Cholinergic mechanisms in spinal locomotion-potential target for rehabilitation approaches

Abstract

Previous experiments implicate cholinergic brainstem and spinal systems in the control of locomotion. Our results demonstrate that the endogenous cholinergic propriospinal system, acting via M2 and M3 muscarinic receptors, is capable of consistently producing well-coordinated locomotor activity in the in vitro neonatal preparation, placing it in a position to contribute to normal locomotion and to provide a basis for recovery of locomotor capability in the absence of descending pathways. Tests of these suggestions, however, reveal that the spinal cholinergic system plays little if any role in the induction of locomotion, because MLR-evoked locomotion in decerebrate cats is not prevented by cholinergic antagonists. Furthermore, it is not required for the development of stepping movements after spinal cord injury, because cholinergic agonists do not facilitate the appearance of locomotion after spinal cord injury, unlike the dramatic locomotion-promoting effects of clonidine, a noradrenergic α-2 agonist. Furthermore, cholinergic antagonists actually improve locomotor activity after spinal cord injury, suggesting that plastic changes in the spinal cholinergic system interfere with locomotion rather than facilitating it. Changes that have been observed in the cholinergic innervation of motoneurons after spinal cord injury do not decrease motoneuron excitability, as expected. Instead, the development of a "hyper-cholinergic" state after spinal cord injury appears to enhance motoneuron output and suppress locomotion. A cholinergic suppression of afferent input from the limb after spinal cord injury is also evident from our data, and this may contribute to the ability of cholinergic antagonists to improve locomotion. Not only is a role for the spinal cholinergic system in suppressing locomotion after SCI suggested by our results, but an obligatory contribution of a brainstem cholinergic relay to reticulospinal locomotor command systems is not confirmed by our experiments.

Keywords: cholinergic mechanisms; chronic spinal cat; chronic spinal rat; decerebrate cat; in vitro neonatal rat; spinal rhythm generation.

Figure 1

Episodes of rhythmic activity are induced after bath application of EDRO. (A) EDRO applied to the bath 30 s after the onset of the recording induced episodes of rhythmic activity with waxing and waning amplitude of the signal in all channels for several minutes. (B) Expanded sample from the same recording showing episodes of rhythmic activity (the area indicated by vertical lines) separated by periods of decreased activity. (C) Expanded sample of the recordings in (B) (the area indicated by vertical lines) showing well-coordinated locomotion characterized by alternating flexor/extensor (L2/L5) and right/left ENG activity (r/l).

Figure 2

Induction of locomotion in vitro by 50 μM EDRO. (A) ENG recordings showing sustained alternating flexor/extensor and left/right activity. (B) Rectified and filtered waveforms of the traces in (A). (C) Polar plots derived from pooled data from 19 experiments showing the circular distribution of mean phase values and mean angles of the right flexor ventral root ENG (r L2) vs. the right extensor ventral root ENG (r L5), and right (r L2) vs. left (l L2) flexor ENGs (green vectors). The superimposed r vectors show the concentration of phase values around the mean angle. Mean rhythm frequency (Hz) of all filtered and rectified r L2 waveforms: 0.213 ± 0.052 Hz (0.13–0.33 Hz). Mean step duration of all rectified and filtered r L2 waveforms: 4.908 ± 1.312 s (4.85–7.87 s). The phase value for ipsilateral flexor/extensor activity (θ = 205°) as well as the bilateral flexor (r L2 vs. l L2) phase value (θ = 201°) for the example shown in (A,B,D) is shown in (C) with the red vector. (D) Overlay of step cycles (triggered on the onset of l L2 activity) of each rectified and filtered waveform shown in (B).

Figure 3

Atropine, telenzepine, and methoctramine effects on the EDRO-induced locomotor rhythm. (A1) Rectified and filtered waveforms showing atropine block of EDRO-induced locomotion. (A2) Baseline locomotor-like activity. (A3) Transient increase in frequency and a decrease in amplitude after 1 μM atropine. (A4) Progressive reduction of EDRO-induced activity by atropine. (B1) Telenzepine (20 μM), an M₁ receptor antagonist, blocks EDRO-induced locomotor-like activity only at high doses, decreasing the amplitude but not the frequency of the ENG activity. (B2–B4) Methoctramine (METHOC, M2 receptor antagonist) produces an increase in frequency of EDRO-induced locomotion at 1–2 μM. (B2) Rectified and filtered waveforms recorded from the right L2 and L5 ventral roots produced by 100 μM EDRO. (B3) Baseline locomotor-like activity recorded during the period indicated by the horizontal bar below the ENG trace in (B1). (B4) Increase in frequency of the ENG bursts resulting from a cumulative dose of two 1 μM METHOC (period indicated in B2).

