Molecular characterization of projection neuron subtypes in the mouse olfactory bulb

Abstract

Projection neurons in the mammalian olfactory bulb (OB) not only receive direct input from the nose but also directly project to diverse cortical and subcortical areas. While morphological and physiological studies have highlighted functional heterogeneity, no molecular markers have been described that could reliably delineate subtypes of projection neurons. Here, we used viral injections into olfactory cortex and fluorescent nucleus sorting to enrich OB neuronal populations for projection neurons. We then performed high-throughput single nucleus and bulk RNA deep sequencing of such enriched populations. Transcriptome analysis together with RNA in situ hybridization allowed us to identify three distinct mitral and five distinct tufted cell populations with characteristic cell adhesion and cell excitability-related gene expression patterns. Using a novel computational approach to integrate bulk and snRNA-seq data we propose that different mitral cell populations selectively project to different regions of olfactory cortex. Together, we have identified potential molecular and gene regulatory mechanisms underlying projection neuron diversity and provide critical new molecular entry points into studying the diverse functional roles of mitral and tufted cell subtypes.

Wiring logic of the early rodent olfactory system revealed by high-throughput sequencing of single neuron projections

“…Our mapping strategy confirmed previous reports on the organization of bulb projections (i.e. mitral vs. tufted cells) 8,9, 13, 14, 47, 90 , as well as biases in intra-piriform connectivity 49, 87 and piriform cortex output projections 86, 91, 92 ( Figs. 1 , 3 ).…”

Piezo2, A Pressure Sensitive Channel Is Expressed in Select Neurons of the Mouse Brain: a Putative Mechanism for Synchronizing Neural Networks by Transducing Intracranial Pressure Pulses

“…The results of this IHC study indicate that the Piezo2 protein is expressed in pyramidal neurons of the NC and HC, Purkinje cells of the cerebellar cortex and mitral cells of the OB. These results are consistent with earlier and more recent studies of Piezo2 expression rodent and human brains [26,27,34,35,40,41]. Although Piezo2 is well established as a pressure sensitive channel in peripheral neurons [5,[17][18][19][20][21][22][23][24][25] further functional studies, using patch clamp [43] and/or single cell Ca 2+ imaging [44] are required to con rm that Piezo2 is functionally active in these different neuron types.…”

“…The expression of Piezo2 in the OB mitral cells was unanticipated, especially given the well-recognized role of mitral cells in processing primary olfactory sensory inputs, without any requirement for intrinsic mechanosensitivity. However, as already noted, a recent study using single nucleus and bulk RNA deep sequencing has con rmed that Piezo2 is a genetic marker of speci c mitral cells within the mouse OB [35].…”

“…In particular, this resonance could serve to increase the robustness of respiration entrained neural network oscillations reported previously in the OB, HC and NC and proposed to be driven by either synaptic inputs from peripheral afferents in the nasal epithelium or efferent copy discharges from brain stem respiratory nuclei [30][31][32] Note after our IHC study was completed [33] another study using two different commercially available anti-PIEZO2 antibodies, reported similar results including near pan expression of Piezo2 in rat DRG neurons and also Piezo2 expression in NC and HC neurons but not astrocytes [34]. Moreover, another recent study using single nucleus and bulk RNA deep sequencing has con rmed that Piezo2 is a genetic marker for speci c mitral cells within the mouse OB [35].…”

Generation and Characterization of a Cell Type-Specific, Inducible Cre-Driver Line to Study Olfactory Processing

“…In the olfactory bulb, labelled MCs were present in most domains of the olfactory bulb, except for a small patch on the medial, anterior olfactory bulb that showed a curious lack of labeling. Whether or not these correspond to subclasses of MCs, for example those that differ in the glomerular association (Li et al, 2017), or cortical projection patterns (Zeppilli et al, 2020), will be intriguing for future investigation. Outside of the olfactory bulb, labeled somata were sparse if not absent, especially in the areas that MCs target, including in the anterior olfactory nucleus, olfactory tubercle, and the anterior and posterior piriform cortices.…”