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Brain nuclei are traditionally defined by their anatomy, activity, and expression of specific markers. The hypothalamus contains discrete neuronal populations that coordinate fundamental behavioral functions, including sleep and wakefulness, in all vertebrates. Particularly, the diverse roles of hypocretin/orexin (Hcrt)-releasing neurons suggest functional heterogeneity among Hcrt neurons. Using single-cell RNA sequencing (scRNA-seq) and high-resolution imaging of the adult male and female zebrafish hypothalamic periventricular zone, we identified 21 glutamatergic and 28 GABAergic cell types. Integration of zebrafish and mouse scRNA-seq revealed evolutionary conserved and divergent hypothalamic cell types. The expression of specific genes, including npvf, which encodes a sleep-regulating neuropeptide, was enriched in subsets of glutamatergic Hcrt neurons in both larval and adult zebrafish. The genetic profile, activity, and neurite processing of the neuronal subpopulation that coexpresses both Hcrt and Npvf (Hcrt+Npvf+) differ from other Hcrt neurons. These interspecies findings provide a unified annotation of hypothalamic cell types and suggest that the heterogeneity of Hcrt neurons enables multifunctionality, such as consolidation of both wake and sleep by the Hcrt- and Npvf-releasing neuronal subpopulation. [ABSTRACT FROM AUTHOR]

Thyroid hormones (THs), thyroxine (T4), and triiodothyronine (T3), regulate growth, metabolism, and neurodevelopment. THs secretion is controlled by the pituitary thyroid-stimulating hormone (TSH) and the hypothalamic–pituitary–thyroid (HPT) axis. The organic anion-transporting polypeptide 1C1 (OATP1C1/SLCO1C1) and the monocarboxylate transporter 8 (MCT8/SLC16A2) actively transport THs, which bind to their nuclear receptors and induce gene expression. A mutation in OATP1C1 is associated with brain hypometabolism, gradual neurodegeneration, and impaired cognitive and motor functioning in adolescent patients. To understand the role of Oatp1c1 and the mechanisms of the disease, we profiled the transcriptome of oatp1c1 mutant (oatp1c1−/−) and mct8−/−xoatp1c1−/− adult male and female zebrafish brains. Among dozens of differentially expressed genes, agouti-related neuropeptide 1 (agrp1) expression increased in oatp1c1−/− adult brains. Imaging in the hypothalamus revealed enhanced proliferation of Agrp1 neurons in oatp1c1−/− larvae and adults, and increased food consumption in oatp1c1−/− larvae. Similarly, feeding and the number of Agrp1 neurons increased in thyroid gland-ablated zebrafish. Pharmacological treatments showed that the T3 analog TRIAC (3,3′,5-tri-iodothyroacetic acid), but not T4, normalized the number of Agrp1 neurons in oatp1c1−/− zebrafish. Since the HPT axis is hyperactive in the oatp1c1−/− brain, we used the CRISPR-Cas9 system to knockdown tsh in oatp1c1−/− larvae, and inducibly enhanced the HPT axis in wild-type larvae. These manipulations showed that Tsh promotes proliferation of Agrp1 neurons and increases food consumption in zebrafish. The results revealed upregulation of both the HPT axis–Agrp1 circuitry and feeding in a zebrafish model for OATP1C1 deficiency. [ABSTRACT FROM AUTHOR]

Background: The thyroid hormones (THs) triiodothyronine (T3) and thyroxine (T4) are crucial regulators of brain development and function. Cell-specific transporter proteins facilitate TH uptake and efflux across the cell membrane, and insufficient TH transport causes hypothyroidism and mental retardation. Mutations in the TH transporters monocarboxylate transporter 8 (MCT8, SLC16A2) and the organic anion-transporting polypeptide 1C1 (OATP1C1, SLCO1C1) are associated with the psychomotor retardation Allan-Herndon-Dudley syndrome and juvenile neurodegeneration, respectively. Methods: To understand the mechanisms and test potential treatments for the recently discovered OATP1C1 deficiency, we established an oatp1c1 mutant (oatp1c1−/−) zebrafish. Results:oatp1c1 is expressed in endothelial cells, neurons, and astrocytes in zebrafish. The activity of the hypothalamic–pituitary–thyroid axis and behavioral locomotor activity increased in oatp1c1−/− larvae. Neuropathological analysis revealed structural alteration in radial glial cells and shorter neuronal axons in oatp1c1−/− larvae and adults. Notably, oatp1c1−/− and oatp1c1−/−Xmct8−/− adults exhibit an enlarged thyroid gland (goiter). Pharmacological assays showed that TH analogs, but not THs, can reduce the size and improve the color of the thyroid gland in adult mutant zebrafish. Conclusion: These results establish a vertebrate model for OATP1C1 deficiency that demonstrates endocrinological, neurological, and behavioral alterations mimicking findings observed in an OATP1C1-deficient patient. Further, the curative effect of TH analogs in the oatp1c1−/− zebrafish model may provide a lead toward a treatment modality in human patients. [ABSTRACT FROM AUTHOR]