Identification of a cellular node for motor control pathways

Abstract

The rich behavioral repertoire of animals is encoded in the CNS as a set of motorneuron activation patterns, also called 'motor synergies'. However, the neurons that orchestrate these motor programs as well as their cellular properties and connectivity are poorly understood. Here we identify a population of molecularly defined motor synergy encoder (MSE) neurons in the mouse spinal cord that may represent a central node in neural pathways for voluntary and reflexive movement. This population receives direct inputs from the motor cortex and sensory pathways and, in turn, has monosynaptic outputs to spinal motorneurons. Optical stimulation of MSE neurons drove reliable patterns of activity in multiple motor groups, and we found that the evoked motor patterns varied on the basis of the rostrocaudal location of the stimulated MSE. We speculate that these neurons comprise a cellular network for encoding coordinated motor output programs.

Figure 1

Labeling of first-order spinal neurons targeting gastrocnemius motorneurons. (a, b) Images of a RabΔG:GFP-labeled spinal cord following injection into the medial gastrocnemius (GS) muscle. (a) Lateral projection of an optically cleared lumbar spinal cord, shows motorneurons (GS MNs) in the ventral horn of L4 and L5 spinal segments. Premotor cells and fibers can be seen in the dorsal funiculus (DF), laminae I–IV, laminae V–VI and laminae VII–IX. (b) Collapsed transverse view of the lumbar spinal cord in a, shows ipsilateral (ipsi) and contralateral (contra) premotor cells. (c) Quantification of total pregastrocnemius spinal neurons (all laminae) and of the subset of pregastrocnemius neurons in medial laminae V–VI along the rostral-caudal axis of the lumbar spinal cord. Neuron counts (IN#) were normalized to the maximum number of neurons in a single section for each spinal cord to control for the variability in labeling. Means and s.d. are shown. Values are listed in Supplementary Table 1. Location 0 indicates the section with the peak number of motorneurons, and is usually in caudal L4. (d) Premotor cell distributions depicted on transverse spinal cord images representing the spinal level with peak motorneurons (0 mm), at a level 1.5 mm rostral (mid-lumbar) and at a level 3.5 mm rostral (upper lumbar/lower thoracic). Laminae were divided into functional regions, and the percentage of total premotor cells at each level are shown for each region, represented by the diameter of the colored circles. These regions are superficial dorsal horn (laminae I–IV, yellow), medial deep dorsal horn (medial laminae V–VI, green), lateral deep dorsal horn (lateral laminae V–VI, gray), ventral horn (laminae VII–IX, blue) and contralateral (gray).

Figure 2

Motorneuron responses to optical stimulation of medial deep dorsal horn premotor neurons or of nonspecific ventral interneurons. (a, c) Experimental setups for optical stimulation of transynaptic MSE neurons (a) or non-specific ventral interneuron (c). Following transynaptic RabΔG:ChR2 labeling (a) or spinal injection of non-transynaptic RabΔG: ChR2 (c), focal blue light was used to optically excite spinal neurons in the deep dorsal horn (a) or ventral horn (c). Electrical recordings were performed on the L5 ventral root that contains gastrocnemius motorneuron axons (red, orange) and the L2 ventral root that contains ileo-psoas and quadriceps motorneuron axons (purple, blue). (b, d) Ventral root recordings after stimulation of L3 MSE neurons (b) and L3 non-specific ventral interneurons (d). Black ticks indicate latency from the onset of light stimulation (blue box) to the first motorneuron action potentials. Five consecutive traces are shown for each example. (e) Mean (±s.e.m.) fraction of stimulus locations with reliable L2 and L5 ventral root activity analyzed in each spinal cord (Online Methods). *P = 0.0034, two-sided t test. Vertical scale bars, 20 μV.

Figure 3

Molecular markers Tcfap2β and Satb1/2 identify medial deep dorsal horn neurons. (a, b) Projected confocal stacks showing immunolabeling of Tcfap2β (a) and Satb1/2 (b) in transverse sections of P8 lumbar spinal cord. Scale bar, 250 μm. (c) Neurotransmitter status of Tcfap2β and Satb1/2 cells determined using in situ hybridization at P10 against vGlut2 (excitatory), Gad65 (inhibitory), and Gad67 (inhibitory), or with antibodies at P2 to identify Tlx3 (excitatory) and Pax2 (inhibitory). Mean percentages ± s.d. are: 26± 4% of Tcfap2β⁺ neurons expressed vGlut2 (n = 570 neurons in five P10 spinal cords), 67 ± 6% of Tcfap2β neurons expressed Gad65 (n = 344 neurons in 4 P10 spinal cords) and 40 ± 8% expressed Gad67 (n = 201 neurons in three P10 spinal cords). It is likely that Gad65 and Gad67 are coexpressed in some cells. Among the Satb1/2⁺ population, 22 ± 5% of all Satb1/2⁺ expressed Pax2 (n = 1,056 neurons in 4 P2 spinal cords, mostly in the ventral subdivision of Satb1/2⁺ medial deep dorsal neurons), and 52 ± 7% of all Satb1/2⁺ expressed Tlx3 (n = 956 neurons in 4 P2 spinal cords, mostly in the dorsal subdivision of Satb1/2⁺ medial deep dorsal horn neurons).

