Lumbar V3 interneurons provide direct excitatory synaptic input onto thoracic sympathetic preganglionic neurons, linking locomotor, and autonomic spinal systems

Abstract

Although sympathetic autonomic systems are activated in parallel with locomotion, the neural mechanisms mediating this coordination are incompletely understood. Sympathetic preganglionic neurons (SPNs), primarily located in the intermediate laminae of thoracic and upper lumbar segments (T1-L2), increase activation of tissues and organs that provide homeostatic and metabolic support during movement and exercise. Recent evidence suggests integration between locomotor and autonomic nuclei occurs within the brainstem, initiating both descending locomotor and sympathetic activation commands. However, both locomotor and sympathetic autonomic spinal systems can be activated independent of supraspinal input, in part due to a distributed network involving propriospinal neurons. Whether an intraspinal mechanism exists to coordinate activation of these systems is unknown. We hypothesized that ascending spinal neurons located in the lumbar region provide synaptic input to thoracic SPNs. Here, we demonstrate that synaptic contacts from locomotor-related V3 interneurons (INs) are present in all thoracic laminae. Injection of an anterograde tracer into lumbar segments demonstrated that 8-20% of glutamatergic input onto SPNs originated from lumbar V3 INs and displayed a somatotopographical organization of synaptic input. Whole cell patch clamp recording in SPNs demonstrated prolonged depolarizations or action potentials in response to optical activation of either lumbar V3 INs in spinal cord preparations or in response to optical activation of V3 terminals in thoracic slice preparations. This work demonstrates a direct intraspinal connection between lumbar locomotor and thoracic sympathetic networks and suggests communication between motor and autonomic systems may be a general function of the spinal cord.

Keywords: motor systems; optical stimulation; propriospinal neurons; spinal interneurons; sympathetic preganglionic neurons.

FIGURE 1

V3 neuronal cell bodies distributed throughout the thoracic and lumbar spinal regions observed in adult mice. (A) Schematic and cartoon demonstrating Rexed’s Laminae and regions from dorsal (green boxes) and ventral (yellow boxes) regions for images shown in panel (C). (B) Summary data showing mean numbers (±SD) of V3 cell bodies from sections in rostral and caudal thoracic and lumbar regions. Similar numbers of V3 cell bodies were observed within rostral and caudal thoracic and lumbar regions but differed between thoracic and lumbar regions (****P < 0.0001, n = 5, one way ANOVA), (C) V3 cell bodies were observed at each spinal level in ventral regions (C, i–l) and in dorsal regions at T1 (Ce), T7 (Cf) and L1/2 (Cg) but were absent at L5 (Ch). Scale bar in a–d = 200 μm, and = 50 μm for remaining images. White arrowhead denote V3 soma.

FIGURE 2

V3 neuron terminals observed within the IML at all thoracic spinal segments, with highest density observed at mid-thoracic levels in adult mice. (A) Schematic demonstrating V3 cell bodies and terminals observed in gray matter of spinal cord. XY coordinates of V3 terminals were exported to create contour maps in Imaris. (B) Representative contour plots of relative V3 terminal densities within gray matter at each thoracic spinal level from one adult mouse. Note the high density of V3 terminals in the IML and throughout gray matter. (C) Graph summarizing numbers of V3 terminals observed in IML ROI (278 μm × 277 μm) at each thoracic spinal level for each animal examined (n = 3 animals, 12 sections per animal). Numbers of terminals within the gray matter ranged from ∼2,500 – ∼17,700, and within the IML, terminal numbers ranged from (∼500 – ∼2,400) per section, depending on the rostrocaudal level of the section (n = 3).

FIGURE 3

V3 synaptic terminals directly appose thoracic SPNs in adult mice. (A) Double labeled VGluT2 and TdTom terminals apposing SPNs in rostral thoracic spinal cord. Double labeled neurons indicated with arrowheads (white in merged image). (B) Double labeled VGluT2 and TdTom terminals apposing SPNs in caudal thoracic spinal cord. Double labeled neurons indicated with arrowheads (white in merged image). (C) Rostrocaudal distribution of percentage of double labeled VGluT2⁺/TdTom⁺ puncta within thoracic spinal cord. Mean + SD shown. (D) Glutamatergic excitatory V3 IN innervation of SPNs averaged ∼20% per section examined, with no significant difference between T1-T6 and T7-T13 regions (p > 0.9999 Kruskal–Wallis test, n = 4 mice). Scale bar = 20 μm for panels (A,B).

FIGURE 4

BDA injection in lumbar SC demonstrates ascending V3 neuronal projections to thoracic SPNs in adult mice. (A) Schematic of BDA injections. (B) Double-labeled BDA and TdTom terminal apposing SPNs in read rostral and caudal thoracic spinal cord (arrowheads and white in merged image). (C) Rostro-caudal distribution of BDA⁺ puncta shown by thoracic segmental level, based on lumbar injection level (note that data for n = 2 mice injected at L2 is mean + SD). (D,E) In animals with BDA injection at L2, higher percentage of BDA⁺/TdTom⁺ terminals were present in sections from rostral thoracic compared to caudal thoracic regions (**p = 0.0067, n = 2 mice, Mann–Whitney). In contrast, BDA injections in L4/5 demonstrated higher percentages of BDA⁺/TdTom⁺ terminals in sections from caudal thoracic compared to rostral thoracic regions (***p = 0.0002, n = 1, Mann–Whitney). L3 BDA injections showed no significant difference in percentage of BDA⁺/TdTom⁺ terminals in sections from either rostral or caudal thoracic regions (p = 0.8568, n = 1, Mann–Whitney). Scale bar = 20 μm for panels (A,B).

FIGURE 5

Channel rhodopsin expression in lumbar V3 neurons and nerve terminals in the IML in neonatal mice. (A) Transverse sections from L2 spinal cord demonstrating V3 neurons (TdTomato Ai, red) also express channel rhodopsin (Aii, green), with merged image in Aiii, yellow. Dorsal (D) and ventral (V) and central canal (CC) shown. (B) Transverse sections of T6 spinal cord demonstrate expression of Tdtomato (Bi) in V3 fibers within IML (white box), channel rhodopsin (Bii), and both in merged images (Biii). (C) Optical stimulation evokes multiple action potential in whole-cell patch clamped V3 neurons located in thoracic SC. Example from n = 10 cells from 6 mice of either sex.

FIGURE 6

Optical stimulation of V3 nerve terminals elicits excitatory post synaptic responses in thoracic SPNs in neonatal mice. Optical stimulation of V3 nerve terminals could elicit EPSPs [(A) n = 2] or action potentials [(B) n = 4] in whole cell patched clamped thoracic SPNs. Examples from n = 10 cells from 4 mice of both sexes.

FIGURE 7

Optical stimulation of lumbar V3 neurons elicits action potentials in thoracic SPNs in neonatal mice. In intact spinal cord preparations, thoracic SPNs labeled with RDA were visualized under fluorescence and whole cell patch clamp recordings were collected in response to optical stimulation of the ventral surface of the L2 spinal segment. In this example, lumbar V3 stimulation consistently evoked action potentials in the patched SPN. Representative example from one of the n = 20 cells recorded from 15 mice of either sex.