with a variety of behavioral and environmental variables. In particular, neuronal responses in the zebrafish hindbrain relate to oculomotor and swimming variables as well as sensory information. However, the precise functional organization of the neurons has been difficult to unravel because neuronal responses are heterogeneous. Here, we used dimensionality reduction methods on neuronal population data to reveal the role of the hindbrain in visually driven oculomotor behavior and swimming. We imaged neuronal activity in zebrafish expressing GCaMP6s in the nucleus of almost all neurons while monitoring the behavioral response to gratings that rotated with different speeds. We then used reduced-rank regression, a method that condenses the sensory and motor variables into a smaller number of "features," to predict the fluorescence traces of all ROIs (regions of interest). Despite the potential complexity of the visuo-motor transformation, our analysis revealed that a large fraction of the population activity can be explained by only two features. Based on the contribution of these features to each ROI's activity, ROIs formed three clusters. One cluster was related to vergent movements and swimming, whereas the other two clusters related to leftward and rightward rotation. Voxels corresponding to these clusters were segregated anatomically, with leftward and rightward rotation clusters located selectively to the left and right hemispheres, respectively. Just as described in many cortical areas, our analysis revealed that single-neuron complexity co-exists with a simpler population-level description, thereby providing insights into the organization of visuo-motor transformations in the hindbrain.

GABA-immunoreactive neurons in the Central Nervous System of the viviparous teleost Poecilia sphenops.

Gamma-aminobutyric acid (GABA) functions as the primary inhibitory neurotransmitter within the central nervous system (CNS) of vertebrates. In this study, we examined the distribution pattern of GABA-immunoreactive (GABA-ir) cells and fibres in the CNS of the viviparous teleost Poecilia sphenops using immunofluorescence method. GABA immunoreactivity was seen in the glomerular, mitral, and granular layers of the olfactory bulbs, as well as in most parts of the dorsal and ventral telencephalon. The preoptic area consisted of a small cluster of GABA-ir cells, whereas extensively labelled GABA-ir neurons were observed in the hypothalamic areas, including the paraventricular organ, tuberal hypothalamus, nucleus recessus lateralis, nucleus recessus posterioris, and inferior lobes. In the thalamus, GABA-positive neurons were only found in the ventral thalamic and central posterior thalamic nuclei, whereas the dorsal part of the nucleus pretectalis periventricularis consisted of a few GABA-ir cells. GABA-immunoreactivity was extensively seen in the alar and basal subdivisions of the midbrain, whereas in the rhombencephalon, GABA-ir cells and fibres were found in the cerebellum, motor nucleus of glossopharyngeal and vagal nerves, nucleus commissuralis of Cajal, and reticular formation. In the spinal cord, GABA-ir cells and fibres were observed in the dorsal horn, ventral horn, and around the central canal. Overall, the extensive distribution of GABA-ir cells and fibres throughout the CNS suggests several roles for GABA, including the neuroendocrine, viscerosensory, and somatosensory functions, for the first time in a viviparous teleost.

Zbtb16 mediates a switch between Fgf signalling regimes in the developing hindbrain.

Developing tissues are sequentially patterned by extracellular signals that are turned on and off at specific times. In the zebrafish hindbrain, fibroblast growth factor (Fgf) signalling has different roles at different developmental stages: in the early hindbrain, transient Fgf3 and Fgf8 signalling from rhombomere 4 is required for correct segmentation, whereas later, neuronal Fgf20 expression confines neurogenesis to specific spatial domains within each rhombomere. How the switch between these two signalling regimes is coordinated is not known. We present evidence that the Zbtb16 transcription factor is required for this transition to happen in an orderly fashion. Zbtb16 expression is high in the early anterior hindbrain, then gradually upregulated posteriorly and confined to neural progenitors. In mutants lacking functional Zbtb16, fgf3 expression fails to be downregulated and persists until a late stage, resulting in excess and more widespread Fgf signalling during neurogenesis. Accordingly, the spatial pattern of neurogenesis is disrupted in Zbtb16 mutants. Our results reveal how the distinct stage-specific roles of Fgf signalling are coordinated in the zebrafish hindbrain.

Small molecule inhibitor of tau self-association in a mouse model of tauopathy: A preventive study in P301L tau JNPL3 mice.

Advances in tau biology and the difficulties of amyloid-directed immunotherapeutics have heightened interest in tau as a target for small molecule drug discovery for neurodegenerative diseases. Here, we evaluated OLX-07010, a small molecule inhibitor of tau self-association, for the prevention of tau aggregation. The primary endpoint of the study was statistically significant reduction of insoluble tau aggregates in treated JNPL3 mice compared with Vehicle-control mice. Secondary endpoints were dose-dependent reduction of insoluble tau aggregates, reduction of phosphorylated tau, and reduction of soluble tau. This study was performed in JNPL3 mice, which are representative of inherited forms of 4-repeat tauopathies with the P301L tau mutation (e.g., progressive supranuclear palsy and frontotemporal dementia). The P301L mutation makes tau prone to aggregation; therefore, JNPL3 mice present a more challenging target than mouse models of human tau without mutations. JNPL3 mice were treated from 3 to 7 months of age with Vehicle, 30 mg/kg compound dose, or 40 mg/kg compound dose. Biochemical methods were used to evaluate self-associated tau, insoluble tau aggregates, total tau, and phosphorylated tau in the hindbrain, cortex, and hippocampus. The Vehicle group had higher levels of insoluble tau in the hindbrain than the Baseline group; treatment with 40 mg/kg compound dose prevented this increase. In the cortex, the levels of insoluble tau were similar in the Baseline and Vehicle groups, indicating that the pathological phenotype of these mice was beginning to emerge at the study endpoint and that there was a delay in the development of the phenotype of the model as originally characterized. No drug-related adverse effects were observed during the 4-month treatment period.

Noradrenergic projections regulate the acquisition of classically conditioned eyelid responses in wild-type and are impaired in kreisler mice.

During embryonic development, heterozygous mutant kreisler mice undergo ectopic expression of the Hoxa3 gene in the rostral hindbrain, affecting the opioid and noradrenergic systems. In this model, we have investigated behavioral and cognitive processes in their adulthood. We confirmed that pontine and locus coeruleus neuronal projections are impaired, by using startle and pain tests and by analyzing immunohistochemical localization of tyrosine hydroxylase. Our results showed that, even if kreisler mice are able to generate eyelid reflex responses, there are differences with wild-types in the first component of the response (R1), modulated by the noradrenergic system. The acquisition of conditioned motor responses is impaired in kreisler mice when using the trace but not the delay paradigm, suggesting a functional impairment in the hippocampus, subsequently confirmed by reduced quantification of alpha2a receptor mRNA expression in this area but not in the cerebellum. Moreover, we demonstrate the involvement of adrenergic projection in eyelid classical conditioning, as clonidine prevents the appearance of eyelid conditioned responses in wild-type mice. In addition, hippocampal motor learning ability was restored in kreisler mice by administration of adrenergic antagonist drugs, and a synergistic effect was observed following simultaneous administration of idazoxan and naloxone.