Transcriptional profiling of identified neurons in leech

Abstract

Background While leeches in the genus Hirudo have long been models for neurobiology, the molecular underpinnings of nervous system structure and function in this group remain largely unknown. To begin to bridge this gap, we performed RNASeq on pools of identified neurons of the central nervous system (CNS): sensory T (touch), P (pressure) and N (nociception) neurons; neurosecretory Retzius cells; and ganglia from which these four cell types had been removed. Results Bioinformatic analyses identified 3565 putative genes whose expression differed significantly among the samples. These genes clustered into 9 groups which could be associated with one or more of the identified cell types. We verified predicted expression patterns through in situ hybridization on whole CNS ganglia, and found that orthologous genes were for the most part similarly expressed in a divergent leech genus, suggesting evolutionarily conserved roles for these genes. Transcriptional profiling allowed us to identify candidate phenotype-defining genes from expanded gene families. Thus, we identified one of eight hyperpolarization-activated cyclic-nucleotide gated (HCN) channels as a candidate for mediating the prominent sag current in P neurons, and found that one of five inositol triphosphate receptors (IP3Rs), representing a sub-family of IP3Rs absent from vertebrate genomes, is expressed with high specificity in T cells. We also identified one of two piezo genes, two of ~ 65 deg/enac genes, and one of at least 16 transient receptor potential (trp) genes as prime candidates for involvement in sensory transduction in the three distinct classes of leech mechanosensory neurons. Conclusions Our study defines distinct transcriptional profiles for four different neuronal types within the leech CNS, in addition to providing a second ganglionic transcriptome for the species. From these data we identified five gene families that may facilitate the sensory capabilities of these neurons, thus laying the basis for future work leveraging the strengths of the leech system to investigate the molecular processes underlying and linking mechanosensation, cell type specification, and behavior.

Activity-Dependent Modulation of Tonic GABA Currents by Endocannabinoids in Hirudo verbana

“…TRPV1 is known to be an important cannabinoid receptor in mammals for both AEA and 2-AG ( Zygmunt et al, 1999 , 2013 ; Gibson et al, 2008 ; De Petrocellis et al, 2011 ). TRPV-encoding genes are present in Hirudo ( Heath-Heckman et al, 2021 ) and pharmacological studies using both activators (capsaicin and resiniferatoxin) and inhibitors (capsazepine and SB366791) support that a TRPV-like channel acts as a cannabinoid receptor in this species ( Yuan and Burrell, 2010 ; Li and Burrell, 2011 ; Higgins et al, 2013 ; Wang and Burrell, 2016 ).…”

“…The Hirudo CNS possesses mechanosensory neurons that are functionally similar to mammals, including rapidly adapting touch (T), slow adapting pressure sensitive (P), and both mechanical and polymodal nociceptive (N) neurons (Nicholls and Baylor, 1968;Blackshaw et al, 1982;Pastor et al, 1996;Lewin and Moshourab, 2004;Abraira and Ginty, 2013;Burrell, 2017). Furthermore, recent studies have found that genes associated with mechanosensation in mammals are also expressed in the Hirudo mechanosensory neurons, e.g., piezo, DeG/ENaC, and trp encoding genes (Heath-Heckman et al, 2021). As in mammals, Hirudo possesses the endocannabinoids 2-AG and AEA, with the former being more abundant (Matias et al, 2001).…”

“…As in mammals, Hirudo possesses the endocannabinoids 2-AG and AEA, with the former being more abundant ( Matias et al, 2001 ). Hirudo possesses genes encoding the proteins required for 2-AG synthesis, [diacylglycerol lipase (DAGL) α and β (accession #s KU500007 & MT610103 ), 2-AG metabolism (monoacylglycerol lipase (MAGL) (KY971276)], AEA metabolism [fatty acid amide hydrolase (FAAH) (pending)], and TRPV ( Heath-Heckman et al, 2021 ). Hirudo MAGL (hirMAGL) has been studied in some detail and exhibits considerable structural and functional conservation at the protein’s catalytic active site ( Kabeiseman et al, 2020 ).…”

A Draft Genome forHirudo verbana, the Medicinal Leech

“…This misunderstanding regarding the taxonomic classification of these leech subspecies has led to some confusion surrounding appropriate cataloging of preliminary leech omics databases [21,22]. Finally, in spite of the advancements in sequencing technology, most of the existing sequence repositories for the medicinal leeches have been comprised of expressed sequence tag [23] and transcriptomic databases [24][25][26], with recent leech genomics work centering around H. medicinalis despite the prominence of H. verbana in neuroscience research [27,28]. Moreover, other leech genomes are also becoming available, including that for the Asian Buffalo leech, Hirudinaria manillensis [29].…”

“…This work presents the first draft genome for H. verbana, which consists of 250 Mbp, 61,282 contigs, an N50 of 8,638 bp, and a GC content of 38%. This draft genome, in addition to the growing transcriptomic resources for H. verbana [24,26,30] and the aforementioned genomic databases for the closely related leech species, will help accelerate studies seeking to link the molecular basis of previous and ongoing functional studies utilizing medicinal leeches.…”