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, whose activity in time relies on retrograde neuromodulator 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-arachydonoylglycerol and anandamide signaling cassettes, with a focus on receptors and metabolic enzymes, in both molecularly-defined neurons and astrocytes. We find type 1 cannabinoid receptors (Cnr1), but neither Cnr2 nor Gpr55, expressed only in neurons of the PVN. A developmental switch from Daglb to Dagla is observed, with minimal Mgll mRNA in all neuronal subtypes of the PVN. Notably, astrocytes also expressed both Dagl isoforms. Napepld, and Faah were sparsely present. 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.