Molecular Fingerprint of Endocannabinoid Signaling in the Developing Paraventricular Nucleus of the Hypothalamus as Revealed by Single-Cell RNA-Seq and In Situ Hybridization

The paraventricular nucleus of the hypothalamus (PVN) regulates, among others, the stress response, sexual behavior, and energy metabolism through its magnocellular and parvocellular neurosecretory cells. Within the PVN, ensemble coordination occurs through the many long-range synaptic afferents, wh...

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Main Authors: Evgenii O. Tretiakov, Zsófia Hevesi, Csenge Böröczky, Alán Alpár, Tibor Harkany, Erik Keimpema
Format: Article
Language:English
Published: MDPI AG 2025-05-01
Series:Cells
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Online Access:https://www.mdpi.com/2073-4409/14/11/788
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Summary:The paraventricular nucleus of the hypothalamus (PVN) regulates, among others, the stress response, sexual behavior, and energy metabolism through its magnocellular and parvocellular neurosecretory cells. Within the PVN, ensemble coordination occurs through the many long-range synaptic afferents, whose activity in time relies on retrograde neuromodulation by, e.g., endocannabinoids. However, the nanoarchitecture of endocannabinoid signaling in the PVN, especially during neuronal development, remains undescribed. By using single-cell RNA sequencing, in situ hybridization, and immunohistochemistry during fetal and postnatal development in mice, we present a spatiotemporal map of both the 2-arachidonoylglycerol (2-AG) and anandamide (AEA) signaling cassettes, with a focus on receptors and metabolic enzymes, in both molecularly defined neurons and astrocytes. We find type 1 cannabinoid receptors (<i>Cnr1</i>), but neither <i>Cnr2</i> nor <i>Gpr55</i>, expressed in neurons of the PVN. <i>Dagla</i> and <i>Daglb</i>, which encode the enzymes synthesizing 2-AG, were found in all neuronal subtypes of the PVN, with a developmental switch from <i>Daglb</i> to <i>Dagla</i>. <i>Mgll</i>, which encodes an enzyme degrading 2-AG, was only found sporadically. <i>Napepld</i> and <i>Faah</i>, encoding enzymes that synthesize and degrade AEA, respectively, were sparsely expressed in neurons throughout development. Notably, astrocytes expressed <i>Mgll</i> and both <i>Dagl</i> isoforms. In contrast, mRNA for any of the three major cannabinoid-receptor subtypes could not be detected. Immunohistochemistry validated mRNA expression and suggested that endocannabinoid signaling is configured to modulate the activity of afferent inputs, rather than local neurocircuits, in the PVN.
ISSN:2073-4409