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Transcriptomic changes induced in one cell type by another mediate many biological processes in the brain and elsewhere; however, achieving artifact-free physical separation of cell types to study them is challenging and generally allows for analysis of only a single cell type. We describe an approach using a co-culture of distinct cell types from different species that enables physical cell sorting to be replaced by in silico RNA sequencing (RNA-seq) read sorting, which is possible because of evolutionary divergence of messenger RNA (mRNA) sequences. As an exemplary experiment, we describe the co-culture of purified neurons, astrocytes, and microglia from different species (12–14 d). We describe how to use our Python tool, Sargasso, to separate the reads from conventional RNA-seq according to species and to eliminate any artifacts borne of imperfect genome annotation (10 h). We show how this procedure, which requires no special skills beyond those that might normally be expected of wet lab and bioinformatics researchers, enables the simultaneous transcriptomic profiling of different cell types, revealing the distinct influence of microglia on astrocytic and neuronal transcriptomes under inflammatory conditions.

Microglia, brain-resident macrophages, require instruction from the CNS microenvironment to maintain their identity and morphology and regulate inflammatory responses, although what mediates this is unclear. Here, we show that neurons and astrocytes cooperate to promote microglial ramification, induce expression of microglial signature genes ordinarily lost in vitroand in age and disease in vivo, and repress infection- and injury-associated gene sets. The influence of neurons and astrocytes separately on microglia is weak, indicative of synergies between these cell types, which exert their effects via a mechanism involving transforming growth factor β2 (TGF-β2) signaling. Neurons and astrocytes also combine to provide immunomodulatory cues, repressing primed microglial responses to weak inflammatory stimuli (without affecting maximal responses) and consequently limiting the feedback effects of inflammation on the neurons and astrocytes themselves. These findings explain why microglia isolated ex vivoundergo de-differentiation and inflammatory deregulation and point to how disease- and age-associated changes may be counteracted.

Cellular hyperexcitability is a salient feature of fragile X syndrome animal models. The cellular basis of hyperexcitability and how it responds to changing activity states is not fully understood. Here, we show increased axon initial segment length in CA1 of the Fmr1−/ymouse hippocampus, with increased cellular excitability. This change in length does not result from reduced AIS plasticity, as prolonged depolarization induces changes in AIS length independent of genotype. However, depolarization does reduce cellular excitability, the magnitude of which is greater in Fmr1−/yneurons. Finally, we observe reduced functional inputs from the entorhinal cortex, with no genotypic difference in the firing rates of CA1 pyramidal neurons. This suggests that AIS-dependent hyperexcitability in Fmr1−/ymice may result from adaptive or homeostatic regulation induced by reduced functional synaptic connectivity. Thus, while AIS length and intrinsic excitability contribute to cellular hyperexcitability, they may reflect a homeostatic mechanism for reduced synaptic input onto CA1 neurons.

The GluN2 subtype (2A versus 2B) determines biophysical properties and signaling of forebrain NMDA receptors (NMDARs). During development, GluN2A becomes incorporated into previously GluN2B-dominated NMDARs. This “switch” is proposed to be driven by distinct features of GluN2 cytoplasmic C-terminal domains (CTDs), including a unique CaMKII interaction site in GluN2B that drives removal from the synapse. However, these models remain untested in the context of endogenous NMDARs. We show that, although mutating the endogenous GluN2B CaMKII site has secondary effects on GluN2B CTD phosphorylation, the developmental changes in NMDAR composition occur normally and measures of plasticity and synaptogenesis are unaffected. Moreover, the switch proceeds normally in mice that have the GluN2A CTD replaced by that of GluN2B and commences without an observable decline in GluN2B levels but is impaired by GluN2A haploinsufficiency. Thus, GluN2A expression levels, and not GluN2 subtype-specific CTD-driven events, are the overriding factor in the developmental switch in NMDAR composition.