Gareth B. Miles

Functional Properties of Motoneurons Derived from Mouse Embryonic Stem Cells

The capacity of embryonic stem (ES) cells to form functional motoneurons (MNs) and appropriate connections with muscle was investigated in vitro. ES cells were obtained from a transgenic mouse line in which the gene for enhanced green fluorescent protein (eGFP) is expressed under the control of the promotor of the MN specific homeobox gene Hb9. ES cells were exposed to retinoic acid (RA) and sonic hedgehog agonist (Hh-Ag1.3) to stimulate differentiation into MNs marked by expression of eGFP and the cholinergic transmitter synthetic enzyme choline acetyltransferase. Whole-cell patch-clamp recordings were made from eGFP-labeled cells to investigate the development of functional characteristics of MNs. In voltage-clamp mode, currents, including EPSCs, were recorded in response to exogenous applications of GABA, glycine, and glutamate. EGFP-labeled neurons also express voltage-activated ion channels including fast-inactivating Na+ channels, delayed rectifier and IA-type K+ channels, and Ca2+ channels. Current-clamp recordings demonstrated that eGFP-positive neurons generate repetitive trains of action potentials and that l-type Ca2+ channels mediate sustained depolarizations. When cocultured with a muscle cell line, clustering of acetylcholine receptors on muscle fibers adjacent to developing axons was seen. Intracellular recordings of muscle fibers adjacent to eGFP-positive axons revealed endplate potentials that increased in amplitude and frequency after glutamate application and were sensitive to TTX and curare. In summary, our findings demonstrate that MNs derived from ES cells develop appropriate transmitter receptors, intrinsic properties necessary for appropriate patterns of action potential firing and functional synapses with muscle fibers.

Spinal cholinergic interneurons regulate the excitability of motoneurons during locomotion

To effect movement, motoneurons must respond appropriately to motor commands. Their responsiveness to these inputs, or excitability, is regulated by neuromodulators. Possible sources of modulation include the abundant cholinergic “C boutons” that surround motoneuron somata. In the present study, recordings from motoneurons in spinal cord slices demonstrated that cholinergic activation of m2-type muscarinic receptors increases excitability by reducing the action potential afterhyperpolarization. Analyses of isolated spinal cord preparations in which fictive locomotion was elicited demonstrated that endogenous cholinergic inputs increase motoneuron excitability during locomotion. Anatomical data indicate that C boutons originate from a discrete group of interneurons lateral to the central canal, the medial partition neurons. These results highlight a unique component of spinal motor networks that is critical in ensuring that sufficient output is generated by motoneurons to drive motor behavior.

Mechanisms underlying the early phase of spike frequency adaptation in mouse spinal motoneurones

Spike frequency adaptation (SFA) is a fundamental property of repetitive firing in motoneurones (MNs). Early SFA (occurring over several hundred milliseconds) is thought to be important in the initiation of muscular contraction. To date the mechanisms underlying SFA in spinal MNs remain unclear. In the present study, we used both whole‐cell patch‐clamp recordings of MNs in lumbar spinal cord slices prepared from motor functionally mature mice and computer modelling of spinal MNs to investigate the mechanisms underlying SFA. Pharmacological blocking agents applied during whole‐cell recordings in current‐clamp mode demonstrated that the medium AHP conductance (apamin), BK‐type Ca2+‐dependent K+ channels (iberiotoxin), voltage‐activated Ca2+ channels (CdCl2), M‐current (linopirdine) and persistent Na+ currents (riluzole) are all unnecessary for SFA. Measurements of Na+ channel availability including action potential amplitude, action potential threshold and maximum depolarization rate of the action potential were found to correlate with instantaneous firing frequency suggesting that the availability of fast, inactivating Na+ channels is involved in SFA. Characterization of this Na+ conductance in voltage‐clamp mode demonstrated that it undergoes slow inactivation with a time course similar to that of SFA. When experimentally measured parameters for the fast, inactivating Na+ conductance (including slow inactivation) were incorporated into a MN model, SFA could be faithfully reproduced. The removal of slow inactivation from this model was sufficient to remove SFA. These data indicate that slow inactivation of the fast, inactivating Na+ conductance is likely to be the key mechanism underlying early SFA in spinal MNs.

