Article Figures & Data
Figures
- Figure 1.
Location and calcium transients of lumbosacral SPNs in E10 embryos. A, Texas Red retrograde labeling from ventral root reveals two populations of neurons. One population is consistent with lateral motor column (LMC), the other population is medial, slightly dorsal of the central canal, consistent with Column of Terni. B, In separate experiments, the medial population of SPNs were also retrogradely labeled from the IGN, which did not result in labeling of limb motoneurons in the LMC. C, Of the 179 total cells imaged on medial surface, 81.0% were in the region between the central canal and halfway to the dorsal edge of the cord (highlighted in yellow), where SPNs reside. D, Calcium transients were observed using cells labeled by the calcium indicator Calcium green. The medial view of the hemicord revealed that both limb MNs and SPNs were active during evoked and spontaneous episodes of network activity (ΔF/F of >20%, N = 3 cords).
- Figure 2.
Intracellular chloride levels of MNs and SPNs were altered after synaptic blockade. A, Clomeleon electroporation into the neural tube at embryonic day 3 (E3). B, YFP, CFP, and merged image of ventral view of spinal cord were used to analyze chloride levels in somatic MNs in E10 spinal cord. Schematic at bottom left shows orientation of ventral view. C, Analysis of MNs showed significant reduction in YFP/CFP ratio in drug-treated group. In order to examine entire dataset while controlling for unbalanced number of cells observed between cords, hierarchical bootstrapping was implemented. This analysis of resampled data revealed lower ratios in GBZ-treated cells (1.79 ± 0.03) compared with vehicle (1.92 ± 0.05, p = 0.035,*). D, Left, Medial view of hemisected spinal cord was imaged for analysis of SPNs. Right, Merged YFP-CFP image, with SPNs designated with white arrow, at E10 in embryos which were labeled with Clomeleon at E3. E, Analysis of SPNs also revealed a reduction in ratio in GBZ-treated cells. Hierarchical bootstrapping test confirmed lower ratios in GBZ-treated cords compared with control cords (GBZ =1.66 ± 0.04, H2O = 1.86 ± 0.07, p < 0.0001,***). Finally, linear mixed effects test was performed on entire dataset, which determined difference between groups is explained by fixed effect of treatment group, not driven by random effects such as embryo number or experiment date (p < 0.005). See Extended Data Figure 2-1 for additional statistical treatment of data.
- Figure 3.
PGNs develop mature synaptic activity between E13 and E17. A, Schematic of PGN extracellular recording set up. B, Example traces at E17 show evoked response, including synaptically-driven discharge in PGNs (highlighted in green box, 10 ms), followed by a reduction in discharge after the addition of 100 μm Hex into the bath. C, The reduction of discharge was calculated across many trials for the following developmental stages: E10, E13, and E17 (gray lines represent individual experiments, green dashed line represents mean). Paired, two tailed t tests revealed the following: at E10, there was no difference in discharge after the addition of Hex (t(3) = 1.8, p = 0.17). At E13, there was a significant reduction in synaptic discharge with Hex (t(3) = 6.6, p < 0.01, **). At the E17 stage, the activity was significantly reduced after addition of 100 μm Hex (t(6) = −4.6, p < 0.005, ***).
- Figure 4.
Nicotinic blockade led to a change in excitability in PGNs that did not appear to be in a compensatory direction. A, Progressively increasing depolarizing current steps (500 ms in duration, intervals of 5 pA) were delivered every other second. B, Frequency/current (f/I) curve reveals the relationship between treatment condition and excitability of cells. Two-way repeated measures ANOVA revealed a main effect of input current on output number of action potentials, as well as a significant between-group effect of treatment (F(14) = 32.8, p < 0.0001). Because cords were not evenly sampled, and because of the hierarchical nature of the dataset, a linear mixed effect (LME) model was performed on a linear portion of the dataset (20 to 55 pA) to verify this effect of treatment group. This analysis showed a significant effect of treatment group (t(15.49) = −2.28, p = 0.037), and eliminated any effect of potential confounding factors such as experiment date. C, Schematic representation of parameters measured from individual spikes, summarized in Table 1. D, To confirm the effect using all input current values, logistic regression model was trained on a subset of data to predict the treatment group. When given naive data from the test dataset, the mean accuracy of the model was significantly greater than chance (mean accuracy = 82.7%, t(28) = 7.29, p < 0.001, ***). In comparison, the model’s accuracy when predicting treatment group for a shuffled dataset, was no better than chance (mean accuracy = 46.6%, not different from chance accuracy of 0.50 (t(28) = 0.63, p = 0.54), further validating the effect of treatment group. Error bars represent standard error.
