Downregulation of M Current Is Coupled to Membrane Excitability in Sympathetic Neurons Before the Onset of Hypertension

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mM KCl, 10 mM HEPES, 10 mM Na + -Phosphocreatine, 4 mM MgATP, 0.3 mM Na2GTP. Internal pH was adjusted to 7.3 with KOH. To study KNa, NaCl was substituted for LiCl to allow an inward gLi via NaV channels but prevent activation of slick and slack channels by intracellular sodium 45 . For Ca 2+ free experiments, Ca 2+ was removed from the external solution and 5 mM EGTA, a Ca 2+ buffer, was added.
qRT-PCR Total RNA from whole flash frozen stellate ganglia was isolated using a RNeasy minikit (Qiagen, US) and immediately stored on dry ice before cDNA library preparation. For cDNA synthesis, Superscript IV VILO with ezDNase genomic DNA depletion (Thermofisher, US) was used, cDNA was then stored at -80 o C until required. Taqman PCR primers were used for the transcript identification of KCNQ2 (Rn00591249_m1), KCNQ3 ( Rn00580995_m1), KCNQ5 (Rn01512013_m1), SCN10A (Rn00568393_m1), where either GAPDH (Rn01775763_g1) or B2M (Rn00560865_m1) were used to normalize values to their age matched controls via the ΔΔCT method 46 . Samples were measured on an ABI Prism 7000 (Thermofisher, US) as per the standard protocol for taqman. The known clinical characteristics of the 4 donor human stellate ganglia samples were as follows: Sample ID 19, Male, 77 years old, 30-35% left ventricular ejection fraction, nonischemic cardiomyopathy and ventricular fibrillation; Sample ID 20, Female, 61 years old, Hypertension and hypothyroidism; Sample ID 23, Male, 19 years old, left ventricular ejection fraction 55-60%, normal cardiac function; Sample ID 24, Male, 62 years old, left ventricular ejection fraction 50%, polymorphic ventricular tachycardia.
Cryosectioning and Immunohistochemistry Freshly isolated stellate ganglia were immediately transferred to 4% paraformaldehyde for 1-2 hours, after which the tissue was incubated overnight in 20% sucrose-PBS at 4 o C, before embedding in OCT compound (Tissue-Tek). Tissue was then frozen and stored at -80 o C until cryosectioning the tissue as 12 µm sections. Slides were then permeabilized in 0.3% triton-X for 30 minutes at room temperature, before blocking for 2 hours in 1% BSA, 5% donkey serum. Sections were then incubated for 24 hours with primary antibodies at 4 o C, followed by five 5 minute washes in PBS and 2 hours incubation with the relevant secondary antibodies. Sections were subsequently washed 3 times in PBS, and incubated with DAPI/PBS for 5 minutes, before a final 2 washes in PBS. Slides were then mounted with 50% glycerol in PBS before imaging. Sections were imaged on a Zeiss LSM 880 Airy Scan Upright laser-scanning confocal microscope with a Plan-Apochromat 20x/0.8 M27 objective. Sections were DAPI stained, labelled with a mouse anti-TH antibody (66334-1-Ig) (ProteinTech, US) and a rabbit antibody against either KCNQ2 (ab22897), KCNQ3 (ab66640) or KCNQ5 (ab66740) (Abcam, UK). For secondary antibodies, 1:200 Donkey anti-mouse Alexa Fluor 555 (A-31570) and 1:200 Donkey anti-rat Alexa Fluor 488 (A-21208) were used (Invitrogen).
Single cell RNA-sequencing A pooled single cell suspension of stellate ganglia cells from six animals per strain was prepared via enzymatic dissociation as described under cell culture methods. Following blockade of enzymatic activity via three washes in blocking solution, the cell solution was transferred to phosphate buffered saline. The cell solutions were immediately transferred to ice and transported to the Wellcome Trust Centre for Human Genetics (WTCHG) for scRNAseq via 10x genomics chromium (10x genomics, US) (Single Cell 3' v3) and Illumina hiseq 4000 (Illumina, US). This approach achieved 66-72K mean reads per cell and a sequencing depth of 53-55% per cell before filtering. Initial analysis was performed by the WTHCG using the cell ranger pipeline (x10 genomics, US) (Cell ranger, v3.0.2) (Rnor6.0) with default parameters, before the data were exported to Seurat (v3.0) 47 and analyzed in house. Cells were excluded in Seurat if the number of counts per cell was less than 4000 or percentage of mitochondrial genes was equal to or less than 0.3. For FindVariableFeatures we used 10000 features and the election method VST. Data was intergrated using 30 dimensions, 30 principle components were using for PCA analysis. UMAP and TSNE, FindNeighbours were ran with 19 dimensions. Findclusters was ran with a resolution of 0.6. Differential expression analysis was performed via MAST 48 within Seurat. To determine multiple neuron populations for supplementary figure 5, findclusters was ran at a resolution of 10, and neuron-like groups were subset for further analysis. Individual groups were then visually identified based upon clear separation from neighbouring subgroups.
Firing rate was taken as the maximum firing rate elicited by a range of 10 pA current injections between 10-200 pA. Membrane potential was monitored for stability during drug wash in and cells with large jumps in membrane potential were discarded.
Action potential parameters were measured from the first sequential 50 pA current step that induced an action potential. Peak amplitude (mV) was taken as the difference between the average baseline and maximum peak response of the action potential. Action potential upstroke (mV/ms) was taken as the maximum velocity from baseline to the peak amplitude.
Input resistance was calculated based upon a series of hyperpolarizing and depolarizing current injections ranging from -200 to 200 pA in amplitude in 10 pA increments 49 . The average value of the final 100 ms was analyzed. As previously classified, small hyperpolarizing pulses were assumed to elicit the least active processes and any points that departed from linearity with these points or contained visible active processes in the final 200 mS of current injection were excluded.
Liquid junction potentials were calculated in JPCalcW 50 in Clampex (v11.0.3) (Molecular Devices, US), where ion availabilities were used instead of concentrations. Free Ca 2+ , ATP, EGTA and Mg 2+ for internal solutions were estimated via MaxChelator (v8) 51 when relevant. For perforated-patch voltage-clamp and current clamp recordings a Liquid junction potential of 24.3 mV was calculated, without correction for the perforated patch Donnan potential 52 . Whole cell current clamp recordings had an estimated liquid junction potential of -15.7 mV. Figure S1 Analysis of the relationship between cell capacitance, days in vitro (DIV) and maximum firing rate in either strain as measured by perforated patch clamp. (A) The number of days in vitro does not affect the maximum neuronal firing rate in Wistar neurons (Median) (DIV1, 2 Hz, n = 19; DIV2, 2 Hz, n = 14; DIV3, 1.5 Hz, n = 28; DIV4, 1 Hz, n = 5) (Kruskal-Wallis test, n = 66, p = 0.48). (B) There is no clear relationship between cell capacitance and firing rate in Wistar neurons. (C) The number of days in vitro does not affect the maximum neuronal firing rate in SHR neurons (Median) (DIV1, 5.5 Hz, n = 18; DIV2, 9 Hz, n = 22; DIV3, 9.5 Hz, n = 18; DIV4, 4 Hz, n = 10; DIV5, 8.5 Hz, n = 2) (Kruskal-Wallis test, n = 70, p = 0.35). (D) There is no clear relationship between cell capacitance and firing rate in SHR neurons. (E) There is not a significant difference in the number of days in vitro between strains (Median) (Wistar, 2.5 days, n = 66; SHR, 2 days, n = 70) (Mann-Whitney test, p = 0.83). (F) There is no significant difference in capacitance between strains, although there is a non-significant trend (Median) (Wistar, 26.74 pF, n = 66; SHR, 23.35 pF, n = 70) (Mann-Whitney, p = 0.27). (G) Dependence of firing rate on membrane potential was determined in SHR neurons by applying a pre-pulse in the range -10 to -100 pA for 1 second before applying a 150 pA positive current injection to elicit cell firing. No difference was found (Friedman test, n = 26, p = 0.165).

