Katharina A. Quinlan

Genetic ablation of V2a ipsilateral interneurons disrupts left-right locomotor coordination in mammalian spinal cord.

The initiation and coordination of activity in limb muscles are the main functions of neural circuits that control locomotion. Commissural neurons connect locomotor circuits on the two sides of the spinal cord, and represent the known neural substrate for left-right coordination. Here we demonstrate that a group of ipsilateral interneurons, V2a interneurons, plays an essential role in the control of left-right alternation. In the absence of V2a interneurons, the spinal cord fails to exhibit consistent left-right alternation. Locomotor burst activity shows increased variability, but flexor-extensor coordination is unaffected. Anatomical tracing studies reveal a direct excitatory input of V2a interneurons onto commissural interneurons, including a set of molecularly defined V0 neurons that drive left-right alternation. Our findings imply that the neural substrate for left-right coordination consists of at least two components; commissural neurons and a class of ipsilateral interneurons that activate commissural pathways.

Segmental, Synaptic Actions of Commissural Interneurons in the Mouse Spinal Cord

Left–right alternation depends on activity in commissural interneurons (CINs) that have axons crossing in the midline. In this study, we investigate the CIN connectivity to local motor neurons using a newly developed preparation of the in vitro neonatal mouse spinal cord that allows us to identify all classes of CINs. Nineteen of 29 short-range CINs with axonal projections <1.5 segments (sCINs) directly excited, directly inhibited, or indirectly inhibited contralateral motor neurons in the quiescent spinal cord. Excitation was glutamatergic and inhibition was mixed glycinergic and/or GABAergic. Long-range CINs were also found to have input to local, contralateral motor neurons. Thirteen of 29 descending CINs had similar synaptic connectivity to contralateral motor neurons as the sCINs, including direct excitation and direct and indirect inhibition. Some (9 of 23) rostrally projecting ascending CINs, and a few (2 of 10) CINs with bifurcating axons that both ascend and descend, indirectly inhibited local, contralateral motor neurons. Rhythmic firing during locomotor-like activity was observed in a number of CINs with segmental synaptic effects on contralateral motor neurons. This study outlines the basic connectivity pattern of CINs in the mouse spinal cord on a segmental level. Our study suggests that, based on observed synaptic connectivity, both short- and long-range CINs are likely involved in segmental left–right coordination and that the CIN system is organized into a dual-inhibitory and single-excitatory system. These systems are organized in a way that they could provide appropriate coordination during locomotion.

Altered postnatal maturation of electrical properties in spinal motoneurons in a mouse model of amyotrophic lateral sclerosis

Non‐technical summary  Our focus was on whether amyotrophic lateral sclerosis (ALS) might be precipitated by early developmental changes in large spinal motoneurons, which are vulnerable to early die‐off in ALS. It has been shown that some electrical properties in motoneurons are profoundly altered soon after birth in mutant superoxide dismutase‐1 (SOD1) mice, a standard animal model of ALS. These same properties undergo rapid developmental changes in normal mice during this time period. Our goal was to compare the development of motoneuron electrical properties in normal and SOD1 mice. Properties were measured from birth to 12 days of age, when the mouse is considered juvenile, but long before symptom onset. Most electrical properties in the SOD1 motoneurons showed an accelerated pace of maturation during this early developmental period compared with the normal motoneurons. If this trend persists, it could, along with other disease factors, hasten the onset of normal motoneuron degeneration due to ageing and result in the development of ALS.

eGFP Expression under UCHL1 Promoter Genetically Labels Corticospinal Motor Neurons and a Subpopulation of Degeneration-Resistant Spinal Motor Neurons in an ALS Mouse Model