- Figure 5.
Acute nicotinic blockade resulted in reduction of synaptic activity which did not recover over several hours. Ex vivo preparations of sympathetic chain tissue were used for extracellular recordings in baseline conditions, and during nicotinic blockade using either 50 or 100 μm hexamethonium (Hex). Synaptic activity was evoked using a train of three stimulations (20-Hz interval) every 5 min, and the resulting synaptic discharge was calculated at each time point. The synaptic discharge remained reduced for several hours, such that baseline levels of synaptic activity did not recover for 4–5 h of nicotinic blockade. In two experiments, washout of the drug was conducted. Synaptic activity returned after washout, demonstrating that the tissue was still healthy.
Tables
- Table 1
Comparison of membrane properties between treatment groups (vehicle and hexamethonium)
Table of membrane properties Vehicle treated Hex treated t statistic p value Capacitance (pF) 25.3 ± 3.5 23.5 ± 4.5 t(15) = 0.31 0.76 Resting membrane potential (mV) −56 ± 2 −59 ± 1.7 t(15) = 0.3 0.77 Rheobase (pA) 30.4 ± 4.9 44.7 ± 6.1 t(14) = 1.82 0.091 Input resistance (MΩ) 1052.7 ± 115 788.6 ± 110 t(12) = 1.72 0.11 Threshold voltage (mV) −54 ± 1.3 −51 ± 2.4 t(15) = −1.19 0.25 Action potential half-width (ms) 4.4 ± 0.5 2.7 ± 0.2 t(13) = 2.15 0.051 After-hyperpolarization amplitude (mV) 10.2 ± 1.6 10.5 ± 1.7 t(14) = −0.11 0.91 Action potential peak amplitude (mV) 12.6 ± 2.3 24.7 ± 3.5 t(14) = −2.94 <0.011* Membrane time constant τ (ms) 26.8 ± 5.2 18.4 ± 2 t(15) = 1.3 0.21 Slope of rising phase (mV/ms) 8.5 ± 3.4 8.1 ± 2.2 t(14) = 0.25 0.81 Mean values are listed with standard error. Statistical values reflect the degrees of freedom for each measure compared using a two-tailed, unpaired t test. p < 0.05 is denoted by *.
Extended Data Figure 2-1
Statistical confirmation of Clomeleon data. A, Motoneuron dataset showed significant difference between control and drug-treated groups. An independent, two sample t test was performed, restricting data to 10 cells per cord to handle uneven sampling between cords. This test revealed that ratio of YFP:CFP intensity was lower in cells from embryos treated with GBZ (1.81 ± 0.01) than from those treated with vehicle (H2O, 1.94 ± 0.02, t(175) = 6.7, p < 0.001). B, Full dataset is shown here, including every cell from each cord. This dataset is not suitable for a t test, however linear mixed effects modeling revealed a significant difference between groups was driven primarily by treatment group, not driven by cord umber or experiment date (p < 0.001). C, Analysis of SPNs also revealed a reduction in ratio in treated cells. An independent, two sample t test using four cells per cord revealed that FRET ratio of YFP:CFP were significantly lower in spinal cords from embryos treated with GBZ (1.65 ± 0.03) compared to those treated with H2O (1.86 ± 0.02, t(49) = 5.9, p < 0.001). D, Full dataset is shown here. As number of cells was imbalanced between cords, data was unsuitable for t test. Therefore, linear mixed effects test was performed on entire dataset. This test determined a significant difference between groups that was explained by treatment group alone, thus not driven the random factors of cord number or experiment date (p < 0.005). Bars represent standard error. Notches in bars represent mean. Download Figure 2-1, TIF file.
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