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.

We report that, in the rat, administering insulin-like growth factor II (IGF-II) significantly enhances memory retention and prevents forgetting. Inhibitory avoidance learning leads to an increase in hippocampal expression of IGF-II, which requires the transcription factor CCAAT enhancer binding protein β and is essential for memory consolidation. Furthermore, injections of recombinant IGF-II into the hippocampus after either training or memory retrieval significantly enhance memory retention and prevent forgetting. To be effective, IGF-II needs to be administered within a sensitive period of memory consolidation. IGF-II-dependent memory enhancement requires IGF-II receptors, new protein synthesis, the function of activity-regulated cytoskeletal-associated protein and glycogensynthase kinase 3 (GSK3). Moreover, it correlates with a significant activation of synaptic GSK3β and expression of GluR1 a-amino-3-hydroxy-5-methyl-4-isoxasoleproprionic acid receptor subunits. In hippocampal slices, IGF-II promotes IGF-II receptor-dependent, persistent long-term potentiation after weak synaptic stimulation. Thus, IGF-II may represent a novel target for cognitive enhancement therapies.

Highly multiplexed single-molecule FISH has emerged as a promising approach to spatially resolved single-cell transcriptomics because of its ability to directly image and profile numerous RNA species in their native cellular context. However, backgroundfrom off-target binding of FISH probes and cellular autofluorescence-can become limiting in a number of important applications, such as increasing the degree of multiplexing, imaging shorter RNAs, and imaging tissue samples. Here, we developed a sample clearing approach for FISH measurements. We identified off-target binding of FISH probes to cellular components other than RNA, such as proteins, as a major source of background. To remove this source of background, we embedded samples in polyacrylamide, anchored RNAs to this polyacrylamide matrix, and cleared cellular proteins and lipids, which are also sources of autofluorescence. To demonstrate the efficacy of this approach, we measured the copy number of 130 RNA species in cleared samples using multiplexed error-robust FISH (MERFISH). We observed a reduction both in the background because of off-target probe binding and in the cellular autofluorescence without detectable loss in RNA. This process led to an improved detection efficiency and detection limit of MERFISH, and an increased measurement throughput via extension of MERFISH into four color channels. We further demonstrated MERFISH measurements of complex tissue samples from the mouse brain using this matrix-imprinting and -clearing approach. We envision that this method will improve the performance of a wide range of in situ hybridization-based techniques in both cell culture and tissues.tissue clearing | fluorescence in situ hybridization | multiplexed imaging | single-cell transcriptomics | brain S ingle-molecule FISH (smFISH) is a powerful technique that allows the direct imaging of individual RNA molecules within single cells (1, 2). In this approach, RNAs are labeled via the hybridization of fluorescently labeled oligonucleotide probes, producing bright fluorescent spots for single RNA molecules, which reveal both the abundance and the spatial distribution of these RNAs inside cells (1, 2). The ability of smFISH to image gene expression at the single-cell level in both cell culture and tissue has led to exciting advances in our understanding of the natural noise in gene expression and its role in cellular response (3, 4), the intracellular spatial organization of RNAs and its role in posttranscriptional regulation (5, 6), and the spatial variation in gene expression within complex tissues and its role in the molecular definition of cell types and tissue functions (6, 7).To extend the benefits of this technique to systems-level questions and high-throughput gene-expression profiling, approaches to increase the multiplexing of smFISH (i.e., the number of different RNA species that can be simultaneously quantified within the same cell) have been developed (8-13). Most of these approaches take advantage of color multiplexing, which has allowed a few tens...