The cellular basis of distinct thirst modalities

We performed single-cell RNA-seq (10x Chromium 3' v.2) on dissociated single cell suspensions from mouse (C57BL/6) osmosensory brain nuclei - subfornical organ (SFO) and organum vasculosum lamina terminalis (OVLT) to determine transcriptomic cell types in each structure. We identified 12 and 13 major cell classes as well as 8 neuron types in each for SFO and OVLT respectively. Furthermore, we studied how different types of thirst signals (osmotic thirst, hypovolemic thirst, chronic water deprivation) engage this cellular diversity by performing single cell RNA-seq based (10x Chromium 3' v.3.0) stimulus to cell-type mapping, where dissociation related transcriptional artifacts were suppressed through a modified tissue preparation protocol involving a transcriptional blocker and dissociation at room temperature. This approach revealed the endogenous immediate early gene expression triggered by distinct thirst stimuli in SFO and OVLT cells. By analyzing immediate early gene expression that is driven by changes in neural activity (Fos) we identified excitatory neuron types in SFO and OVLT that are uniquely tuned to distinct thirst states. To determine transcriptomic cell types in the SFO and OVLT we used 8 and 12 male mice respectively. For stimulus to cell-type mapping experiments we used 12 animals per condition. Both male and female animals were used in equal measure. All animals were 7.5-8 weeks of age. Prior to tissue extraction, animals were subjected to one of the following experimental treatments: a) wated satiation: ad libitum access to food and water); b) osmotic thirst: intraperitoneal (i.p.) injection of 2 M NaCl (5 μl/g bodyweight) followed by sacking 1 hour after injection; c) hypovolemic thist: i.p. injection of furosemide at a dose of 50 mg/kg bodyweight followed by sacking 4 hours after injection; d) chronic water deprivation: 36H water deprivation with food.