Figure 4

Molecular markers Tcfap2β and Satb1/2 identify medial deep dorsal horn premotor neurons. (a–c) Projected confocal stacks showing combined immunolabeling and RabΔG labeling in transverse sections of P8 lumbar spinal cords. Distribution of gastrocnemius motorneurons and pregastrocnemius spinal neurons (RabΔG: GFP) at four rostral-caudal levels, together with immunolabeling of Tcfap2β and Satb1/2 (white) (a). The levels are at the peak of gastrocnemius motorneurons (0 mm), and 1.5 mm, 2 mm and 3.5 mm rostral. Higher-magnification images of the RabΔG-labeled premotor spinal neurons in the medial deep dorsal horn (green), positive for Tcfap2β (yellow, filled arrowheads) and Satb1/2 (light blue, unfilled arrowheads), that are directly presynaptic to the gastrocnemius (b) or wrist extensors (c). (d) Fraction of total pregastrocnemius spinal neurons (top) and medial laminae V–VI premotor spinal neurons (bottom) identified by Tcfap2β, Satb1/2 and other previously described premotor spinal neuron classes^,. Scale bars, 250 μm (a) and 25 μm (b, c).

Figure 5

MSE neurons receive sensory and corticospinal inputs. (a) RabΔG labeling of pregastrocnemius MSE (green) and genetic labeling of proprioceptive afferent synaptic terminals (Parvalbumin::synaptophysin-tdTomato (PV-Syn-Tomato), red). (b) RabΔG labeling of pre-tibialis anterior (TA) MSE (green) and genetic labeling of corticospinal terminations from the caudal motor cortex, following focal unilateral injection of AAV: Cre into the caudal motor cortex of cre-dependent synaptophysin-tdTomato (MCtx-Syn-Tomato, red) pups. (c) RabΔG labeling of MSE and immunolabeled capsaicin induced c-fos expression. (d, e) To stringently identify synaptic inputs onto RabΔG:GFP⁺ (green) and Satb1/2⁺ (white) MSE neurons from Parvalbumin::synaptophysin-tdTomato (d) or Emx1::synaptophysin-tdTomato (Ctx-Syn-Tomato) (e) neurons, colocalized GFP⁺ and Tomato⁺ pixels were identified, pseudocolored yellow and projected onto the GFP⁺ neuron. As a result of this analysis, Syn-Tomato that was not colocalized with the GFP⁺ neuron is not shown. Insets show single optical slices and also depict the total Syn-Tomato (blue) so that sites of synaptic contacts appear white. (f) High-magnification projected confocal image of a RabΔG-pre-TA (green)/Satb1/2⁺ (blue) MSE neuron boxed in c activated by a painful heel stimulus (c-fos, red), arrowhead. Scale bars, 250 μm (a–c) and 10 μm (d–f).

Figure 6

Rostral-caudal position of MSE neurons maps to distinct functional outputs. (a) Stereoscope image of the medial surface of a P8 lumbar spinal cord revealing the MSE column in the medial deep dorsal horn, labeled by pre-gastrocnemius RabΔG:ChR2-Cherry. The approximate location of gastrocnemius motorneurons (GS MNs), lumbar segments (L1–L5) and stimulation sites for data in d and e (blue circles) are shown. Scale bar, 1 mm. (b, c) Average latency of the first action potentials in the L2 and L5 ventral roots in one spinal cord (b) and as composite data from four spinal cords (c) after optical stimulation at multiple locations along the rostral-caudal axis (position). Error bars, s.d. for 10 traces for each location in b. Locations and latency points for data in d and e are boxed in gray. 0 position represents the caudal end of L5. The relationships between latency and location in the composite data were fit with a linear model (lines in c). (d, e) L5 and L2 ventral root traces (representative single traces are shown in red (L5) or purple (L2) and individual traces are in gray) after optical stimulation (blue boxes) of pregastrocnemius medial deep dorsal neurons in the L2 spinal segment (d) and the L3–L4 border (e). For a full set of additional traces, see Supplementary Figure 8.