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Interaction between hypothalamic dorsomedial nucleus and the suprachiasmatic nucleus determines intensity of food anticipatory behavior.

ABSTRACT: Food anticipatory behavior (FAA) is induced by limiting access to food for a few hours daily. Animals anticipate this scheduled meal event even without the suprachiasmatic nucleus (SCN), the biological clock. Consequently, a food-entrained oscillator has been proposed to be responsible for meal time estimation. Recent studies suggested the dorsomedial hypothalamus (DMH) as the site for this food-entrained oscillator, which has led to considerable controversy in the literature. Herein we demonstrate by means of c-Fos immunohistochemistry that the neuronal activity of the suprachiasmatic nucleus (SCN), which signals the rest phase in nocturnal animals, is reduced when animals anticipate the scheduled food and, simultaneously, neuronal activity within the DMH increases. Using retrograde tracing and confocal analysis, we show that inhibition of SCN neuronal activity is the consequence of activation of GABA-containing neurons in the DMH that project to the SCN. Next, we show that DMH lesions result in a loss or diminution of FAA, simultaneous with increased activity in the SCN. A subsequent lesion of the SCN restored FAA. We conclude that in intact animals, FAA may only occur when the DMH inhibits the activity of the SCN, thus permitting locomotor activity. As a result, FAA originates from a neuronal network comprising an interaction between the DMH and SCN. Moreover, this study shows that the DMH-SCN interaction may serve as an intrahypothalamic system to gate activity instead of rest overriding circadian predetermined temporal patterns.

SUBMITTER: Acosta-Galvan G

PROVIDER: S-EPMC3078408 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Interaction between hypothalamic dorsomedial nucleus and the suprachiasmatic nucleus determines intensity of food anticipatory behavior.

Acosta-Galvan Guadalupe G Yi Chun-Xia CX van der Vliet Jan J Jhamandas Jack H JH Panula Pertti P Angeles-Castellanos Manuel M Del Carmen Basualdo María M Escobar Carolina C Buijs Ruud M RM

Proceedings of the National Academy of Sciences of the United States of America 20110314 14

Food anticipatory behavior (FAA) is induced by limiting access to food for a few hours daily. Animals anticipate this scheduled meal event even without the suprachiasmatic nucleus (SCN), the biological clock. Consequently, a food-entrained oscillator has been proposed to be responsible for meal time estimation. Recent studies suggested the dorsomedial hypothalamus (DMH) as the site for this food-entrained oscillator, which has led to considerable controversy in the literature. Herein we demonstr ...[more]

PMID: 21402951

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Project description:Single-cell RNA-seq data from the mouse Dorsomedial Hypothalamic Nucleus (DMH) was generated following a modified protocol from https://www.nature.com/articles/s41586-020-2821-8. Briefly, six 7.5 week old C57B6 mice (3 male, 3 female) were anaesthetized with isoflurane. Brains were rapidly extracted and dropped into ice-cold carbogenated NMDG-HEPES-ACSF. Brain sections (2 mm) containing DMH were cut with a razor blade on a stainless steel brain matrix (51392, Stoelting) and transferred to a dissection dish on ice containing NMDG-HEPES-ACSF. DMH was bilaterally microdissected in ~700·700·350 micron tissue block. Microdissected tissue was aggregated in a collection tube on ice containing NMDG-HEPES-ACSF and 30 μM actinomycin D. For enzymatic tissue digestion, NMDG-HEPES-ACSF was replaced by trehalose-HEPES-ACSF containing papain (50 U/ml; P3125, Sigma Aldrich, pre-activated with 2.5 mM cysteine and a 30-min incubation at 34 °C and supplemented with 0.5 mM EDTA) and 15 μM actinomycin D. Extracted DMH tissue was incubated at room temperature with gentle carbogenation for 55 min. During enzymatic digestion the tissue was pipetted periodically every 10 min. At the end of enzymatic digestion, the medium was replaced with 200μl of room temperature trehalose-HEPES-ACSF containing 3 mg/ml ovomucoid inhibitor (OI-BSA,Worthington)25 U/ml DNase I (90083, Thermo Scientific) and 15 μM actinomycin D and tissue was gently triturated into a uniform single-cell suspension with fire-polished glass Pasteur pipettes. The resulting cell suspension volume was brought up to 1 ml with trehalose-HEPES-ACSF with 3 mg/ml ovomucoid inhibitor and pipetted through a 40μm cell strainer. Single-cell suspension was centrifuged down at 300g for 5 min at 4 °C and the supernatant was replaced with 1 ml fresh ice-cold trehalose-HEPES-ACSF and the cell pellet was resuspended. Cells were pelleted again and resuspended in 100 μl of ice-cold resuspension-ACSF. Cell suspensions were kept on ice while cell densities were quantified with a haemocytometer and the final cell densities were verified to be in the range of 300–1,000 cells/μl. Cell suspension volumes estimated to retrieve ~10,000 single-cell transcriptomes were added to the 10x Genomics RT reaction mix and loaded to the 10X Next GEM Chip G (2000177, 10x Genomics) per the manufacturer’s protocol. We used the Chromium Next GEM Single Cell 3’ Reagents Kit v3.1 (1000128, 10x Genomics) and the Single Index Kit Set A (100213) to prepare Illumina sequencing libraries downstream of reverse transcription following the manufacturer’s protocol. Resulting scRNA-seq sequencing library was sequenced on an Illumina NovaSeq 6000 sequencers (paired-end 150). Sequencing data were aligned to optimized pre-mRNA reference transcriptome52 and digital gene-cell matrices were generated with the 10x Genomics Cell Ranger v.6.0.0 count pipeline. The resulting scRNA-seq data were analyzed in R (4.1.2) using Seurat (v.4.1.0.9007) as previously described51. Briefly, expression data were filtered to exclude cells with fewer than 1,000 unique transcripts as well as cells exhibiting more than 15% of mitochondrial transcripts. We followed the standard Seurat workflow to identify transcriptomic cell-types and assigned cel class identities based on canonical cell-type markers: Ndrg4 for neurons, Ntsr2 for astrocytes, Slco1c1 for endothelial cells, Fcer1g for microglia and Mag for oligodendrocytes. We digitally extracted neuronal clusters from our dataset resulting in expression data for 4587 DMH neurons that we used to establish an unbiased neuronal nomenclature for DMH.

Dataset Information

Interaction between hypothalamic dorsomedial nucleus and the suprachiasmatic nucleus determines intensity of food anticipatory behavior.

Publications

Interaction between hypothalamic dorsomedial nucleus and the suprachiasmatic nucleus determines intensity of food anticipatory behavior.

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