Whole-cell patch-clamp was performed similarly to previously published work (Quinlan et al., 2011) from P0–5. Briefly, horizontal spinal cord slices 350 μm thick were obtained using a Leica 1,000 vibratome. Slices were incubated for 1 h at 30°C and recordings were performed at room temperature. During recording, slices were perfused with oxygenated (95% O₂ and 5% CO₂) modified Ringer’s solution containing (in mM): 111 NaCl, 3.09 KCl, 25.0 NaHCO₃, 1.10 KH₂PO₄, 1.26 MgSO₄, 2.52 CaCl₂, and 11.1 glucose at 2 ml/min. Whole-cell patch electrodes (1–3 MΩ) contained (in mM) 138 K-gluconate, 10 HEPES, 5 ATP-Mg, 0.3 GTP-Li and Texas Red dextran (150 μM, 3,000 MW). PICs were measured in voltage-clamp mode with holding-potential of −90 mV and depolarizing voltage ramps of both 36 mV/s and 11.25 mV/s bringing the cell to 0 mV in 2.5 s or 8 s, respectively and then back to the holding potential in the following 2.5 s or 8 s. Input resistance was measured from the slope of the leak current near the holding potential. Capacitance was measured with Multiclamp’s whole-cell capacitance compensation function. Resting membrane potential was measured in voltage-clamp as the voltage at which there is 0 pA of injected current in the descending ramp. In current clamp, frequency—current measurements were obtained from current ramps. The first spike on the current ramp was used to measure the properties of action potentials. The threshold voltage was defined as the voltage at which the action potential slope exceeds 10 V/s. Rate of rise and fall of the action potential were measured as peak and trough of the first derivative of the action potential. The duration of the action potential was measured at half-peak (defined as the midpoint between overshoot and threshold voltages). Depolarizing current steps of varying amplitude were used to find maximum firing rates (near depolarization block) and to measure after-spike afterhyperpolarization (in single spikes elicited near-threshold). Hyperpolarizing current steps (typically between −850 and −1,250 pA) were used to measure hyperpolarization-activated sag currents (I_H). Neuron selection: neurons were targeted in MN pools mainly from cervical and lumbar regions of the cord and were removed from the data set if their resting membrane potential was more depolarized than −45 mV in current clamp.

After electrophysiological measurements were obtained, MNs were imaged to assess anatomical development, and photos were obtained of the electrode placement within the spinal cord slice, as shown in Figure 1. Images were acquired with a Nikon microscope fitted with a 40× water-dipping objective lens and two-photon excitation fluorescence microscopy performed with a galvanometer-based Coherent Chameleon Ultra II laser. To optimize the excitation of red/green fluorophores, the laser was tuned to 900 nm. 3D reconstructions of MNs were created using Neurolucida 360° software. It is likely that some processes extended past the surface of the slice and were excluded from reconstructions. However, since this was the case for all MNs in this study it is unlikely to have an impact on the findings.

All variables were checked for normality and homogeneity (using Shapiro–Wilk and Levene’s tests). The variables that were parametric (normal and homogenous) were run with one-way Analysis of Variance (ANOVA) and then assessed post hoc with a Tukey test for between-group significance. The non-parametric variables were run with the Kruskal–Wallis test followed by Dunn’s test as a post hoc analysis to assess significance between groups, adjusting the p-value for multiple comparisons. The analysis was done using R software for determining the significance of parameters over groups, according to their injury classification (sham, HI unaffected, HI mildly affected, and HI severely affected). Injury classification, age of the kit (P0–P5), and spinal cord region (cervical, thoracic, lumbar or sacral) were all tested. Significance was determined by p-values ≤ 0.05.

