Spontaneous rhythmic activity in early chick spinal cord influences distinct motor axon pathfinding decisions

Abstract

During embryonic development chick and mouse spinal cords are activated by highly rhythmic episodes of spontaneous bursting activity at very early stages, while motoneurons are still migrating and beginning to extend their axons to the base of the limb. While such spontaneous activity has been shown to be important in refining neural projections once axons have reached their targets, early pathfinding events have been thought to be activity independent. However, in-ovo pharmacological manipulation of the transmitter systems that drive such early activity has shown that early motor axon pathfinding events are highly dependent on the normal pattern of bursting activity. A modest decrease in episode frequency resulted in dorsal-ventral pathfinding errors by lumbar motoneurons, and in the downregulation of several molecules required to successfully execute this guidance decision. In contrast, increasing the episode frequency was without effect on dorsal-ventral pathfinding. However, it prevented the subsequent motoneuron pool specific fasciculation of axons and their targeting to appropriate muscles, resulting in marked segmental pathfinding errors. These observations emphasize the need to better evaluate how such early spontaneous electrical activity may influence the molecular and transcription factor pathways that have been shown to regulate the differentiation of motor and interneuron phenotypes and the formation of spinal cord circuits. The intracellular signaling pathways by which episode frequency affects motor axon pathfinding must now be elucidated and it will be important to more precisely characterize the patterns with which specific subsets of motor and inter-neurons are activated normally and under conditions that alter spinal circuit formation.

Figure 1. Major pathfinding decisions of lumbar spinal motor axons

A. Drawing that illustrates the sorting of LMCl dorsally projecting femorotibialis (red) and sartorius (green) motor axons from LMCm ventrally projecting adductor (blue) motoneurons in the crural pleuxus. After this dorso-ventral (D-V) sorting, the two dorsal pools segregate into discrete fascicles. The medially located adductor pool is not shown in cord for clarity. Femoro, femorotibialis; sart, sartorius; adduct, adductor. B,C. patterns of defasiculation of motor axons in the plexus in control and picrotoxin treated embryos respectively. Top-thick vibratome slices transverse to cord with motor axons visualized by eGFP under the control of a motoneuron specific promotor. Bottom- single color coded axon trajectories traced with neurolucida software from slices above.

Figure 2. Altered A-P/motoneuron-pool specific pathfinding following sarcosine treatment

A. Whole mounts of axons retrogradely labeled with fluorescent dextrans injected into the sartorius (green) and the femorotibialis (red) muscles in control and sarcosine treated embryos. No dually labeled axons were ever observed in the control or sarcosine treated cases. The slight yellow glow in the sarcosine treared example is due to glare from the injection site. B. Histograms showing the A-P localization of motoneuron somas for the sartorius and femorotibialis pools in control and sarcosine treated embryos, expressed as per cent of pool/section. C. Compound action potentials from EMG recordings from the sartorius and femorotibialis muscles following electrical stimulation of lumbosacral (LS) spinal nerves 1, 2, or 3. A-P, anterior-posterior.

Figure 3. Possible signaling pathways that may mediate the effects of altered frequencies of spontaneous activity on motor axon pathfinding

During bursts Ca²⁺ may enter through voltage dependent Ca²⁺ channels (VDCC), with L, P/Q, N, and T type channels all present. The transmitters GABA, glycine, and acetylcholine (ACh) will all be released and can act via their receptors which are present on motoneurons at early stages of development. α7 and non-α7 nicotinic receptors as well as muscarinic receptors are present. Motoneurons also express neurotrophin (NT) receptors such as TrkB which can be activated by activity dependent BDNF (brain derived neurotophic factor) release. During asynchronous firing of motoneurons between bursts Ca²⁺ influx would occur but motoneurons would not be as strongly activated by the transmitters and neurotrophins.