Spinal motoneurons respond aberrantly to serotonin in a rabbit model of cerebral palsy.

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-ischaemic (HI) injury in utero (at 70%-83% gestation) are born with muscle stiffness, hyperreflexia and, as recently discovered, increased 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 the application of α-methyl 5-HT (a 5-HT1 /5-HT2 receptor agonist) and citalopram (a selective 5-HT reuptake inhibitor) with increased amplitude and hyperpolarization of persistent inward currents and hyperpolarized threshold voltage for action potentials, whereas control MNs did not exhibit any of these responses. Although 5-HT similarly modulated MN properties of HI motor-unaffected and motor-affected kits, it affected sag/hyperpolarization-activated cation current (Ih ) and spike frequency adaptation only in HI motor-affected MNs. To further explore the differential sensitivity of MNs to 5-HT, we performed immunostaining for inhibitory 5-HT1A receptors in lumbar spinal MNs at P5. Fewer HI MNs expressed the 5-HT1A receptor compared to age-matched control MNs. This suggests that HI MNs may lack a normal mechanism of central fatigue, mediated by 5-HT1A receptors. Altered expression of other 5-HT receptors (including 5-HT2 ) likely also contributes to the robust increase in HI MN excitability. In summary, by directly exciting MNs, the increased concentration of spinal 5-HT in HI-affected 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: We used whole cell patch clamp electrophysiology to test the responsivity of spinal motoneurons (MNs) from neonatal control and hypoxia-ischaemia (HI) rabbits to 5-HT, which is elevated in the spinal cord after prenatal HI injury. HI rabbit MNs showed a more robust excitatory response to 5-HT than control rabbit MNs, including hyperpolarization of the persistent inward current and threshold voltage for action potentials. Although most MN properties of HI motor-unaffected and motor-affected kits responded similarly to 5-HT, 5-HT caused larger sag/hyperpolarization-activated cation current (Ih ) and altered repetitive firing patterns only in HI motor-affected MNs. Immunostaining revealed that fewer lumbar MNs expressed inhibitory 5-HT1A receptors in HI rabbits compared to controls, which could account for the more robust excitatory response of HI MNs to 5-HT. These results suggest that elevated 5-HT after prenatal HI injury could trigger a cascade of events that lead to muscle stiffness and altered motor unit development.

Inhibitory interneurons show early dysfunction in a SOD1 mouse model of amyotrophic lateral sclerosis.

Few studies in amyotrophic lateral sclerosis (ALS) measure effects of the disease on inhibitory interneurons synapsing onto motoneurons (MNs). However, inhibitory interneurons could contribute to dysfunction, particularly if altered before MN neuropathology, and establish a long-term imbalance of inhibition/excitation. We directly assessed excitability and morphology of glycinergic (GlyT2 expressing) ventral lumbar interneurons from SOD1G93AGlyT2eGFP (SOD1) and wild-type GlyT2eGFP (WT) mice on postnatal days 6-10. Patch clamp revealed dampened excitability in SOD1 interneurons, including depolarized persistent inward currents (PICs), increased voltage and current threshold for firing action potentials, along with a marginal decrease in afterhyperpolarization duration. Primary neurites of ventral SOD1 inhibitory interneurons were larger in volume and surface area than WT. GlyT2 interneurons were then divided into three subgroups based on location: (1) interneurons within 100 µm of the ventral white matter, where Renshaw cells (RCs) are located, (2) interneurons interspersed with MNs in lamina IX, and (3) interneurons in the intermediate ventral area including laminae VII and VIII. Ventral interneurons in the RC area were the most profoundly affected, exhibiting more depolarized PICs and larger primary neurites. Interneurons in lamina IX had depolarized PIC onset. In lamina VII-VIII, interneurons were least affected. In summary, inhibitory interneurons show very early region-specific perturbations poised to impact excitatory/inhibitory balance of MNs, modify motor output and provide early biomarkers of ALS. Therapeutics like riluzole that universally reduce CNS excitability could exacerbate the inhibitory dysfunction described here. KEY POINTS: Spinal inhibitory interneurons could contribute to amyotrophic lateral sclerosis (ALS) pathology, but their excitability has never been directly measured. We studied the excitability and morphology of glycinergic interneurons in early postnatal transgenic mice (SOD1G93A GlyT2eGFP). Interneurons were less excitable and had marginally smaller somas but larger primary neurites in SOD1 mice. GlyT2 interneurons were analysed according to their localization within the ventral spinal cord. Interestingly, the greatest differences were observed in the most ventrally located interneurons. We conclude that inhibitory interneurons show presymptomatic changes that may contribute to excitatory/inhibitory imbalance in ALS.

Altered Motoneuron Properties Contribute to Motor Deficits in a Rabbit Hypoxia-Ischemia Model of Cerebral Palsy.

Cerebral palsy (CP) is caused by a variety of factors attributed to early brain damage, resulting in permanently impaired motor control, marked by weakness and muscle stiffness. To find out if altered physiology of spinal motoneurons (MNs) could contribute to movement deficits, we performed whole-cell patch-clamp in neonatal rabbit spinal cord slices after developmental injury at 79% gestation. After preterm hypoxia-ischemia (HI), rabbits are born with motor deficits consistent with a spastic phenotype including hypertonia and hyperreflexia. There is a range in severity, thus kits are classified as severely affected, mildly affected, or unaffected based on modified Ashworth scores and other behavioral tests. At postnatal day (P)0-5, we recorded electrophysiological parameters of 40 MNs in transverse spinal cord slices using whole-cell patch-clamp. We found significant differences between groups (severe, mild, unaffected and sham control MNs). Severe HI MNs showed more sustained firing patterns, depolarized resting membrane potential, and fired action potentials at a higher frequency. These properties could contribute to muscle stiffness, a hallmark of spastic CP. Interestingly altered persistent inward currents (PICs) and morphology in severe HI MNs would dampen excitability (depolarized PIC onset and increased dendritic length). In summary, changes we observed in spinal MN physiology likely contribute to the severity of the phenotype, and therapeutic strategies for CP could target the excitability of spinal MNs.

Ontogeny of Feeding Mechanics in Smoothhound Sharks: Morphology and Cartilage Stiffness.

The diet of dusky smoothhound sharks, Mustelus canis, shifts over ontogeny from soft foods to a diet dominated by crabs. This may be accompanied by changes in the skeletal system that facilitates the capture and processing of large and bulky prey. The hyoid arch, for example, braces the jaws against the cranium, and generates suction for prey capture and intraoral transport. In this study, ontogenetic changes in the hyoid arch were investigated by quantifying size, mineralization, and stiffness to determine whether increasingly stiffer cartilages are associated with the dietary switch. Total length and length of the hyomandibula and ceratohyal cartilages over ontogeny were the proxy for body size. Cross-sectional area, percent mineralization, and second moment of area were quantified in 28 individuals spanning most of the natural size range. Mechanical compression tests were conducted to compare flexural stiffness to size. Our results show that the morphological characters tested for the hyomandibular and ceratohyal cartilages scales isometrically with length. While stiffness of the hyomandibular and ceratohyal cartilages scales isometrically with length when assessed on morphological characters alone (second moment of area), this relationship becomes allometric when mechanical properties are included (flexural stiffness). Thus, while the hyoid arch elements grow isometrically, the mechanical properties dictate a scaling relationship that dwarfs morphological characteristics. The various combinations of morphologies and ontogenetic trajectories of chondrichthyan species illustrate the tremendous flexibility that they possess in the functional organization of the feeding apparatus.