In rabbit kits severely injured by HI, MNs had significantly increased sustained firing. The frequency current (F-I) relationship was measured using the current ramps, as shown in Figure 2. Depolarizing current ramps are used to evoke firing, and current at onset and offset of firing (I_ON and I_OFF) determine ΔI. In sham control MNs, ΔI was larger and always a positive value, indicating firing ceased at a higher current amplitude on the descending ramp than the current level that elicited firing on the ascending ramp (see Figure 2A). Severe HI MNs had a smaller, and usually negative ΔI, revealing increasingly sustained firing (see Figure 2B). Also, resting membrane potential was significantly more depolarized in HI MNs than sham controls (see Figure 2F). Another significant difference in severe HI MNs is the instantaneous firing rate, as shown in Figure 3. At the start of a depolarizing current step, the peak (instantaneous) firing rate is higher in severe HI MNs than sham controls (see Figure 3B). Sustained firing is also apparent in Figure 3C, in the second current step which evokes a brief burst of action potentials followed by depolarization block in both MNs. The severe HI MN recovers and resumes firing while the sham control MN remains in depolarization block. Mean values for both instantaneous and steady-state parameters are shown in bar graphs (Figures 3D,E). Significant changes in MN physiology were present in severely affected animals: post hoc analysis showed significant changes between sham and severe HI MNs in ΔI, RMP, and instantaneous firing rate. The instantaneous firing rate also reached significance in mild HI MNs vs. sham. No significant changes were present in HI unaffected MNs in any properties. While these parameters (ΔI, RMP, and instantaneous firing rate) suggest increased excitability in HI-injured MNs, there was no significant change in the threshold, I_ON, or I_OFF in HI MNs. Thus, MNs from severely affected kits should not be classified as hyperexcitable per se, since they begin firing with the same depolarizing input and at the same voltage threshold.

A complete analysis of I_H (sag and rebound currents), action potential parameters, and after-spike after-hyperpolarization (AHP) was performed and no significant differences in these parameters were found between groups. All data is included in Supplementary Tables S1, S2.

PICs were significantly affected by hypoxia-ischemia (HI), revealing that intrinsic excitability may be dampened. PICs were measured using both short (5 s) and long (16 s) protocols, which can preferentially activate and inactivate Na⁺ and Ca²⁺ mediated PICs. The different protocols yielded different results. For example, as shown in Figure 4, voltage dependence (PIC onset and PIC Max) was unchanged in the PICs evoked using a short 5-s voltage ramp HI severe MNs. Using longer voltage ramps (16 s), PIC onset was significantly depolarized in HI severe MNs compared to sham (see Tables 2, 3). Since the change in PIC onset was more pronounced in longer ramps this could suggest an altered balance of Na⁺ and Ca²⁺ channel activation or altered activation/inactivation of these channels (see “Discussion” section). Change in the magnitude of the PIC was not observed outright in either of the protocols: the magnitude of the currents was similar between groups (see Figure 4 and Tables 2, 3). Intrinsic properties including capacitance (which significantly increased in HI MNs compared to sham) and input resistance of all MNs are included in Table 2.

Morphology of MNs was assessed in all patched neurons, as shown in Figure 5. As suggested by the significantly larger whole-cell capacitance, there were changes in MN morphology after HI injury. The soma size was unchanged: there were no significant differences between groups in soma’s largest cross-sectional area (Figure 5D) or other measurements of soma size (Table 4). There was, however, a significant increase in dendrite length in HI injured MNs compared to sham controls (Figure 5E), which could account for changes in electrical properties. Since, we recorded from motor pools throughout the spinal cord (cervical through sacral), there was a large amount of variability within our data set. Future studies in our lab focus on the analysis of specific motor pools. All data on dendritic morphology is included in Table 5.

No significant changes in MN properties were found due to the spinal region. Postnatal age had a significant effect on the following properties: input resistance (decrease with age), action potential size (mV), rate of rise and rate of fall (all increase with age), 5 s PIC amplitude (increase with age), and normalized PIC (PIC/Cap; current density increased with age for both 5 and 16 s ramps). These results are in line with previous studies on embryonic and postnatal maturation of MNs.

Electrophysiological properties of spinal MNs are altered by developmental HI injury, and the magnitude of changes is correlated to the severity of motor deficits. Specifically, these changes include increased sustained firing and a higher firing rate. These changes could indicate increased excitability and would contribute to muscle stiffness that is common in spastic CP. However, concomitant changes in PIC onset and longer dendritic length could serve to dampen excitability and could contribute to weakness. Since traditional views of CP largely view motor dysfunction as a result of the damaged motor cortex improperly signaling to spinal neurons, our new evidence suggests this is only part of the problem. Spinal MNs are not developing the same after HI and show an overall change in intrinsic properties. Whether these changes are directly due to the HI insult or indirectly due to downstream effects must be determined by future work.

