We show that the G protein‐coupled receptor GPR37‐like 1 (GPR37L1) is expressed in most astrocytes and some oligodendrocyte precursors in the mouse central nervous system. This contrasts with GPR37, which is mainly in mature oligodendrocytes. Comparison of wild type and Gpr37l1–/– mice showed that loss of GPR37L1 did not affect the input resistance or resting potential of astrocytes or neurons in the hippocampus. However, GPR37L1‐mediated signalling inhibited astrocyte glutamate transporters and – surprisingly, given its lack of expression in neurons – reduced neuronal NMDA receptor (NMDAR) activity during prolonged activation of the receptors as occurs in ischemia. This effect on NMDAR signalling was not mediated by a change in the release of D‐serine or TNF‐α, two astrocyte‐derived agents known to modulate NMDAR function. After middle cerebral artery occlusion, Gpr37l1 expression was increased around the lesion. Neuronal death was increased by ∼40% in Gpr37l1–/– brain compared to wild type in an in vitro model of ischemia. Thus, GPR37L1 protects neurons during ischemia, presumably by modulating extracellular glutamate concentration and NMDAR activation.

Astrocytes and one of their products, IL-6, not only support neurons but also mediate inflammation in the brain. Retinoidrelated orphan receptor-␣ (ROR␣) transcription factor has related roles, being neuro-protective and, in peripheral tissues, antiinflammatory. We examined the relation of ROR␣ to astrocytes and IL-6 using normal and ROR␣ loss-of-function mutant mice. We have shown ROR␣ expression in astrocytes and its up-regulation by pro-inflammatory cytokines. We have also demonstrated that ROR␣ directly trans-activates the Il-6 gene. We suggest that this direct control is necessary to maintain IL-6 basal level in the brain and may be a link between the neuro-supportive roles of ROR␣, IL-6, and astrocytes. Furthermore, after inflammatory stimulation, the absence of ROR␣ results in excessive IL-6 up-regulation, indicating that ROR␣ exerts an indirect repression probably via the inhibition of the NF-B signaling. Thus, our findings indicate that ROR␣ is a pluripotent molecular player in constitutive and adaptive astrocyte physiology.inflammation ͉ staggerer ͉ microglia

RNA analysis at the cellular resolution in the human brain is challenging. Here, we describe an optimised approach for detecting single RNA transcripts in a cell-type specific manner in frozen human brain tissue using multiplexed fluorescent RNAscope probes. We developed a new robust analytical approach for RNAscope quantification. Our method shows that low RNA integrity does not significantly affect RNAscope signal, recapitulates bulk RNA analysis and provides spatial context to transcriptomic analysis of human post-mortem brain at single-cell resolution. In summary, our optimised method allows the usage of frozen human samples from brain banks to perform quantitative RNAscope analysis. Molecular characterisation of different cell-types in complex human tissues such as the brain is of crucial importance for both fundamental and applied research. The human brain is composed of potentially hundreds of different cell types displaying complex morphologies and often long, ramified and entwined processes, making its dissociation into single cells a difficult process. RNA-seq of immunopanned human brain cell types has been performed from fresh biopsies obtained from neurosurgical site 1 , but these samples are non-existent for neurodegenerative diseases; instead, neuroscientists normally access frozen or fixed post-mortem human brain samples from brain banks. However, single-cell preparation from snap frozen tissue is extremely difficult, thus making single-cell sorting virtually impossible. To overcome these limitations, several laboratories have performed single-nucleus RNA-seq. This method can be readily applied to frozen tissue samples and it has been shown to well recapitulate the cellular transcriptome 2,3. Nonetheless, this method has its own limitations, notably the loss of any compartmentalised cytoplasmic RNA 3. Single-cell or single-nucleus-based transcriptomics fail to capture spatial context, cell-to-cell relationships and circuitry structure. In situ hybridisation (ISH), which visualises specific mRNA molecules at a cellular level, is ideal to study gene expression in different cell types because it uses intact tissue sections, therefore maintaining tissue integrity, cell structure and morphology 4,5. Additionally, ISH has been successfully used for systematic analyses of RNA expression in the rodent brain (Allen Brain Atlas). Over the last decade, new and more sensitive ISH methods have been developed 4 , including single molecule RNA fluorescent in situ hybridization (smFISH) 6,7 , enabling quantification of RNA expression levels in specific cell types at single-cell resolution. Crucially, post-mortem human brain tissue presents higher rates of RNA degradation 8 , making bulk and single-cell or single-nuclei RNA analyses more complex. Alzheimer's disease (AD) is a devastating neurodegenerative disorder that accounts for two thirds of total dementia cases, and is characterised by progressive cognitive impairment and memory loss leading to difficulties in daily tasks 9. The AD brain displays extracellular...