Principles of interneuron development learned from Renshaw cells and the motoneuron recurrent inhibitory circuit

Abstract

Renshaw cells provide a convenient model to study spinal circuit development during the emergence of motor behaviors with the goal of capturing principles of interneuron specification and circuit construction. This work is facilitated by a long history of research that generated essential knowledge about the characteristics that define Renshaw cells and the recurrent inhibitory circuit they form with motoneurons. In this review, we summarize recent data on the specification of Renshaw cells and their connections. A major insight from these studies is that the basic Renshaw cell phenotype is specified before circuit assembly, a result of their early neurogenesis and migration. Connectivity is later added, constrained by their placement in the spinal cord. Finally, different rates of synapse proliferation alter the relative weights of different inputs on postnatal Renshaw cells. Based on this work some general principles on the integration of spinal interneurons in developing motor circuits are derived.

Figure 1

Diagram of basic connectivity in the recurrent and reciprocal inhibitory circuit controlling motor output, superimposed on a Nissl stained section of the lumbar spinal cord. Motoneurons are arranged in pools that innervate different muscles. While Renshaw cells receive inputs from certain pools and provide feedback inhibition to the same motoneurons and its synergists, Ia inhibitory interneurons mediate reciprocal inhibition, such that they inhibit motor pools with antagonist actions to the muscle of origin of the Ia afferent, thus permitting smooth flexor extension around individual joints. Ia inhibitory interneurons and motoneurons receiving common Ia inputs also receive similar excitatory drive from other systems, such that activation of motor pools is always coupled with relaxation of antagonists. However, Ia inhibitory interneuron activation is tightly controlled, in part, by the Renshaw cells themselves, so that the amount of relaxation or cocontraction of antagonist muscles is finely modulated. Excitatory drive onto motoneurons acts on this last order circuit involved in the last step of motor control. Each of the two interneurons display specific placement in the ventral horn and in connectivity with target motoneurons and incoming Ia afferents, as depicted in the diagram.

Figure 2

Early formation of the recurrent inhibitory circuit in mouse embryos. In E10.5 embryos, Renshaw cell precursors (RCs) are identified as the first calbindin (CB)-IR interneurons generated in the spinal cord. Other CB-expressing cells are present in the floor plate (FP) and dorsal root ganglion (DRG). This E10.5 section was counterstained with Tuj1 antibodies to depict the location of differentiating neurons and their axons. At this age, ventral roots are already formed and motor axons are entering the limb buds. CB-IR Renshaw cells are exiting the progenitor zone (negative for Tuj1 immunoreactivity) and position themselves laterally. By E11.5, two populations of CB-IR interneurons are present in the ventral horn. Most CB-IR interneurons in central regions are not RCs; they correspond with interneurons that, in their majority, will downregulate CB during embryonic and early postnatal development. RC precursors are located at the lateral edge and actively migrate toward the ventral root. This pathway surrounds the motoneurons (labeled by EGFP, driven by the Hb9 promoter), which have not yet started to separate into discrete columns (although they already express different EGFP levels). At this time, the ventral funiculus is formed (axons heavily immunoreactive for SV2) and CB-IR RC axons appear for the first time at this location. Between E12 and E13, the major motor columns of the lumbar spinal cord (MMC, LMCd, LMCv) can be distinguished by different levels of EGFP expression and they start to segregate spatially. During this time, we find the first evidence of immunoreactivity against the vesicular acetylcholine transporter (VAChT). VAChT-IR axons are restricted to locations containing motor pools, motor axons, and RCs. The first evidence of EGFP and VAChT-IR processes in contact with RCs (arrows) is also apparent at this time, suggesting the possibility of early motor axon synaptic interactions. RCs emit an ascending axon located in the ventral funiculus, but this has no synaptic collaterals entering the spinal cord. A large invasion of the motor pools by synaptophysin-containing axons does not occur until E15. Before that time, all synaptic markers are restricted to the developing white matter. The presence of synaptophysin processes coincides with the presence of CB-IR axons putatively originating from RCs around motoneurons. It is also at this time that the spatial relationships of different motor columns mature and a tight cluster of RCs is formed between the LMC and MMC in front of the ventral root, now placed more ventrally instead of more laterally, due to spinal cord morphogenetic maturation. Some CB-IR processes are in contact with EGFP-labeled motoneurons and contain synaptophysin, suggesting the presence of synapses. Finally, other inputs, like Ia afferents, are added later. In the image, dorsal root (DR) axons were anterogradely labeled by filling the dorsal roots with FITC-conjugated dextrans in a section that was also immunostained for CB. CB-IR RCs are at the very bottom of the ventral horn, which explains the relatively late formation of Ia afferent synapses on them.

Figure 3

Landmark steps in the specification of Renshaw cells and their connections (events above the time line) in the context of other developmental processes in the spinal cord (events below the time line), and the corresponding motor output. Dates are approximations because of differences associated with spinal cord rostrocaudal level and even small differences within litters, with embryos showing more or less advanced maturation.