ABSTRACT
Cerebral palsy (CP) is caused by a variety of factors that damage the developing central nervous system. Impaired motor control, including muscle stiffness and spasticity, is the hallmark of spastic CP. Rabbits that experience hypoxic-ischemic (HI) injury in utero (at 70-80% gestation) are born with muscle stiffness, hyperreflexia, and, as recently discovered, increased serotonin (5-HT) in the spinal cord. To determine whether serotonergic modulation of spinal motoneurons (MNs) contributes to motor deficits, we performed ex vivo whole cell patch clamp in neonatal rabbit spinal cord slices at postnatal day (P) 0-5. HI MNs responded to application of α-methyl 5-HT (a 5-HT 1 /5-HT 2 receptor agonist) and citalopram (a selective 5-HT reuptake inhibitor) with hyperpolarization of persistent inward currents and threshold voltage for action potentials, reduced maximum firing rate, and an altered pattern of spike frequency adaptation while control MNs did not exhibit any of these responses. To further explore the differential sensitivity of MNs to 5-HT, we performed immunohistochemistry for inhibitory 5-HT 1A receptors in lumbar spinal MNs at P5. Fewer HI MNs expressed the 5-HT 1A receptor compared to age-matched controls. This suggests many HI MNs lack a normal mechanism of central fatigue mediated by 5-HT 1A receptors. Other 5-HT receptors (including 5-HT 2 ) are likely responsible for the robust increase in HI MN excitability. In summary, by directly exciting MNs, the increased concentration of spinal 5-HT in HI rabbits can cause MN hyperexcitability, muscle stiffness, and spasticity characteristic of CP. Therapeutic strategies that target serotonergic neuromodulation may be beneficial to individuals with CP. Key points: After prenatal hypoxia-ischemia (HI), neonatal rabbits that show hypertonia are known to have higher levels of spinal serotoninWe tested responsivity of spinal motoneurons (MNs) in neonatal control and HI rabbits to serotonin using whole cell patch clampMNs from HI rabbits showed a more robust excitatory response to serotonin than control MNs, including hyperpolarization of the persistent inward current and threshold for action potentials, larger post-inhibitory rebound, and less spike frequency adaptation Based on immunohistochemistry of lumbar MNs, fewer HI MNs express inhibitory 5HT 1A receptors than control MNs, which could account for the more robust excitatory response of HI MNs. These results suggest that after HI injury, the increased serotonin could trigger a cascade of events leading to muscle stiffness and altered motor unit development.
ABSTRACT
Spinal motoneuron dysfunction and loss are pathological hallmarks of the neuromuscular disease spinal muscular atrophy (SMA). Changes in motoneuron physiological function precede cell death, but how these alterations vary with disease severity and motoneuron maturational state is unknown. To address this question, we assessed the electrophysiology and morphology of spinal motoneurons of presymptomatic Smn2B/- mice older than 1 wk of age and tracked the timing of motor unit loss in this model using motor unit number estimation (MUNE). In contrast to other commonly used SMA mouse models, Smn2B/- mice exhibit more typical postnatal development until postnatal day (P)11 or 12 and have longer survival (~3 wk of age). We demonstrate that Smn2B/- motoneuron hyperexcitability, marked by hyperpolarization of the threshold voltage for action potential firing, was present at P9-10 and preceded the loss of motor units. Using MUNE studies, we determined that motor unit loss in this mouse model occurred 2 wk after birth. Smn2B/- motoneurons were also larger in size, which may reflect compensatory changes taking place during postnatal development. This work suggests that motoneuron hyperexcitability, marked by a reduced threshold for action potential firing, is a pathological change preceding motoneuron loss that is common to multiple models of severe SMA with different motoneuron maturational states. Our results indicate voltage-gated sodium channel activity may be altered in the disease process.NEW & NOTEWORTHY Changes in spinal motoneuron physiologic function precede cell death in spinal muscular atrophy (SMA), but how they vary with maturational state and disease severity remains unknown. This study characterized motoneuron and neuromuscular electrophysiology from the Smn2B/- model of SMA. Motoneurons were hyperexcitable at postnatal day (P)9-10, and specific electrophysiological changes in Smn2B/- motoneurons preceded functional motor unit loss at P14, as determined by motor unit number estimation studies.
