Article Figures & Data
Figures
- Figure 1.
Electrophysiological properties of CC and Atoh1-lineage medial and lateral neurons. A–B’’’, Proprioceptive afferent terminals are close to the soma of Clarke's column (CC) and Atoh1-lineage medial (M) and lateral (L) neurons. A, Representative lower thoracic image of CC neurons (GdnfTOM). A’,A’’, Proprioceptive afferents (PV+VG1+, arrows) are in close apposition to the soma of CC neurons (TOM). B, Representative lower thoracic image of M and L neurons in Atoh1TOM mice. B’–B’’’, Proprioceptive afferents (PV+VG1+, arrows) are in close apposition to the soma of Atoh1-lineage M (B’’) and L cells (B’’’). C, CC and Atoh1-lineage neurons (GdnfTOM; Atoh1GFP) cluster into distinct populations within laminae V–VII of the intermediate spinal cord. D, Top Row. Some cells fire action potentials (APs) at their resting membrane potential Vmrest (i.e., no holding currents). Vmrest is shown to the left of each sample trace. Each sample trace contains five traces corresponding to five 2 s sweeps. Only one trace is highlighted in black, and the rest are shown as background in light gray. Bottom row, The protocol schematic to the left indicates that cells were held at −70 mV. After an initial estimation of the rheobase, a more precise determination was done by current steps of 1–3 pA where the first step was a few pAs below the initially estimated threshold. The schematic and traces show the sweep at the threshold in black and sweeps 3 pA below and 9 pA above the threshold in light gray. E, Quantification for capacitance, input resistance (Rm), Vmrest, firing frequency at rest, and rheobase. PV, parvalbumin; VG1, VGLUT1; TOM, tdTomato. Scale bars: 100 µm (A, A’, B, B’, C), 10 µm (A’’, B’’, B’’’).
- Figure 2.
Firing types for CC and Atoh1-lineage medial and lateral neurons. A, Current steps in increments of 50 pA were injected up to 500 pA. Steps at 50, 150, and 250 pA are shown. The columns show the main firing types found for all cell groups. The tonic-firing type (T) showed an increase in frequency at higher steps. The fading-firing type (F) showed increased firing frequency upon the first few depolarization steps, but further depolarization failed to sustain firing. The “single”-firing type (S) showed less than three APs, even at the highest currents injected. The blue lines on the top traces highlight the slope differences among the firing types, namely, the decrease in maximal AP amplitude over the time course of the current injection and the degree of baseline depolarization. The slope is shallow for the T-firing type but very steep for F- and S-firing cells. B, Percentages of firing types for each cell group. M and L neurons that changed firing type depending on the level of current injection were classified as undefined (U). C, The relation for current injected and # of APs fired is shown for T- and F-firing types for each cell group.
- Figure 3.
Ih currents and sag potentials are present in CC cells. A, Hyperpolarizing −10 mV voltage steps (left) and −50 pA current steps (right) induced Ih currents and sag potentials in CC cells. The inset in the middle shows the quantification for the amplitude of the Ih current induced by steps at −30, −60, and −90 mV, after the subtraction of the instantaneous leak current. B, To confirm the nature of the hyperpolarization-activated currents in CC neurons, we used ZD-7288 to block Ih currents. Except for one cell, a nearly complete blockage of the hyperpolarization-activated current was achieved with an average 70% blockage (left traces, bar graph to the right). The sag potential (Δ) is blocked with ZD-7288 and membrane hyperpolarization continues (−Δ; right traces). The difference in sag potential is >100% compared with control when Ih is blocked (right traces, bar graph to the right). C, Neither Ih currents nor sag potentials were induced in Atoh1-lineage neurons. Instead, Atoh1-lineage neurons showed high susceptibility to damage when subjected to hyperpolarization (right). D, Compared to Atoh1-lineage medial neurons, CC cells showed a relatively large after depolarization (orange-shaded area) following the first rebound AP fired. E, Number of rebound APs fired following a hyperpolarization step. F, Blockage of Ih current in CC cells almost eliminates rebound firing, strongly suggesting that upon repolarization, residual Ih depolarizes CC cells inducing rebound firing. Paired one-tailed t test: 0.0486. G, Transcript counts for Hcn1, Hcn2, Hcn3, and Hcn4 transcripts in CC neurons from single-cell RNA-seq data published in Baek et al. (2019). H, Hcn1 and Hcn3 mRNA transcripts are present in CC neurons (RNAscope+TOM+, arrowheads, left and right panels). Hcn2 mRNA is weakly expressed in CC neurons (RNAscope+TOM+, arrowheads, middle panel), while nearby neurons express Hcn2 mRNA (arrows). Scale bars: 100 µm (H), 10 µm (H, insets).
- Figure 4.
