Claire Francesca Meehan

Precise motor control relies on continuous sensory feedback from muscles, a process in which gamma motoneurons play a key role. These specialized spinal neurons innervate intrafusal muscle fibres, modulating their sensitivity to stretch and maintaining proprioceptive signalling during movement. Gamma motoneurons are characterized by a distinct biophysical profile, including low recruitment thresholds and high firing rates that enable rapid activation of intrafusal fibres at contraction onset. Despite their importance, the intrinsic mechanisms that underlie these properties remain poorly understood. In the present study, we analysed published and unpublished data to identify a population of low‐threshold, high‐gain motoneurons with features consistent with gamma motoneurons, emerging during the third postnatal week in mice. Their low recruitment threshold was linked to lower membrane capacitance, higher input resistance, a more hyperpolarized activation of persistent inward currents (PICs) and a narrower axon initial segment. By contrast, higher firing rates were associated not with PIC amplitude but with shorter action potential durations and smaller medium afterhyperpolarizations. Notably, 92% of putative gamma motoneurons exhibited a sodium pump‐mediated ultra‐slow afterhyperpolarization, which was absent in slow alpha motoneurons. This difference could not be attributed to h‐current activity or expression of the alpha 3 subunit of the sodium‐potassium ATPase. These findings reveal key intrinsic properties that support the unique excitability of gamma motoneurons, offering new insight into their contribution to motor control. This work provides a foundation for future studies into their development, regulation and involvement in neuromuscular disorders. image Key points: A distinct cluster of motoneurons with low recruitment current and high firing gain, characteristic of gamma motoneurons, emerges in the third week of postnatal development. Gamma motoneurons have a low recruitment current as a result of lower capacitance, higher input resistance and a more hyperpolarized activation voltage for persistent inward currents. Their high firing rates are not driven by differences in persistent inward current amplitude but are instead attributed to shorter duration action potentials and smaller amplitude medium afterhyperpolarizations. A narrower axon initial segment in gamma motoneulrons may contribute to their increased excitability compared to alpha motoneurons. Gamma motoneurons present with a higher prevalence of ultra slow afterhyperpolarization than slow alpha motoneurons that cannot be accounted for by differences in h‐current or expression of alpha 3 subunits of the sodium potassium ATPase pump.

A hyperexcitability of the motor system is consistently observed in Amyotrophic Lateral Sclerosis (ALS) and has been implicated in the disease pathogenesis. What drives this hyperexcitability in the vast majority of patients is unknown. This is important to know as existing treatments simply reduce all neuronal excitability and fail to distinguish between pathological changes and important homeostatic changes. Understanding what drives the initial pathological changes could therefore provide better treatments. One challenge is that patients represent a heterogeneous population and the vast majority of cases are sporadic. One pathological feature that almost all (~97%) cases (familial and sporadic) have in common are cytoplasmic aggregates of the protein TDP-43 which is normally located in the nucleus. In our experiments we investigated whether this pathology was sufficient to increase neuronal excitability and the mechanisms by which this occurs. We used the TDP-43(ΔNLS) mouse model which successfully recapitulates this pathology in a controllable way. We used in vivo intracellular recordings in this model to demonstrate that TDP-43 pathology is sufficient to drive a severe hyper-excitability of spinal motoneurones. Reductions in soma size and a lengthening and constriction of axon initial segments were observed, which would contribute to enhanced excitability. Resuppression of the transgene resulted in a return to normal excitability parameters by 6–8 weeks. We therefore conclude that TDP-43 pathology itself is sufficient to drive a severe but reversible hyperexcitability of spinal motoneurones.

A hyperexcitability of the motor system is consistently observed in Amyotrophic Lateral Sclerosis (ALS) and has been implicated in the disease pathogenesis. What drives this hyperexcitability in the vast majority of patients is unknown. This is important to know as existing treatments simply reduce all neuronal excitability and fail to distinguish between pathological changes and important homeostatic changes. Understanding what drives the initial pathological changes could therefore provide better treatments. One challenge is that patients represent a heterogeneous population and the vast majority of cases are sporadic. One pathological feature that almost all (∼97%) cases (familial and sporadic) have in common is cytoplasmic aggregates of the protein TDP-43 which is normally located in the nucleus. In our experiments we investigated whether this pathology was sufficient to increase neuronal excitability and the mechanisms by which this occurs. We used the TDP-43(ΔNLS) mouse model which successfully recapitulates this pathology in a controllable way. We used in vivo intracellular recordings in this model to demonstrate that TDP-43 pathology is sufficient to drive a severe hyper-excitability of spinal motoneurones. Reductions in soma size and a lengthening and constriction of axon initial segments were observed, which would contribute to enhanced excitability. Resuppression of the transgene resulted in a return to normal excitability parameters by 6-8 weeks. We therefore conclude that TDP-43 pathology itself is sufficient to drive a severe but reversible hyperexcitability of spinal motoneurones.