Figure 4

EDRO-induced locomotion is blocked by 4-DAMP. (A) 50 μM EDRO induced a sustained pattern of locomotion. (A, right panel) Polar Plots of flexor-extensor and left/right alternation show highly coordinated locomotion prior to the application of 4-DAMP. (B) 4-DAMP applied to the bath decreased rhythm frequency from 0.178 to 0.063 Hz followed by complete blockade of rhythmic activity. An additional dose of EDRO (100 μM) failed to induce locomotion (data not shown), but adding 1 μM 5-HT to the bath produced tonic firing followed by locomotor-like activity. The fact that 5-HT can “rescue” locomotor-like activity despite continued block of the EDRO effect shows that the preparation remained viable and that M₃ receptor blockade did not interfere with locomotion induced by 5-HT. (C,D) Comparison of the effects of 4-DAMP on EDRO-induced locomotion with hind limbs attached (C) or removed (D). The right panels show the results from the application of 4-DAMP in the two preparations.

Figure 5

Effect of mecamylamine and atropine on locomotion induced by electrical stimulation of the MLR. (A) Site of stimulation was localized to the ventrolateral border of the cuneiform nucleus (CNF) near the brachium conjunctivum (BC) at P2, L4, H-1.5 (coordinates according to Berman, 1968). (B) Treadmill locomotion evoked from this brainstem site prior to any drugs (stimulus strength of 135 μA); (C) following the infusion of mecamylamine (3 mg/kg, stimulation strength 60 μA) and (D) following atropine (2.5 mg/kg, stimulation strength 70 μA). Thresholds for the initiation of locomotion for (B–D) were 150, 150, and 75 μA, respectively. The gain of each muscle's EMG is the same throughout all trials.

Figure 6

Neither EDRO nor ACh in the presence of EDRO facilitated the onset of locomotor activity in a spinal cat. (A) Pre-drug records of a cat walking on a treadmill with strong exteroceptive stimulation used to activate the locomotor CPG. Stick figures of the limb trajectory over 5 s of activity, along with the corresponding joint angles and EMG recordings are illustrated. (B) Similar records taken 35 min. after 4 mM EDRO administration. (C) Recordings taken 1 h after administration of 2 mM ACh and 2 h after the original dose of edrophonium. (D) Subsequent dose of clonidine (3.8 mM) elicited typical stepping movements on the treadmill, as illustrated by joint angles and EMG, showing that the animal was capable of producing locomotion.

Figure 7

Carbachol, a cholinergic agonist, produced co-contractions and interfered with locomotor activity early after injury. (A) Pre-drug trial with EMG activity produced by perineal stimulation. (B) 1 h 45 after i.t. carbachol (1 mM, 100 μl) administration, perineal stimulation produced uncoordinated rhythmic activity.

Figure 8

Intrathecal atropine facilitated locomotion in spinal cats. (A–D) show kinematic and EMG recordings of a cat walking on a treadmill at increasing time after atropine (1 mM, 100 μl) administration: stick figures of one step cycle (top), averaged angular excursion of the four joints over N > 12 step cycles (middle) and corresponding averaged rectified EMG traces (bottom). The cycle is normalized to 1 and is repeated twice (separated by doted vertical lines) for clarity at turning points. The average is synchronized with the contact of the left foot (LC). (A) Pre-drug recordings at a treadmill speed of 0.4 m/s; (B) 12 min after atropine administration at a treadmill speed of 0.6 m/s; (C) 36 min after atropine at a treadmill speed of 0.8 m/s; (D) 3 h after Atropine at a treadmill speed of 0.7 m/s. (E) Cycle duration as a function of treadmill speed, showing a maximal increase in treadmill speed the cat could follow after atropine at approximately 30 min, with a slight decrease at 3 h after drug administration.

