A stochastic RNA editing process targets a limited number of sites in individual Drosophila glutamatergic motoneurons.

RNA editing is a post-transcriptional source of protein diversity and occurs across the animal kingdom. Given the complete profile of mRNA targets and their editing rate in individual cells is unclear, we analyzed single cell RNA transcriptomes from Drosophila larval tonic and phasic glutamatergic motoneuron subtypes to determine the most highly edited targets and identify cell-type specific editing. From ∼15,000 genes encoded in the genome, 316 high confidence A-to-I canonical RNA edit sites were identified, with 102 causing missense amino acid changes in proteins regulating membrane excitability, synaptic transmission, and cellular function. Some sites showed 100% editing in single neurons as observed with mRNAs encoding mammalian AMPA receptors. However, most sites were edited at lower levels and generated variable expression of edited and unedited mRNAs within individual neurons. Together, these data provide insights into how the RNA editing landscape alters protein function to modulate the properties of two well-characterized neuronal populations in Drosophila .

Molecular Logic of Synaptic Diversity Between Drosophila Tonic and Phasic Motoneurons.

Although neuronal subtypes display unique synaptic organization and function, the underlying transcriptional differences that establish these features is poorly understood. To identify molecular pathways that contribute to synaptic diversity, single neuron PatchSeq RNA profiling was performed on Drosophila tonic and phasic glutamatergic motoneurons. Tonic motoneurons form weaker facilitating synapses onto single muscles, while phasic motoneurons form stronger depressing synapses onto multiple muscles. Super-resolution microscopy and in vivo imaging demonstrated synaptic active zones in phasic motoneurons are more compact and display enhanced Ca 2+ influx compared to their tonic counterparts. Genetic analysis identified unique synaptic properties that mapped onto gene expression differences for several cellular pathways, including distinct signaling ligands, post-translational modifications and intracellular Ca 2+ buffers. These findings provide insights into how unique transcriptomes drive functional and morphological differences between neuronal subtypes.

Molecular logic of synaptic diversity between Drosophila tonic and phasic motoneurons.

Although neuronal subtypes display unique synaptic organization and function, the underlying transcriptional differences that establish these features are poorly understood. To identify molecular pathways that contribute to synaptic diversity, single-neuron Patch-seq RNA profiling was performed on Drosophila tonic and phasic glutamatergic motoneurons. Tonic motoneurons form weaker facilitating synapses onto single muscles, while phasic motoneurons form stronger depressing synapses onto multiple muscles. Super-resolution microscopy and in vivo imaging demonstrated that synaptic active zones in phasic motoneurons are more compact and display enhanced Ca2+ influx compared with their tonic counterparts. Genetic analysis identified unique synaptic properties that mapped onto gene expression differences for several cellular pathways, including distinct signaling ligands, post-translational modifications, and intracellular Ca2+ buffers. These findings provide insights into how unique transcriptomes drive functional and morphological differences between neuronal subtypes.

Interactions with Astroglia Influence the Shape of the Developing Dendritic Arbor and Restrict Dendrite Growth Independent of Promoting Synaptic Contacts.

Astroglia play key roles in the development of neurons, ranging from regulating neuron survival to promoting synapse formation, yet basic questions remain about whether astrocytes might be involved in forming the dendritic arbor. Here, we used cultured hippocampal neurons as a simple in vitro model that allowed dendritic growth and geometry to be analyzed quantitatively under conditions where the extent of interactions between neurons and astrocytes varied. When astroglia were proximal to neurons, dendrites and dendritic filopodia oriented toward them, but the general presence of astroglia significantly reduced overall dendrite growth. Further, dendritic arbors in partial physical contact with astroglia developed a pronounced pattern of asymmetrical growth, because the dendrites in direct contact were significantly smaller than the portion of the arbor not in contact. Notably, thrombospondin, the astroglial factor shown previously to promote synapse formation, did not inhibit dendritic growth. Thus, while astroglia promoted the formation of presynaptic contacts onto dendrites, dendritic growth was constrained locally within a developing arbor at sites where dendrites contacted astroglia. Taken together, these observations reveal influences on spatial orientation of growth as well as influences on morphogenesis of the dendritic arbor that have not been previously identified.