Ascending propriospinal modulation of thoracic sympathetic preganglionic neurons during lumbar locomotor activity

Figure 1. Calcium responses in thoracic motoneurons (MNs) during tonic and rhythmic ventral root (VR) activity. (A) Schematic of the retrograde dye labeling procedure and lumbar VR recording set-up (left). Transverse section of thoracic spinal cord with retrogradely labeled MNs and SPNs (right). (B) VR recordings at baseline, and during tonic and rhythmic activity. (C) Calcium imaging responses of 22 motoneurons at T10–11 during rhythmic activity induced by 20 μM 5-HT, 5 μM NMDA, and 50 μM DA. (D) Ca-FI traces from four representative MNs (MNs 1–4 in C), shown during baseline, tonic, and rhythmic VR activity. Ca-FI increased during tonic activity with some transitioning to rhythmic oscillations. Note that Ca-Fl intensity levels were higher during tonic versus rhythmic activity for each of these 4 MNs and that rhythmic oscillations were clearly evident during rhythmic VR activity. Calcium intensity is shown in arbitrary units (AU, scale bar top right). (E) Individual Ca-RI (left), and violin plots (right) for all 22 MNs during tonic and rhythmic VR activity. Most MNs showed an initial increase in Ca-RI during tonic activity compared to normalized baseline, which may further increase during…

Figure 2. Calcium responses of thoracic SPNs during VR activity induced by whole-bath application of 5-HT, NMDA, and DA. (A) Schematic showing transverse section of spinal cord with rectangle highlighting IML region of interest (top left); image below shows masks over cell bodies of four CGDA-retrogradely labeled thoracic SPNs at T6–7 (bottom left); and calcium imaging set up with lumbar VR recordings is shown in accompanying schematic (top right). (B) Violin plots show changes in calcium relative intensity (Ca-RI) corresponding to VR activity in (C). Baseline, tonic, and rhythmic VR activity shown for a single L5 VR. Violin plot Ca-RI values are normalized to baseline background fluorescence (marked by line at 1.0). Recordings were obtained during 12 s calcium imaging episodes sampled at 7.8 Hz. Note that Ca-RI increased more during tonic (median = 1.065) versus rhythmic (median 1.035) activity.

Figure 3. Increased recruitment of thoracic SPNs during tonic and rhythmic VR activity. (A) Schematic showing transverse section of spinal cord with rectangle highlighting IML region of interest (left) and experimental set-up for SPN imaging at T4–5 within the whole-bath configuration for applying locomotor-inducing neurochemicals to the entire spinal preparation. (B) Representative calcium images of SPNs at the T4–5 level in a GCaMP6s P2 mouse with VR recordings at baseline (i), and during tonic (ii) and rhythmic (iii) VR activity induced by 20 μM 5-HT and 5 μM NMDA. Images below each electroneurogram show clusters of SPNs with images captured during baseline, tonic, and rhythmic activity. As highlighted by the white box in the magnified images on the right, two SPNs exhibit visibly progressive increases in Ca-Fl during tonic and then rhythmic activity. (C) Left panel: Individual Ca-RI values for the 9 recorded SPNs during tonic and rhythmic VR activity. Most SPNs (8/9) had increased Ca-RI compared to the normalized baseline (shown as the solid black line at 1.0) during tonic activity. Similarly, most SPNs (8/9) had increased Ca-RI during rhythmic VR activity compared to normalized…

Figure 4. Thoracic SPN Ca-RI responses recorded from a variety of thoracic segmental levels in response to whole cord drug application. (A) Experimental set up for the whole-bath configuration, as in Figure 3. (B) Electroneurogram recordings during baseline, tonic and rhythmic L5 VR activity with timing of simultaneous imaging of SPNs at T6-7 shown by colored bars above each trace. (C) Ca-RI responses for 3 different experiments recorded T4-5, T6-7, and T10-11 (colors reference segments shown in schematic in A). Upper panels show individual Ca-RI responses during tonic and rhythmic VR activity, normalized to baseline, with corresponding violin plots shown in lower panels. The majority of SPNs at T4-5 in this experiment (left) showed increased Ca-RI during tonic VR activity whereas fewer (~ half) had increased Ca-RI during rhythmic VR activity. Violin plots indicate a slight increase in Ca-RI during tonic VR activity (median = 1.03), with SPN responses during rhythmic VR activity more variable, with 5 increasing, 2 decreasing, and most remaining similar to baseline (median = 1.01). At T6-7 (middle), SPNs showed greater increases in Ca-RI during rhythmic (median = 1.06) compared to tonic VR…

Figure 5. Thoracic SPN Ca-RI responses recorded from a variety of thoracic segmental levels in response to lumbar spinal cord drug application. (A) Schematic showing split-bath configuration with barrier to physically separate thoracic and lumbar regions for selective pharmacological activation of lumbar spinal neurons (gray rectangle, bottom), and photomicrograph showing SPNs imaged at T6-7 level in a GCaMP6s P4 mouse (top). (B) Corresponding neurogram recordings during baseline, tonic, and rhythmic L5 VR activity with timing of simultaneous imaging of SPNs at T6-7 shown by coloured bars above each trace. (C) Ca-RI responses for 3 different representative experiments recorded at T4-5, T6-7, and T8-9 [colors reference segments shown in schematic in (A)]. Upper panels represent the individual Ca-RI responses, with corresponding violin plots shown in lower panels. Most SPNs at T4-5 (left) showed decreased Ca-RI during tonic VR activity, as represented by a median change to 0.95 in the violin plot, whereas about half had increased Ca-RI during rhythmic VR activity with the averaged response near baseline (median = 1.01). SPNs at T6-7 segment (middle), exhibited a larger increase in Ca-RI…

Figure 6. Distinct thoracic SPN Ca-RI responses are observed at different rostrocaudal levels during locomotor activity evoked by either whole cord or lumbar-only drug application. Schematic of the whole-bath design (A) and split-bath design for lumbar only drug application (B). Ca-RI changes are summarized for all SPNs recorded at each segmental level examined (color-coded by segment to match schematics). SPN responses to whole bath application are shown on the left and split-bath application of drugs to lumbar-only regions on the right, with responses during tonic VR shown on the top and rhythmic VR activity on the bottom. The gray line is the normalized baseline (1.0), and the gray shadowed region indicates Ca-RI changes < or > 3%, with SPNs in this range depicted by clear circles. During tonic VR activity (Ai,Bi), SPNs showing increases or decreases in Ca-RI > 3% are depicted by dark or light blue circles, respectively. (Aii,Bii): During rhythmic VR activity, SPNs showing increases or decreases in Ca-RI > 3% are depicted by dark or light pink circles, respectively. In the whole-bath configuration during either tonic or rhythmic VR activity, greater numbers of SPNs had increased Ca-RI…

Figure 7. 3-way ANOVA graphic showing main interactions for mean Ca-RI as the measured variable and nominative variables thoracic level (T4–T5, T6–T7, T8–T9), lumbar VR activity (tonic, rhythmic) and bath configuration (whole, split). Main interactions within bath configuration, and between thoracic region, during either tonic (A) or rhythmic (B) lumbar VR activity. Main interactions between tonic and rhythmic activity, and between bath configurations at each thoracic level is shown in (C). Median values are plotted for the whole-bath (solid rectangle) and split-bath (half-filled rectangle) configurations.