Human iPSC-derived motoneurons harbouring TARDBP or C9ORF72 ALS mutations are dysfunctional despite maintaining viability

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease for which a greater understanding of early disease mechanisms is needed to reveal novel therapeutic targets. We report the use of human induced pluripotent stem cell (iPSC)-derived motoneurons (MNs) to study the pathophysiology of ALS. We demonstrate that MNs derived from iPSCs obtained from healthy individuals or patients harbouring TARDBP or C9ORF72 ALS-causing mutations are able to develop appropriate physiological properties. However, patient iPSC-derived MNs, independent of genotype, display an initial hyperexcitability followed by progressive loss of action potential output and synaptic activity. This loss of functional output reflects a progressive decrease in voltage-activated Na+ and K+ currents, which occurs in the absence of overt changes in cell viability. These data implicate early dysfunction or loss of ion channels as a convergent point that may contribute to the initiation of downstream degenerative pathways that ultimately lead to MN loss in ALS.

Motoneurons Derived from Embryonic Stem Cells Express Transcription Factors and Develop Phenotypes Characteristic of Medial Motor Column Neurons

Embryonic stem (ES) cells differentiate into functional motoneurons when treated with a sonic hedgehog (Shh) agonist and retinoic acid (RA). Whether ES cells can be directed to differentiate into specific subtypes of motoneurons is unknown. We treated embryoid bodies generated from HBG3 ES cells with a Shh agonist and RA for 5 d in culture to induce motoneuron differentiation. Enhanced green fluorescent protein (eGFP) expression was used to identify putative motoneurons, because eGFP is expressed under the control of the Hb9 promoter in HBG3 cells. We found that 96 ± 0.7% of the differentiated eGFP+ motoneurons expressed Lhx3, a homeobox gene expressed by postmitotic motoneurons in the medial motor column (MMCm), when the treated cells were plated on a neurite-promoting substrate for 5 d. When the treated embryoid bodies were transplanted into stage 17 chick neural tubes, the eGFP+ motoneurons migrated to the MMCm, expressed Lhx3, projected axons to the appropriate target for MMCm motoneurons (i.e., epaxial muscles), and contained synaptic vesicles within intramuscular axonal branches. In ovo and in vitro studies indicated that chemotropic factors emanating from the epaxial muscle and/or surrounding mesenchyme likely guide Lhx3+ motoneurons to their correct target. Finally, whole-cell patch-clamp recordings of transplanted ES cell-derived motoneurons demonstrated that they received synaptic input, elicited repetitive trains of action potentials, and developed passive membrane properties that were similar to host MMCm motoneurons. These results indicate that ES cells can be directed to form subtypes of neurons with specific phenotypic properties.

Transplanted Mouse Embryonic Stem-Cell-Derived Motoneurons Form Functional Motor Units and Reduce Muscle Atrophy

Prolonged muscle denervation resulting from motor neuron (MN) damage leads to atrophy and degeneration of neuromuscular junctions (NMJs), which can impart irreversible damage. In this study, we ask whether transplanted embryonic stem (ES) cells differentiated into MNs can form functional synapses with host muscle, and if so what effects do they have on the muscle. After transplantation into transected tibial nerves of adult mice, ES-cell-derived MNs formed functional synapses with denervated host muscle, which resulted in the ability to produce average tetanic forces of 44% of nonlesioned controls. ES-cell-derived motor units (MUs) had mean force values and ranges similar to control muscles. The number of type I fibers and fatigue resistance of the MUs were increased, and denervation-associated muscle atrophy was significantly reduced. These results demonstrate the capacity for ES-cell-derived MNs not only to incorporate into the adult host tissue, but also to exert changes in the target tissue. By providing the signals normally active during embryonic development and placing the cells in an environment with their target tissue, ES cells differentiate into MNs that give rise to functional MU output which resembles the MU output of endogenous MNs. This suggests that these signals combined with those present in the graft environment, lead to the activation of a program intended to produce a normal range of MU forces.