Here, we show that MNs show altered excitability after HI injury, including elevated resting potential, and more sustained firing. Previous work showed that after HI injury in rabbits there were also fewer spinal MNs, and spinal interneurons in lamina VII were undergoing apoptosis (Drobyshevsky and Quinlan, 2017). After the loss of corticospinal projections, it was recently found that the spinal cholinergic interneurons which give rise to C boutons on MNs are lost (Jiang et al., 2016, 2018, 2019). Taken together this data suggests spinal circuits are: (1) just as vulnerable to HI injury as the developing cortex; and (2) potentially functioning with fewer neurons and altered circuitry. In addition to fewer neurons, there is also atrophy of the muscles which could contribute to motor deficits in CP. In mice, rabbits, and humans, muscle atrophy appears along with losses in numbers of MNs (Han et al., 2013; Marciniak et al., 2015; Drobyshevsky and Quinlan, 2017; Brandenburg et al., 2018). Recent work has shown similar changes to muscle architecture in the rabbit HI model of CP to humans, including atrophy, muscle shortening, and longer sarcomere length. Increased muscle stiffness in rabbits affected by HI was found even after administration of anesthetic—indicating some muscle stiffness is derived from mechanical changes in the muscles, though a large component of the muscle stiffness was diminished with anesthetic thus was driven neurally (Synowiec et al., 2019). During development, both feedback and feed-forward signaling can regulate growth and maturation, processes that may be disrupted in CP in both MNs and muscle fibers. It is likely the loss of spinal interneurons, MNs and muscle fibers reduce coordination and strength in those with CP. Altered size and excitability of MNs is also a feature of other motor disorders including amyotrophic lateral sclerosis and spinal muscular atrophy (Quinlan et al., 2011, 2019; Gogliotti et al., 2012; Shoenfeld et al., 2014; Dukkipati et al., 2018). Since our data were collected from multiple MN pools throughout the spinal cord, from control and HI injured rabbit kits, relatively large variability in parameters was expected (Kanning et al., 2010), however consistent differences in electrical properties emerged in this data set. This suggests consistent changes are present across motor pools after injury and support further exploration of interventions for CP and other motor disorders that target spinal MNs. These treatments could include neuromodulators and therapies aimed at restoring balance between excitation and inhibition within spinal circuits for alleviation of spasticity.

The exact causes of the changes in MN physiology observed here are unclear, but they could result from the increase in spinal monoamines that occurs after developmental HI injury in both rodents and rabbits (Bellot et al., 2014; Drobyshevsky et al., 2015). Serotonin is generally thought of as a neurotransmitter and neuromodulator, but developmental disruption in 5HT is associated with neurological disorders including autism, Rett syndrome, Down’s syndrome and, more recently, CP (Bar-Peled et al., 1991; Whittle et al., 2007; Bellot et al., 2014; Yang et al., 2014; De Filippis et al., 2015; Drobyshevsky et al., 2015; Muller et al., 2016; Wirth et al., 2017). Serotonin increases MN excitability in neonatal and juvenile mice, rats and guinea pigs (Wang and Dun, 1990; Ziskind-Conhaim et al., 1993; Hsiao et al., 1997, 1998), and likely has the same effect on rabbit MNs. Depolarization of the resting membrane potential, increased action potential firing through hyperpolarization of the voltage threshold and enhanced PIC, increased action potential height and reduction of high-voltage activated Ca²⁺ entry are all associated with 5HT receptor activation in neonatal and adult MNs (Elliot and Wallis, 1992; Larkman and Kelly, 1992; Lindsay and Feldman, 1993; Bayliss et al., 1995; Hsiao et al., 1997, 1998; Inoue et al., 1999; Gilmore and Fedirchuk, 2004; Li et al., 2006). Therefore increased 5HT could have a direct impact on excitability, though in HI rabbits the increase in 5HT was accompanied by decreased mRNA for 5HT₂ receptors and increased mRNA for the SERT serotonin transporter (Drobyshevsky et al., 2015). In light of that finding, it is not clear that neurons remain responsive to 5HT. In the experiments here, all MNs were recorded in spinal cord slices incubated and perfused in standard oxygenated aCSF without any serotonergic drugs present. Therefore, HI MNs in vivo could show different levels of excitability since they would be in the presence of elevated 5HT, while our MNs were all recorded in the same standard physiological solution. Thus, the contribution of 5HT to the altered excitability observed here is restricted to its chronic effects on neuron development, namely morphological changes. Serotonin 5HT_1A and 5HT_2A receptor activation increases neurite outgrowth, dendritic branching, and spine formation (Bou-Flores et al., 2000; Fricker et al., 2005; Mogha et al., 2012), findings that align well with the present finding of increased dendritic length in the HI MNs. Future experiments will be needed to address the role of 5HT in enhancing MN excitability and its effects on synaptically-evoked action potentials. Synaptic events in dendrites would more strongly evoke PICs, though both altered dendritic morphology and elevated 5HT could dampen them.

