Molecular profiling of single neurons of known identity in two ganglia from the crab Cancer borealis

Abstract

Understanding circuit organization depends on identification of cell types. Recent advances in transcriptional profiling methods have enabled classification of cell types by their gene expression. While exceptionally powerful and high throughput, the ground-truth validation of these methods is difficult: If cell type is unknown, how does one assess whether a given analysis accurately captures neuronal identity? To shed light on the capabilities and limitations of solely using transcriptional profiling for cell-type classification, we performed 2 forms of transcriptional profiling—RNA-seq and quantitative RT-PCR, in single, unambiguously identified neurons from 2 small crustacean neuronal networks: The stomatogastric and cardiac ganglia. We then combined our knowledge of cell type with unbiased clustering analyses and supervised machine learning to determine how accurately functionally defined neuron types can be classified by expression profile alone. The results demonstrate that expression profile is able to capture neuronal identity most accurately when combined with multimodal information that allows for post hoc grouping, so analysis can proceed from a supervised perspective. Solely unsupervised clustering can lead to misidentification and an inability to distinguish between 2 or more cell types. Therefore, this study supports the general utility of cell identification by transcriptional profiling, but adds a caution: It is difficult or impossible to know under what conditions transcriptional profiling alone is capable of assigning cell identity. Only by combining multiple modalities of information such as physiology, morphology, or innervation target can neuronal identity be unambiguously determined.

Regulation of Metabolism by an Ensemble of Different Ion Channel Types: Excitation-Secretion Coupling Mechanisms of Adipokinetic Hormone Producing Cells in Drosophila

“…We also cannot rule out the possibility that some of our transcriptome samples stem from synaptic terminals projecting to AKH cells that may express a diversity of ion channels. Nonetheless, our transcriptome analysis represents a first requisite step in gauging the complement of genes expressed in APCs and is consistent with other neuronal sc-transcriptomes (Fuzik et al, 2016;Northcutt et al, 2019) and mammalian pancreatic sctranscriptomes (Muraro et al, 2016).…”

“…We note that most of the ion channel genes within the Drosophila genome are expressed in this secretory tissue. However, we note that single cell (sc) transcriptomes of individual neurons also show a diversity of ion channels present (Fuzik et al, 2016;Northcutt et al, 2019). While many of these sc-RNA studies attempt to elucidate potential genetic heterogeneity of either a brain area or groups of neurons that share a transmitter phenotype, it is clear that a meta-analysis of sc-transcriptomes from mammalian and invertebrate neuronal tissue show multiple FIGURE 3 | Locomotor activity in animals expressing the Sur-RNAi, sei-RNAi, and Ca-beta RNAi elements under replete and starvation conditions.…”

Single cell transcriptomes reveal expression patterns of chemoreceptor genes in olfactory sensory neurons of the Caribbean spiny lobster, Panulirus argus

“…Our study demonstrates that the single cell transcriptomics approach can be applied to crustacean chemoreception. In fact, to our knowledge, this is the first report of the application of single cell transcriptomics to crustacean chemoreception and only the second of any cells in crustaceans [ 169 ]. To understand olfactory coding in spiny lobsters, we need to apply this technique to a much larger set of OSNs.…”

Refining the Identity and Role of Kv4 Channels in Mouse Substantia Nigra Dopaminergic Neurons

“…While the expression of only two types of voltage-gated ion channels in the squid giant axon allowed Hodgkin and Huxley to dissect the biophysical processes underlying action potential (AP) genesis and conduction ( Hodgkin and Huxley, 1952 ), most neuronal types express a multitude of ion channel subtypes underlying their electrical activity ( Cembrowski et al, 2016 ; Fuzik et al, 2016 ; Tapia et al, 2018 ; Northcutt et al, 2019 ). In spontaneously active neurons, a variety of voltage-gated and calcium-gated ion channels are not only responsible for the AP, but also govern the subthreshold oscillations leading to AP firing, determine firing frequency and control its regularity ( Atherton and Bevan, 2005 ; Swensen and Bean, 2005 ; Bean, 2007 ; Gantz et al, 2018 ).…”