Cell segmentation-free inference of cell types from in situ transcriptomics data

Multiplexed fluorescence in situ hybridization techniques have enabled cell-type identification, linking transcriptional heterogeneity with spatial heterogeneity of cells. However, inaccurate cell segmentation reduces the efficacy of cell-type identification and tissue characterization. Here, we present a method called Spot-based Spatial cell-type Analysis by Multidimensional mRNA density estimation (SSAM), a robust cell segmentation-free computational framework for identifying cell-types and tissue domains in 2D and 3D. SSAM is applicable to a variety of in situ transcriptomics techniques and capable of integrating prior knowledge of cell types. We apply SSAM to three mouse brain tissue images: the somatosensory cortex imaged by osmFISH, the hypothalamic preoptic region by MERFISH, and the visual cortex by multiplexed smFISH. Here, we show that SSAM detects regions occupied by known cell types that were previously missed and discovers new cell types.

Gonadotropin Releasing Hormone (Gnrh) Triggers Neurogenesis in the Hypothalamus of Adult Zebrafish

Recently, it has been shown in adult mammals that the hypothalamus can generate new cells in response to metabolic changes, and tanycytes, putative descendants of radial glia, can give rise to neurons. Previously we have shown in vitro that neurospheres generated from the hypothalamus of adult zebrafish show increased neurogenesis in response to exogenously applied hormones. To determine whether adult zebrafish have a hormone-responsive tanycyte-like population in the hypothalamus, we characterized proliferative domains within this region. Here we show that the parvocellular nucleus of the preoptic region (POA) labels with neurogenic/tanycyte markers vimentin, GFAP/Zrf1, and Sox2, but these cells are generally non-proliferative. In contrast, Sox2+ proliferative cells in the ventral POA did not express vimentin and GFAP/Zrf1. A subset of the Sox2+ cells co-localized with Fezf2:GFP, a transcription factor important for neuroendocrine cell specification. Exogenous treatments of GnRH and testosterone were assayed in vivo. While the testosterone-treated animals showed no significant changes in proliferation, the GnRH-treated animals showed significant increases in the number of BrdU-labeled cells and Sox2+ cells. Thus, cells in the proliferative domains of the zebrafish POA do not express radial glia (tanycyte) markers vimentin and GFAP/Zrf1, and yet, are responsive to exogenously applied GnRH treatment.

Transcriptome Sequencing in the Preoptic Region of Rat Dams Reveals a Role of Androgen Receptor in the Control of Maternal Behavior

(1) Background: Preoptic region of hypothalamus is responsible to control maternal behavior, which was hypothesized to be associated with gene expressional changes. (2) Methods: Transcriptome sequencing was first applied in the preoptic region of rat dams in comparison to a control group of mothers whose pups were taken away immediately after parturition and did not exhibit caring behavior 10 days later. (3) Results: Differentially expressed genes were found and validated by quantitative RT-PCR, among them NACHT and WD repeat domain containing 1 (Nwd1) is known to control androgen receptor (AR) protein levels. The distribution of Nwd1 mRNA and AR was similar in the preoptic area. Therefore, we focused on this steroid hormone receptor and found its reduced protein level in rat dams. To establish the function of AR in maternal behavior, its antagonist was administered intracerebroventricularly into mother rats and increased pup-directed behavior of the animals. (4) Conclusions: AR levels are suppressed in the preoptic area of mothers possibly mediated by altered Nwd1 expression in order to allow sustained high-level care for the pups. Thus, our study first implicated the AR in the control of maternal behaviors.

Molecular, spatial, and functional single-cell profiling of the hypothalamic preoptic region

The hypothalamus controls essential social behaviors and homeostatic functions. However, the cellular architecture of hypothalamic nuclei—including the molecular identity, spatial organization, and function of distinct cell types—is poorly understood. Here, we developed an imaging-based in situ cell-type identification and mapping method and combined it with single-cell RNA-sequencing to create a molecularly annotated and spatially resolved cell atlas of the mouse hypothalamic preoptic region. We profiled ~1 million cells, identified ~70 neuronal populations characterized by distinct neuromodulatory signatures and spatial organizations, and defined specific neuronal populations activated during social behaviors in male and female mice, providing a high-resolution framework for mechanistic investigation of behavior circuits. The approach described opens a new avenue for the construction of cell atlases in diverse tissues and organisms.

Role of an excitatory preoptic-raphé pathway in febrile vasoconstriction of the rat's tail

Heat dissipation from the rat's tail is reduced in response to cold and during fever. The sympathetic premotor neurons for this mechanism, located in the medullary raphé, are under tonic inhibitory control from the preoptic area. In parallel with the inhibitory pathway, an excitatory pathway from the rostromedial preoptic region (RMPO) to the medullary raphé mediates the vasoconstrictor response to cold skin. Whether this applies also to the tail vasoconstrictor response in fever is unknown. Single- or a few-unit tail sympathetic nerve activity (SNA) was recorded in urethane-anesthetized, artificially ventilated rats. Experimental fever was induced by PGE2 injected into the lateral cerebral ventricle (50 ng in 1.5 μl icv) or into the RMPO (0.2 ng in 60 nl); in both cases, there was a robust increase in tail SNA and a delayed rise in core temperature. Microinjection of glutamate receptor antagonist kynurenate (50 mM, 120 nl) into the medullary raphé completely reversed the tail SNA response to intracerebroventricular or RMPO PGE2 injection. Inhibiting RMPO neurons by microinjecting glycine (0.5 M, 60 nl) or the GABAA receptor agonist, muscimol (2 mM, 30–60 nl), reduced the tail SNA response to PGE2 injected into the same site by approximately half. Vehicle injections into the medullary raphé or RMPO were without effect. These results suggest that the tail vasoconstrictor response during experimental fever depends on a glutamatergic excitatory synaptic relay in the medullary raphé and that an excitatory output signal from the RMPO contributes to the tail vasoconstrictor response during fever.