Neuromodulation of Vertebrate Locomotor Control Networks

Vertebrate locomotion must be adaptable in light of changing environmental, organismal, and developmental demands. Much of the underlying flexibility in the output of central pattern generating (CPG) networks of the spinal cord and brain stem is endowed by neuromodulation. This review provides a synthesis of current knowledge on the way that various neuromodulators modify the properties of and connections between CPG neurons to sculpt CPG network output during locomotion.

GluR2 AMPA Receptor Subunit Expression in Motoneurons at Low and High Risk for Degeneration in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder that results in selective degeneration of most, but not all, groups of motoneurons. The greater susceptibility of vulnerable motoneurons to glutamate excitotoxicity and neurodegeneration has been hypothesized to result from their lower expression of the GluR2 AMPA receptor subunit under control conditions, which renders these receptors permeable to calcium. To address the question of whether there is differential expression of the GluR2 subunit in motoneurons, we compared in normal adult rats expression of GluR2 mRNA and protein within two cranial motor nuclei that are either resistant (III; oculomotor nucleus) or vulnerable (XII; hypoglossal nucleus) to degeneration in ALS. RT-PCR analysis of tissue punched from III and XII motor nuclei detected mRNA for all AMPA subunits (GluR1-R4). In situ hybridization demonstrated no significant difference in GluR2 mRNA expression between III and XII nuclei. Immunohistochemical examination of GluR2 (and GluR4) protein levels demonstrated a similar pattern of the subunit expression in both motor nuclei. This equivalent expression of GluR2 mRNA and protein in motoneurons that differ in their vulnerability to degeneration in ALS suggests that reduced expression of GluR2 is not a factor predisposing motoneurons to degeneration.

Calcium binding proteins in motoneurons at low and high risk for degeneration in ALS.

Recent reports challenge the hypothesis that expression of calcium binding proteins contributes to the greater resistance of some motoneurons to degeneration in amyotrophic lateral sclerosis (ALS). We therefore re-examined, using immunohis tochemistry, the expression of calbindin, calretinin and parvalbumin in vulnerable (hypoglossal, XII; and cervical spinal) and resistant (oculomotor, III) motoneurons of adult rats. Calbindin immunoreactivity was lacking in motor nuclei but strong in the dorsal horn. Calretinin was expressed in spinal, but not III or XII, motoneurons. Parvalbumin immunoreactivity, tested with a polyclonal antibody, was intense in spinal and III, but not XII, motoneurons; however, no staining in the ventral horn was observed with a monoclonal antibody. Differential expression of calretinin and parvalbumin within vulnerable motoneurons suggests that immunoreactivity for these proteins is not a reliablemarker for resistance to degeneration in ALS.

Gender-specific perturbations in modulatory inputs to motoneurons in a mouse model of amyotrophic lateral sclerosis

The fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) is characterised by loss of motoneurons of the brainstem and spinal cord, and corticospinal neurons of the motor cortex. There is also increasing evidence of involvement of glial cells and interneurons, with non-cell autonomous disease mechanisms now thought to contribute to motoneuron degeneration in ALS. Given the apparent involvement of altered motoneuron excitability in ALS and the recent demonstration that motoneuron excitability is controlled by C-boutons, a specific class of synaptic input recently shown to originate from a small cluster of spinal interneurons, we hypothesised that perturbations in C-bouton inputs to motoneurons may contribute to altered excitability and the eventual degeneration of motoneurons in ALS. To begin to assess this we performed a detailed, developmental study of the anatomy of C-boutons in a mouse model of ALS (G93A SOD1 mutant). We found that C-bouton number is unchanged in ALS mice compared to wildtype littermates at any age. In contrast, we found that the size of C-boutons increases in ALS mice between postnatal day (P)8 and P30, with boutons remaining larger throughout symptomatic stages (P120-P140). Interestingly, we found that C-boutons are only enlarged in male mice. We found no evidence of concomitant changes in clusters of postsynaptic proteins known to align with C-boutons (Kv2.1 K(+) channels and m(2)-type muscarinic receptors). In conclusion, these data support the involvement of pre-symptomatic changes in C-bouton anatomy in ALS pathogenesis and in particular mechanisms underlying the male bias of this disease.