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Colleen S Peterson , Alastair Ferguson , W Mark

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Colleen S Peterson , Alastair Ferguson , W Mark The verified ion channelome of the rat subfornical organ. Colleen S Peterson1, Alastair Ferguson2, W Mark Fry1 1.Department of Biological Sciences, University of Manitoba 2. Centre for Neuroscience, Queen's University Background Results Sensory circumventricular organs (CVOs) lack a blood- Voltage-gated Na+ channels 3 Not present Scn9a brain-barrier, and they express a high density and wide Only detected by RNAseq Scn1a Scn2a variety of receptors. Therefore, CVOs can detect numerous 2 Only detected by microarray Table 1. Voltage-gated Na+ Present Scn3a circulating hormones and signalling molecules, and +60 mV 1 500 pA channels in the SFO. communicate this information to homeostatic control 0 Scn8a 100 ms -70 mV Present Absent centres. -1 -100 mV SCN1A SCN4A Scn10a -2 The subfornical organ (SFO) is an AV3V CVO which is well 1 SCN2A SCN5A -3 Scn5a known to play critical roles in osmoregulation , Scn11a cardiovascular regulation2, and energy balance3. The SCN3A SCNA10A -4 electrical behaviour of SFO neurons is determined by the log2(intensity) Microarray -5 Scn4a SCN8A SCN11A ion channels and receptors present. With the aim of -6 improving our understanding this unique and important -5 -4 -3 -2 -1 0 1 2 3 4 5 structure, we have characterised the transcriptome of the SCN9A RNAseq log2 (FPKM) Figure 1. Voltage-gated Na+ *bold indicates only detected by RNAseq SFO using RNA sequencing. currents from SFO neurons. Figure 2. Correlation between RNAseq FPKM and microarray intensity for Here we present data outlining the ion channel and G- Voltage-gated K+ channels voltage-gated Na+ channels. protein coupled receptors (GPCR) expressed. R2: 0.9247. n = 10. A Table 2. Voltage-gated K+ channels Methods +60 mV 500 pA in the SFO. Animals: All procedures complied with CCAC and were 100 ms -70 mV Present Absent 4 Not present approved by the University of Manitoba. -100 mV Only detected by RNAseq KCNA 1, 2, 3-6 KCNA 7, 10 3 Only detected by microarray Pups were from timed-pregnant Sprague Dawley dams and ITotal Present randomly assigned to dams in litters of 12. At age 6 weeks, KCNB 1, 2 2 rats were sacrificed, SFO carefully dissected out and stored KCNC 1-4 1 B +60 mV KCND 1, 3 in RNAlater at -20 °C until time of RNA extraction. Six +40 mV 0 -70 mV KCNF 1 samples from the same litter were pooled. -1 KCNG 1, 2, 4 KCNG3 -2 Library Prep, RNAseq, and Analysis: KCNH 1, 2, 4, 5, KCNH3, 5, 8 I -3 RNA was extracted using PureLink RNA Mini Kit (Thermo K 6, 7 V Fisher), and analysed using a Bioanalyzer (Agilent). Two µg KCNJ 2, 3, 5, 8- KCNJ1, 4, 15 -4 of RNA were prepared for library contruction using the C 10, 12-14, 16 log2(intensity) Microarray -5 TruSeq Standard mRNA Library Prep Kit (Illumina), then KCNK 1-3, 5, 9, KCNK4, 6, 7, 12, 15, -6 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 sequenced on the NextSeq 500 platform with 2x 75 bp end ITO 10, 13,16 17 sequencing. Reads aligned to RNOR 6.0 genome assembly KCNMA1 RNAseq log2 (FPKM) Figure 3. Voltage-gated K+ (Hisat2). FPKM was used as measure of transcript levels KCNN 1-3 Figure 4. Correlation between RNAseq currents from SFO neurons. (Stringtie). Ion channels and G protein coupled receptors KCNQ 1, 2-4 KCNQ1 FPKM and microarray intensity for (A) Total K+ current. were identified by comparison to the IUPHAR-DB. Data KCNS2 KCNS1, 3 voltage-gated K+ channels. R2: 0.2619. (B) Non-inactivating K+ current. were compared to previously published microarray data for KCNT 1, 2 n = 69. validation, where possible7. (C)Transient K+ current. (B-A) KCNV2 HCN 1, 3, 4 Not present Neuron Culture and Electrophysiology *bold indicates only detected by RNAseq Only detected by RNAseq 8 Only detected by microarray Rats were sacrificed, brain removed, and cultures of G-protein-coupled receptors Present dissociated cells prepared. Cells were plated on 35mm 7 glass culture dishes in B-27 supplemented Neurobasal 1 uM SP 100 nM NT 6 media and cultured up to 5 days at 37 ̊C with 5% CO2. Whole cell patch-clamp of dissociated neurons was 5 conducted in the current clamp and voltage clamp (intensity) 4 2 configuration using using HEKA EPC 10 amplifier and 20 mV 3 PatchMaster V2x90 software. 2 min 2 Figure 5. Response of a Figure 6. Response of dissociated 1 Literature SFO neuron to 100nM neurotensin. dissociated SFO neuron to 1µM 0 1. Felix, D. Naunyn-Schmiedebergs Arch Pharmacol. 292 15–20 substance P. TACR1 is NTSR2 and SORT1 are highly log Microarray (1976) expressed in rat SFO. -1 2. Mangiapane, M.L., and Simpson, J.B. AJP Regul Integr Comp expressed in rat SFO. -2 -1 0 1 2 3 4 5 Physiol. 244 R508-13 (1983) RNAseq log2 (FPKM) 3. Smith, P.M., et al. 2010. Physiol Behav. 99 534–7 (2010) Figure 7. Correlation between RNA- 4. Miselis, R.R. Peptides. 3 501–502. (1982) For a full list of GPCRs and ion channels, 5. Whyment, A., et al. Nat Neurosci. 7 493–494 (2004) seq FPKM and microarray intensity for please visit this website: GPCRs. R2: 0.6274. n = 73. 6. Burton, M.J., et al. Exp Neurol. 51 668–677 (1976) sites.google.com/site/sfochannelome 7. Hindmarch, C. et al. Am J Physiol. 295 R1914-R1920 (2008) .
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