Date of Award
Master's Thesis - Open Access
Master of Science
Biochemistry and Molecular Biology
Joseph M. Wu, Ph.D.
Christopher S. Leonard, Ph.D.
A loss of neurons that synthesize the neuropeptide orexin produces the sleep disorder narcolepsy with cataplexy in humans and other animals. How symptoms of this disorder arise is not well understood, but selectively restoring orexin actions at 5-HT DR neurons rescues key symptoms (cataplexy), suggesting normal orexin signaling is important at these neurons. To better understand how orexin acts on these neurons, our lab identified a set of novel orexin actions that appear mediated by unidentified cation- permeable ion channels. To narrow down the list of possible channels, we used a bioinformatics approach to compare published gene expression profiles of 5-HT neurons and cerebellar Purkinje neurons. These neurons, despite having different electrical properties and different orexin responses, have cation permeable ion channels.
By using the Amigo2 database, we selected calcium channels, glutamate receptors, histamine receptors, inositol 1,4,5-triphosphate (IP3) receptors, ryanodine receptors, transient receptor potential (TRP) channels, potassium channels and sodium channels for examination. We then processed single-cell RNA sequencing (scRNAseq) data available on NCBI for 32 serotonergic neurons (SRP064626; from the Dymecki lab at Harvard) and 32 Purkinje cells (Series GSE78424). Single-cell RNA sequencing for both 5-HT neurons and Purkinje neurons were conducted using Illumina HiSeq 2500.
After normalization of gene expression profiles of these 64 single cells and comparisons of gene expression levels between cell clusters and cell sub-clusters, we identified channels enriched in a subset of 5-HT neurons. Based on published channel properties and experimental data from our lab, we isolated 39 candidate channel genes, from which we selected the Nalcn gene for initial testing. Nalcn encodes a sodium leak and G protein-coupled receptor activated channel that regulates the resting membrane potential and neuronal excitability. It was up-regulated in 5-HT neurons compared to Purkinje cells (p = 1.6e-06) and it produces a membrane current with some similarities to the current activated by orexin in these neurons.
Using a commercially available antibody, we used immunocytochemistry to determine if the channel protein is expressed in 5-HT DR neurons. We identified an effective concentration of the anti-Nalcn antibody to visualize the channel with light microscopy (using VIP as a chromogen) and immunofluorescence. We first confirmed there was Nalcn immunostaining in the DR, and then in 5-HT DR neurons using the mice that express tdTomato fluorescence in 5-HT neurons (Sert-Cre/dTom mice). To conduct functional studies, we intend to knockout the channel since there are no selective Nalcn antagonists available. We utilized NalcnFL/FL mice in which the Nalcn gene was flanked by loxP sites that enables gene knockout by Cre-recombinase expression. We first confirmed that DR 5-HT neurons appear normal in these mice using immunofluorescence staining of tryptophan hydroxylase (TPH), the rate-limiting enzyme for serotonin biosynthesis, and then that Nalcn immunoreactivity was present in the DR of these mice.
Preliminary experiments to knock out Nalcn by delivering Cre-recombinase via viral injection (AAV-Cre) into the DR of NalcnFL/FL mice are also described. Success with using this antibody to monitor the loss of Nalcn immunostaining will confirm antibody specificity and encourage future whole cell patch clamp experiments to functionally test if Nalcn mediates the Orexin A action on 5-HT DR neurons. Collectively, these results will advance our understanding of the neural mechanisms underlying cataplexy.
Javed, Saqlain, "Ion channel candidates mediating orexin neuropeptide actions on brainstem serotonergic neurons" (2021). NYMC Student Theses and Dissertations. 18.
Available for download on Monday, April 25, 2022