Maturation of persistent and hyperpolarization-activated inward currents shapes the differential activation of motoneuron subtypes during postnatal development

Abstract

The fine control of movement is a prerequisite for complex behaviour and is mediated by the orderly recruitment of motor units composed of slow and fast twitch muscle fibres. The size principle was initially proposed to account for orderly recruitment; however, motoneuron size is a poor predictor of recruitment amongst functionally defined motor unit subtypes. While intrinsic properties of motoneurons are key regulators of motoneuron recruitment, the underlying currents involved are not well defined. Whole-cell patch-clamp electrophysiology was deployed to study intrinsic properties, and the underlying currents, that contribute to the differential recruitment of fast and slow motoneurons. Motoneurons were studied during the first three postnatal weeks in mice to identify key properties that establish orderly recruitment and contribute to the emergence of fine motor control. We find that fast and slow motoneurons are functionally homogeneous during the first postnatal week and are recruited based on size, irrespective of motoneuron subtype. The recruitment of fast and slow motoneurons becomes staggered during the second postnatal week due to the differential maturation of passive and active properties, particularly persistent inward currents (PICs). The current required to recruit fast motoneurons increases further in the third postnatal week, despite no additional changes in passive properties or PICs. This further staggering of recruitment currents reflects development of a hyperpolarization-activated inward current during week 3. Our results suggest that motoneuron recruitment is multifactorial, with recruitment order established during postnatal development through the differential maturation of passive properties and sequential integration of persistent and hyperpolarization-activated inward currents.

In vitro longitudinal lumbar spinal cord preparations to study sensory and recurrent motor microcircuits of juvenile mice

“…Proliferation of Ia afferents onto motoneurons and other spinal interneurons such as Renshaw cells and Ia inhibitory interneurons, is also attained in 2 weeks old mice (Alvarez et al, 2013; Mentis et al, 2006, 2010, 2012; Siembab et al, 2010). Maturation of motoneuron properties also occurs in the second postnatal week (Sharples & Miles, 2021; Smith & Brownstone, 2020), which coincides with increased levels of myelination found in the lumbar cord at this stage (Foran & Peterson, 1992). Innervation patterns from descending pathways, such as corticospinal tract terminations (Donatelle, 1977) and brainstem projections (Bregman, 1987; Vinay et al, 2005) also matures by the second postnatal week.…”

“…We then tested the electrophysiological viability of motoneurons from longitudinal preparations. Motoneuron recordings from transverse slices have been already successfully performed in 2-3 weeks old pre-weaned juvenile mice (Bhumbra & Beato, 2018; Sharples & Miles, 2021; Smith & Brownstone, 2020). At this age myelination has progressed (Foran & Peterson, 1992) and the tissue becomes extremely dark, allowing poor detection of transmitted light under low magnification (Fig.…”

“…The identification of neuronal populations based on the expression of specific transcription factors (Jessell, 2000), allowed to selectively target or manipulate spinal neurons, prompting ground-breaking work into the understanding of spinal networks controlling movement. All these achievements and improvements are somewhat diminished by the ‘elephant in the room’ among spinal cord electrophysiologists: in vitro recordings from the spinal cord were mostly performed in embryonic and neonatal preparations, when motoneurons and motor circuits are only partially developed (Clarac et al, 2004; Sharples & Miles, 2021; Smith & Brownstone, 2020; Whelan, 2003). In vitro spinal cord preparations, and especially recordings from motoneurons, from late postnatal mice, have been challenging: motoneurons are very sensitive to anoxia and the thick layer of white matter enveloping the spinal cord prevents sufficient exchange of oxygenated solution within the tissue, leading to motoneuron death.…”

“…Despite technical difficulties (Jensen et al, 2021; Manuel, 2021; Manuel & Zytnicki, 2021), sharp recordings can be used to record membrane and firing properties, but they are not suitable for voltage clamp recordings, thus making it difficult to characterize synaptic conductances in an intact preparation. Recently, researchers have been able to successfully extend the age of in vitro motoneuron recordings from mice, to their juvenile period (P14-30), by tweaking the composition of the solutions used, relying on a very fast dissection and slicing at cold temperatures (Bhumbra & Beato, 2018; Sharples & Miles, 2021; Smith & Brownstone, 2020). These studies have been done in transverse spinal cord slices, an useful preparation to study motoneuron physiology, but that largely ablates motoneuron dendritic arborization and pre-motor networks (Mousa & Elbasiouny, 2021).…”

“…We used: 1) a preparation in which we removed part of the ventral horn thus exposing dorsolateral L3-L5 motoneurons, allowing the study of sensory-related circuits through dorsal root stimulation; and 2) a longitudinal spinal cord section with part of the dorsal horn removed, allowing to record inputs from recurrent motor efferent pathways to motoneurons following ventral root stimulation. Since the mice used here (P14-36) are past the weight-bearing stages and display motor behaviours associated with adulthood (Altman & Sudarshan, 1975), with motoneurons and their sensory and motor pathways fully developed (Alvarez et al, 2013; Sharples & Miles, 2021; Siembab et al, 2010; Smith & Brownstone, 2020), the longitudinal in vitro spinal cord preparations we describe here will provide a useful tool to anyone interested in studying mammalian spinal microcircuits at more mature stages of development.…”