An altered neuronal excitability of spinal motoneurones has consistently been implicated in Amyotrophic Lateral Sclerosis (ALS) leading to several investigations of synaptic input to these motoneurones. One such input that has repeatedly been shown to be affected is a population of large cholinergic synapses terminating mainly on the soma of the motoneurones referred to as C-boutons. Most research on these synapses during disease progression has used transgenic Superoxide Dismutase 1 (SOD1) mouse models of the disease which have not only produced conflicting findings, but also fail to recapitulate the key pathological feature seen in ALS; cytoplasmic accumulations of TAR DNA-binding protein 43 (TDP-43). Additionally, they fail to distinguish between slow and fast motoneurones, the latter of which have more C-boutons but are lost earlier in the disease. To circumvent these issues, we quantified the frequency and volume of C-boutons on traced soleus and gastrocnemius motoneurones, representing predominantly slow and fast motor pools respectively. Experiments were performed using the TDP-43ΔNLS mouse model that carries a transgenic construct of TDP-43 devoid of its nuclear localization signal, preventing its nuclear import. This results in the emergence of pathological TDP-43 inclusions in the cytoplasm, modelling the main pathology seen in this disorder, accompanied by a severe and lethal ALS phenotype. Our results confirmed changes in both the number and volume of C-boutons with a decrease in number on the more vulnerable, predominantly fast gastrocnemius motoneurones and an increase in number on the less vulnerable, predominantly slow soleus motoneurones. Importantly, these changes were only found in male mice. However, both sexes and motor pools showed a decrease in C-bouton volume. Our experiments confirm that cytoplasmic TDP-43 accumulation is sufficient to drive C-bouton changes.

Key points: Although in vitro recordings using neonatal preparations from mouse models of amyotrophic lateral sclerosis (ALS) suggest increased motoneurone excitability, in vivo recordings in adult ALS mouse models have been conflicting. In adult G93A SOD1 models, spinal motoneurones have previously been shown to have deficits in repetitive firing, in contrast to the G127X SOD1 mouse model. Our in vivo intracellular recordings in barbiturate‐anaesthetized adult male G93A SOD1 mice reveal that the incidence of failure to fire with current injection was equally low in control and ALS mice (∼2%). We show that failure to fire repetitively can be a consequence of experimental protocol and should not be used alone to classify otherwise normal motoneurones as hypo‐excitable. Motoneurones in the G93A SOD1 mice showed an increased response to inputs, with lower rheobase, higher input–output gains and increased activation of persistent inward currents. In vitro studies from transgenic amyotrophic lateral sclerosis models have suggested an increased excitability of spinal motoneurones. However, in vivo intracellular recordings from adult amyotrophic lateral sclerosis mice models have produced conflicting findings. Previous investigations using barbiturate anaesthetized G93A SOD1 mice have suggested that some motoneurones are hypo‐excitable, defined by deficits in repetitive firing. Our own previous recordings in G127X SOD1 mice using different anaesthesia, however, showed no repetitive firing deficits and increased persistent inward currents at symptom onset. These discrepancies may be a result of differences between models, symptomatic stage, anaesthesia or technical differences. To investigate this, we repeated our original experiments, but in adult male G93A SOD1 mice, at both presymptomatic and symptomatic stages, under barbiturate anaesthesia. In vivo intracellular recordings from antidromically identified spinal motoneurones revealed that the incidence of failure to fire with current injection was equally low in control and G93A SOD1 mice (∼2%). Motoneurones in G93A SOD1 mice fired significantly more spontaneous action potentials. Rheobase was significantly lower and the input resistance and input–output gain were significantly higher in both presymptomatic and symptomatic G93A SOD1 mice. This was despite a significant increase in the duration of the post‐spike after‐hyperpolarization in both presymptomatic and symptomatic G93A SOD1 mice. Finally, evidence of increased activation of persistent inward currents was seen in both presymptomatic and symptomatic G93A SOD1 mice. Our results do not confirm previous reports of hypo‐excitability of spinal motoneurones in the G93A SOD1 mouse and demonstrate that the motoneurones show an increased response to inputs.

Amyotrophic lateral sclerosis is a neurodegenerative disease preferentially affecting motoneurones. Transgenic mouse models have been used to investigate the role of abnormal motoneurone excitability in this disease. Whilst an increased excitability has repeatedly been demonstrated in vitro in neonatal and embryonic preparations from SOD1 mouse models, the results from the only studies to record in vivo from spinal motoneurones in adult SOD1 models have produced conflicting findings. Deficits in repetitive firing have been reported in G93A SOD1 mice but not in presymptomatic G127X SOD1 mice despite shorter motoneurone axon initial segments (AISs) in these mice. These discrepancies may be due to the earlier disease onset and prolonged disease progression in G93A SOD1 mice with recordings potentially performed at a later sub-clinical stage of the disease in this mouse. To test this, and to explore how the evolution of excitability changes with symptom onset we performed in vivo intracellular recording and AIS labelling in G127X SOD1 mice immediately after symptom onset. No reductions in repetitive firing were observed showing that this is not a common feature across all ALS models. Immunohistochemistry for the Na ⁺ channel Nav1.6 showed that motoneurone AISs increase in length in G127X SOD1 mice at symptom onset. Consistent with this, the rate of rise of AIS components of antidromic action potentials were significantly faster confirming that this increase in length represents an increase in AIS Na ⁺ channels occurring at symptom onset in this model. Highights: In vivo electrophysiological recordings were made in symptomatic G127X SOD1 mice. There were no deficits in repetitive firing in motoneurones in G127X mice. Increased persistent inward currents were still present in the symptomatic mice. Results suggest increases in Na ⁺ currents at axon initial segments (AISs). Immunohistochemistry showed that motoneurone AISs were longer and thinner.