Figure 9

Carbachol disruption of locomotion in spinal cats is reversed by atropine. (A–C) regular stepping movements before drug administration 14 days after the lesion represented by raw EMG traces (A), averaged (N = 12 cycles) joint angular displacements (B) and stick figures (C). (D) Raw EMG recordings after intrathecal carbachol (1 mM, 100 μl) showing deterioration of stepping as soon as 7 min post drug administration even with strong perineal stimulation. (E,F) 55 min post carbachol, the EMG is still not organized to produce a good stepping pattern (E). There is no foot placement as represented by the stick figures (F). (G–I) Five minutes after intrathecal administration of atropine (1 mM, 100 μl), a good pattern of stepping was observed without perineal stimulation as was the case before carbachol administration [compare (G–I) with (A–C) same display]. (J) Cycle duration expressed in terms of treadmill speed, showing disruption of locomotion by carbachol, and facilitation of locomotion with atropine.

Figure 10

Carbachol disruption of locomotion in spinal rats is reversed by atropine. The left panels show representative frames from the videos taken during the time of the corresponding EMG activity (right panels). (A) Irregular stepping movements produced with tail stimulation prior to drug administration. As shown in the left panel, the EMG activity did not give rise to plantar stepping. The toes were always dragging on the treadmill surface during rhythmic alternating activity. (B) Intrathecal carbachol (1 mM, 20 μl) eliminated all stepping movements immediately, and only a few sporadic episodes of largely synchronous bursting occurred (recording taken 1 min after administration). (C) The intrathecal administration of atropine (1 mM, 20 μl) 10 min after carbachol treatment restored alternating movement and led to occasional plantar stepping (left panel). The EMG activity was more consistent, and occurred even in the absence of tail stimulation. The recording was made 30 min after atropine was given.

Figure 11

Atropine facilitates the cutaneous reflex responses evoked by stimulation of superficial peroneal (SP) nerve. (A,B) SP facilitation by intrathecal atropine (70 μg, 100 μl) 1 day after the complete section of the spinal cord: (A) averaged (n = 25) rectified response in ST, GL, and TA muscles after stimulation of SP (1.5T, 3 pulses, 300 Hz) 300 Hz before (dotted line), 53 (thin gray line) and 92 (thick black line) min post atropine; (B) illustrates the evolution of the response amplitude of the SP reflexes as a function of control responses in 4 muscles on the side of the stimulation. (C,D) SP facilitation by atropine 1 week after SCI. Atropine 1 and 2 correspond to intrathecal atropine (70 μg, 100 μl) whereas Bath Atropine 3 corresponds to a bath application of 1.5 ml of a solution containing 1 mg/ml of atropine.

Figure 12

The effect of a sub-threshold dose of clonidine is facilitated by atropine. Clonidine was first given i.v. (20 μg/kg) and then atropine (1.5 ml of a solution containing 1 mg/ml of atropine) was given through a bath covering the L3-L4 segments of the spinal cord 40 min later in a 4 days spinal cat. (A) EMG signals from hindlimbs muscles 80 min after clonidine injection and 40 min after atropine application. Some bursts of activity appear after atropine especially in the hip flexors (St); (B) 60 min after atropine some alternating activity appears between flexor and extensor muscles but it was not organized enough to trigger strong locomotion; (C,D) strong pattern of locomotion was observed 110 and 150 min post atropine.

Figure 13

Sub-threshold clonidine is facilitated by atropine in an acute spinal cat. (A) Pre-drug records of EMG signals from hindlimbs muscles; (B) 20 min after administration of a small volume of clonidine i.v. (20 μg/kg); (C) 20 min after bath atropine (1.5 ml of a solution containing 1 mg/ml of atropine) over L3-L4 and 40 min post clonidine; (D) 55 min after atropine application, a vigorous pattern of locomotion is represented by the EMG signals.

Figure 14

Cartoon showing different functional groups of cholinergic neurons that are suggested by our results. A population for afferent input control (AIC) is illustrated, some of which are known to have direct presynaptic terminals on cutaneous afferents, as well as terminals on GABAergic interneruons (not shown) that may also be responsible for suppressing afferent input. AIC neurons act on their targets through both muscarinic and nicotinic receptors. A population of cholinergic neurons (LRC) that can control the locomotor rhythm by activating the central pattern generator for locomotion (CPG) are also shown. Their precise positions are unknown. They act on their target neurons via M3 receptors. Pitx2 neurons give rise to C terminals on motoneurons and increase motoneruron excitability by reducing the AHP amplitude via M2 receptors. They are also known as V0c neurons. Commissural cholinergic interneurons (CIN) are illustrated, many of which terminate on contralateral motoneurons and produce EPSPs in motoneurons via M-currents blocked by atropine. Other partition or central canal cluster cells with unknown function are likely also present in the spinal cord.