Organization of primary afferent inputs to CC and Atoh1-lineage medial neurons in the intermediate spinal cord. A, Schematic of electrophysiological recordings in sagittal hemisected (left) or horizontally sectioned (right) spinal cords. Stimulation suction electrodes were used either at the lumbar L2–L4 roots or the thoracic T11–T13 roots while recording from CC or Atoh1-lineage neurons in spinal cord segments T10–L2. B, C, The overall threshold stimulation to any response type and the number of segments between the recorded cell and stimulated dorsal root are shown for both lumbar (distal) or thoracic (local) dorsal root stimulation. These include cells that responded to any stimulation intensity. In general, thresholds were higher upon local stimulation for all cell types. D, Samples of the four types of evoked EPSCs recorded upon dorsal root stimulation: Single, multiple, barrage, and Si + Ba. Note that the multiple category has eEPSCs whose peak amplitudes are within 10 ms of stimulation and the barrage category has several eEPSCs that occur within hundreds of ms from stimulation. The representative Ba sample was taken from an M cell. All other examples are from CC cells. E, Percentages for each type of eEPSC for CC and Atoh1-lineage M and L cells. Nonresponsive (NR) cells are included. Upon lumbar stimulation (distal), Si eEPSCs were the most frequent type in CC cells, followed by Ba eEPSCs. Only 27% of M cells and 14% of the L cells showed any response (i.e., 73 and 86% were NR, respectively). No Multi eEPSCs were present in M cells. Only two L cells responded and were either the Si or Ba type. Upon thoracic stimulation (local), CC and Atoh1-lineage medial neurons had several responsive cells (CC, 67%; medial, 82%), whereas 47% of Atoh1-lineage lateral neurons were responsive to local stimulation. F–I, Characteristics of CC single eEPSCs. F–G, Amplitude and first latency for Si eEPSCs are shown for distal (lumbar) or local (thoracic) stimulation. H, Si eEPSCs in CC cells did not show any significant failures when stimulated at a frequency of 20 Hz. I, When amplitudes of the first versus the last EPSC in a train were compared, most of them showed depression above 50%.
- Figure 5.
Optogenetic activation of Pv-lineage inputs onto Atoh1-lineage neurons. A, ChR2 is expressed in Pv-lineage sensory neurons [PvIRES-Cre; R26LSL-ChR2(H134R)-EYFP (Ai32)] and tdTomato in Atoh1-lineage spinal cord neurons (Atoh1P2A-FLPo; R26FSF-tdTom). VGLUT1 antibody labels afferent terminals of proprioceptive and low-threshold mechanoreceptors (LTMRs). Pv-lineage neurons expressing ChR2-EYFP (GFP Ab) have very few axons labeled in the LTMR recipient zone (arrowheads) indicating restricted expression to proprioceptors at P14. B, Representative traces of a medial (7 sweeps) and lateral (10 sweeps) cell with low jitter and of a lateral cell with high jitter (10 sweeps). ChR2 is activated with blue light for ∼12 ms, the limit of the shutter speed of the X-Cite lamp. C, The percentage of responsive M and L cells. D, Apparent latencies for responsive M and L cells. E, Apparent latencies of each M and L cell that responded to optogenetic stimulation (7 sweeps for M, 10 sweeps for L). Only one L cell had high jitter (H). Scale bar: 100 µm (A).
- Figure 6.
Diverse presynaptic inputs to Atoh1-lineage neurons. A, Diagram of the monosynaptic rabies virus approach used to identify cells presynaptic to thoracolumbar Atoh1-lineage neurons. Cells containing both CRE and FLPo recombinase express the HTB allele (histone-GFP, TVA receptor, and B19G glycoprotein). Starter cells expressing the HTB allele and infected with RVΔG-mCherry-EnvA are GFP+ mCherry+. Presynaptic cells in the spinal cord and dorsal root ganglion (DRG) are mCherry+. B, Infection efficiency at the injection site (GFP+mCherry+/total GFP+) and percentage of presynaptic cells in the spinal cord (mCherry+-only/total mCherry+) are shown. C–D’’’, Two examples of unilateral spinal cord rabies virus injections. C,C’, D,D’, Atoh1-lineage starter cells (arrows) and local spinal cord presynaptic cells (arrowheads) can be seen at the site of injection. C’’, An example of an mCherry+ cell in the DRG that expresses PV (arrow). D’’, D’’’, An example of two mCherry+ cells in the DRG that are myelinated (NF200+, arrows). An example of an mCherry+ cell in the DRG that expresses TRKC (arrow). All panels show endogenous mCherry fluorescence except for panels D and D’, which shows amplification of the mCherry signal with dsRed antibody. Scale bars: 100 µm (C–C’’, D–D’’’).
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