Fig 1.
AIS distance differs in motor pools distinguished by their activity patterns.
(A, C) MG and SOL motoneurons pre-labeled with the retrograde tracer cholera toxin subunit B conjugated to Alexa fluor 488 (CTB) and immunolabeled for ankyrin-G (AnkG), a marker for the AIS. Our definition of AIS distance (AISd) and AIS length (AISl) is identified by white arrows and corresponding text in panel C. (A) white arrow indicates the start of axon from soma and first red arrow indicates initiation of AIS. The two red arrowheads identify the start and end of the AIS. The two red arrowheads in C point to lipofuscin granules that is common in mature motoneurons. (B1-B3) Single z-steps through a confocal image stack. (B1) Initial indication of the axon emerging from the soma. The soma-axon boundary is indicated by the solid white line. (B2) The white lines were used to determine the apex of the angle in which the axon emerged from the soma and is marked by a white dot. This was used as the starting point for measuring distance. (B3) The red arrows indicate the start of the AIS as defined by expression of AnkG. (D) Box and whisker plots of AISd and AISl compared between MG (n = 65) and SOL (n = 82) motoneurons. Blue horizontal line inside the plot represents the median value. Black dots represent a single data point and red dots are designated outliers. Based on the distribution of these data, a nonparametric test (Mann-Whitney U test) was used for an initial statistical comparison (AISd: p <0.001, AISl: p = 0.38).
Table 1.
Population data measurements from retrogradely labeled MG and SOL motoneurons.
Fig 2.
Morphological and in vivo electrophysiological parameters measured in individual Neurobiotin filled motoneurons.
(A1) Collapsed confocal image (10x) of a Neurobiotin filled motoneuron in the ventral horn of the spinal cord. Solid white line indicates white/gray matter boundary. (A2-3) High magnification confocal image of AnkG immunoreactivity in ventral horn of the spinal cord (A1). (A3) Image of Neurobiotin filled MG motoneuron revealed with streptavidin-488 merged with AnkG. White arrow heads indicate AIS. (B) Rheobase generated by 50ms square pulse of depolarizing current injection. Blue trace indicates a current injection that did not elicit an action potential. (C) After hyperpolarization (ahp) was elicited via 0.5ms suprathreshold current injection. (D) Input resistance measured by hyperpolarizing currents (-1/3nA) for 50ms. Input conductance is reported as the inverse of resistance. (E) Muscle twitch results from ahp current injection. Twitch force is defined as the maximum amplitude (vertical dashed red line). Contraction time (CT) is measured between the twitch onset to the maximum amplitude (horizontal dashed red line). (F1-2) A small percent (11% in our sample) of axons are of dendritic origin. (F1) Neurobiotin filled motoneuron with a parent dendrite indicated with the red arrowhead and the protruding axon indicated with the white arrowhead. (F2) Merge Imaris rendition of image displayed in F1.
Table 2.
Morphological characteristics of motoneurons and the AIS.
Table 3.
Biophysical measurements from intracellularly recorded MG motoneurons.
Fig 3.
Comparisons were performed using a Bayesian analytical approach to calculate a posterior probability distribution. Plots are based on a generative model conditioned on previous reports and the current data set. Each grey line represents a single trial from 4,000 generative samples and each black dot is an observed data point (n = 16). From the 4,000 samples we provide a 95% high density interval (HDI). The median slope from the generative sample is represented as a blue line (β1). From the slopes and generative model, we compute an R2 equivalent, and this is presented with an HDI and a median. (A) Rheobase: AIS distance. As rheobase increases the AIS distance increased from the motoneuron soma. Model slope: 95% HDI 0.463–1.21, β1 = 0.846; R2: 95% HDI 0.364–0.732, median R2 0.631. (B) Close similarity in rheobase and input conductance between two samples of MG motoneurons. A hierarchical Bayesian model was constructed for 44 MG motoneurons (white filled circles, grey lines) pooled from a larger MG motoneuron database produced by the Cope lab and for 10 MG Neurobiotin filled motoneurons (black circles, blue lines) pooled from the 16 cells presented in Fig 3. Models were conditioned on previous reports and datasets from this study [44]. The positive correlation between rheobase and conductance is representative of prior reports and our small sample of 10 motoneurons fell within the expected range produced from our larger dataset. 44 motoneurons—Model slope: 95% HDI 15.5–25.0, β1 = 20.2; R2: 95% HDI 0.522–0.728, median R2 0.644. Neurobiotin filled motoneurons—Model slope: 95% HDI 10.9–42.2, β1 = 26.0; R2: 95% HDI 0.239–0.743, median R2 0.648.
Fig 4.
AIS geometry relationship to markers of motor unit recruitment order.
3D plot shows motoneurons with relatively lower rheobase and twitch force (grey highlight), which are typically recruited early during muscle contraction also exhibited the shortest AISd.