The mechanism for increased MN activity and thus muscle stiffness may be due to delayed Na⁺ channel inactivation. In neonatal MNs, Na⁺ channels generate the majority of the PIC and account for action potential initiation/repetitive firing. Specifically, Nav 1.1, 1.2 and 1.6 type Na⁺ channels (Boiko et al., 2001, 2003; Rush et al., 2005) inactivate faster than Ca²⁺ channels that contribute to PICs (Perrier and Hounsgaard, 2003; Li et al., 2006). Short voltage ramps preferentially measure the Na⁺ PIC for this reason: Na⁺ channels inactivate quickly enough that even on the descending ramp of the short protocol, there is no longer a region of negative slope (see Figure 4). Changes in Na⁺ channel inactivation in adult MNs along with postnatal development of the longer-lasting Ca²⁺ PIC makes typical adult MNs display more negative ΔI values and longer-lasting PICs (Harvey et al., 2006; Li et al., 2006; Quinlan et al., 2011). In the neonatal rabbit MNs, sham controls showed positive ΔI values that are quite typical for neonates, while HI MNs showed significantly more negative values. This could be due to slower Na⁺ channel inactivation or increased contribution of Ca²⁺ channels to the PICs after injury. Interestingly the more depolarized resting potential found in HI severe MN would serve to increase Na⁺ channel inactivation. To fully determine the altered mechanism of aberrant firing after HI injury future studies into the biophysical properties of Na⁺ channels and maturation of Ca²⁺ channel expression must be pursued.

Generally, unaffected and mildly affected MNs showed parameters that were intermediate between control MNs and severe MNs. There was only one category in which mildly affected MNs become statistically significantly different from sham controls (instantaneous firing frequency). Thus, electrophysiological changes were overwhelmingly in line with phenotype, suggesting aberrant MN properties contribute to the severity of the phenotype. It cannot be ruled out, however, that HI “unaffected” MNs may have a subtle phenotype that is not readily evident based on testing we performed here. Or perhaps abnormalities in these rabbits would develop in later in life: in humans CP patients, diagnosis of CP is not made until 18–24 months of life and the peak of spasticity occurs around 4 years of age (Hägglund and Wagner, 2008; Hadders-Algra, 2014; Novak et al., 2017). In the rabbit model, deficits have not been characterized past P18, and a detailed analysis of the progression of motor deficits from P0–18 is lacking. Future work is needed to assess the maturation of the MN properties in different groups, the potential contribution of delayed Na⁺ channel inactivation in CP, the progression of motor deficits with age, and the development of new therapeutic strategies that could target MNs.

The present study only extended from postnatal day 0–5, but even within this narrow window, significant changes were apparent in MN electrical parameters. As MNs undergo postnatal maturation, they grow larger, with more complex dendritic arborizations, and gain the ability to fire action potentials at higher rates, as reviewed in Carrascal et al. (2005). We found that age had a significant effect on 5 s PIC amplitude (increasing with age), normalized PIC amplitude (both 5 and 16 s PIC/Cap increased with age), input resistance (decreasing with age), and action potential size, rate of rise and rate of fall (all increase with age). While neuron size increases during this period, the amplitude of the PIC typically increases more than proportionally (Quinlan et al., 2011). In other words, voltage-gated ion channels are being inserted into the membrane at a faster rate than the cell is increasing in size, resulting in an increased normalized PIC amplitude with age. We suspect this parallels the ability of MNs to fire action potentials at higher rates during postnatal development, and the acquisition of coordinated motor control and weight-bearing in developing animals.