Figure S2
Single cell RNA-sequencing and a panel of pharmacological inhibitors were used to determine remaining channels involved in SHR enhanced firing, that may be targetable for the reduction of aberrant sympathetic hyperactivity or key to the SHR sympathetic pathology. Single-cell RNA-sequencing was used to identify the cell specific expression patterns of a range of channel subunits which are typically implicated in the control of firing rate in other neuronal populations. Here percentage expressed indicates the number of cells per cluster identity exhibiting transcript expression and average expression refers to the average expression per cluster identity. The table highlights the effect of selective inhibitors on firing rate for channels not shown in the main manuscript. Data shown for 1s 50 pA, 100 pA, 150 pA current injections and at maximum firing frequency between 0-200 pA current amplitude. Paired comparisons were made by Wilcoxon tests.  Upstroke velocity for phasic 1, phasic 2 and tonic neurons revealed a higher Tonic velocity (Median ± IQR) (Phasic 1, 34.71 mV/ms, n = 20; Phasic 2, 63.65 mV/ms, n = 32; Tonic, 85.38 mV/ms, n = 37) (Kruskal-Wallis test; p = 0.0018) (Dunn's multiple comparisons test; Phasic 1 vs Tonic, p = 0.0012). (D) Action potential amplitude was significantly higher for phasic 2 and tonic neurons than in phasic 1 neurons (Mean ± SEM) (Phasic 1, 68.30 ± 4.49 mV, n = 21; Phasic 2, 78.25 ± 2.25 mV, n = 32; Tonic, 81.96 ± 2.14mV, n = 37) (One-way ANOVA; p = 0.0071) (Holm-sidak's multiple comparisons test; Phasic 1 vs Phasic 2, p = 0.049; Phasic 1 vs Tonic, p = 0.0054).

Figure S6
Evidence of multiple populations of sympathetic neurons. (A) Visually identified cell clusters of potential sympathetic neurons. (B) Expression of key transcripts for noradrenaline synthesis and breakdown are shown for each neuron cluster. Of these groups only Neurons 1 and Neurons 2 appear to adequately express the full noradrenaline synthesis pathway. (C) Sympathetic Neurons 1 are shown to be have low expression of CHRM2 and NPY, two physiologically important genes, in contrast to high expression in Sympathetic Neurons 2. For reference, these groups are termed Type A sympathetic neurons (Sympathetic Neurons 1), and type B sympathetic neurons (Sympathetic Neurons 2). Importantly, we also demonstrate that both groups have similar ion channel profiles, with no significant differences observed between groups.