Understanding mechanisms that lead to selective motor neuron degeneration requires visualization and cellular identification of vulnerable neurons. Here we report generation and characterization of UCHL1-eGFP and hSOD1G93A-UeGFP mice, novel reporter lines for cortical and spinal motor neurons. Corticospinal motor neurons (CSMN) and a subset of spinal motor neurons (SMN) are genetically labeled in UCHL1-eGFP mice, which express eGFP under the UCHL1 promoter. eGFP expression is stable and continues through P800 in vivo. Retrograde labeling, molecular marker expression, electrophysiological analysis, and cortical circuit mapping confirmed CSMN identity of eGFP+ neurons in the motor cortex. Anatomy, molecular marker expression, and electrophysiological analysis revealed that the eGFP expression is restricted to a subset of small-size SMN that are slow-twitch α and γ motor neurons. Crossbreeding of UCHL1-eGFP and hSOD1G93A lines generated hSOD1G93A-UeGFP mice, which displayed the disease phenotype observed in a hSOD1G93A mouse model of ALS. eGFP+ SMN showed resistance to degeneration in hSOD1G93A-UeGFP mice, and their slow-twitch α and γ motor neuron identity was confirmed. In contrast, eGFP+ neurons in the motor cortex of hSOD1G93A-UeGFP mice recapitulated previously reported progressive CSMN loss and apical dendrite degeneration. Our findings using these two novel reporter lines revealed accumulation of autophagosomes along the apical dendrites of vulnerable CSMN at P60, early symptomatic stage, suggesting autophagy as a potential intrinsic mechanism for CSMN apical dendrite degeneration.

Links between Electrophysiological and Molecular Pathology of Amyotrophic Lateral Sclerosis

Multiple deficits have been described in amyotrophic lateral sclerosis (ALS), from the first changes in normal functioning of the motoneurons and glia to the eventual loss of spinal and cortical motoneurons. In this review, current results, including changes in size, and electrical properties of motoneurons, glutamate excitotoxicity, calcium buffering, deficits in mitochondrial and cellular transport, impediments to proteostasis which lead to stress of the endoplasmic reticulum (ER), and glial contributions to motoneuronal vulnerability are recapitulated. Results are mainly drawn from the mutant SOD1 mouse model of ALS, and emphasis is placed on early changes that precede the onset of symptoms and the interplay between molecular and electrical processes.

Design and evaluation of a chronic EMG multichannel detection system for long-term recordings of hindlimb muscles in behaving mice

Mouse models are commonly used for identifying the behavioral consequences of genetic modifications, progression or recovery from disease or trauma models, and understanding spinal circuitry. Electromyographic recordings (EMGs) are recognized as providing information not possible from standard behavioral analyses involving gross behavioral or kinematic assessments. We describe here a method for recording from relatively large numbers of muscles in behaving mice. We demonstrate the use of this approach for recording from hindlimb muscles bilaterally in intact animals, following spinal cord injury, and during the progression of ALS. This design can be used in a variety of applications in order to characterize the coordination strategies of mice in health and disease.

Cellular and Synaptic Actions of Acetylcholine in the Lamprey Spinal Cord

This study investigated cellular and synaptic mechanisms of cholinergic neuromodulation in the in vitro lamprey spinal cord. Most spinal neurons tested responded to local application of acetylcholine (ACh) with depolarization and decreased input resistance. The depolarization persisted in the presence of either tetrodotoxin or muscarinic antagonist scopolamine and was abolished with nicotinic antagonist mecamylamine, indicating a direct depolarization through nicotinic ACh receptors. Local application of muscarinic ACh agonists modulated synaptic strength in the spinal cord by decreasing the amplitude of unitary excitatory and inhibitory postsynaptic potentials. The postsynaptic response to direct application of glutamate was unchanged by muscarinic agonists, suggesting a presynaptic mechanism. Cholinergic feedback from motoneurons was assessed using stimulation of a ventral root in the quiescent spinal cord while recording intracellularly from spinal motoneurons or interneurons. Mainly depolarizing potentials were observed, a portion of which was insensitive to removal of extracellular Ca2+, indicating electrotonic coupling. Hyperpolarizing potentials were also observed and were attenuated by the glycinergic antagonist strychnine, whereas depolarizing responses were potentiated by strychnine. Mecamylamine also reduced hyperpolarizing responses. The pharmacology of these responses suggests a Renshaw-like feedback pathway in lamprey. Immunohistochemistry for choline acetyltransferase, performed in combination with retrograde filling of motoneurons, demonstrated a population of nonmotoneuron cholinergic cells in the lamprey spinal cord. Thus endogenous cholinergic modulation of the lamprey spinal locomotor network is likely produced by both motoneurons and cholinergic interneurons acting via combined postsynaptic and presynaptic actions.

Effect of fluoxetine on disease progression in a mouse model of ALS

Selective serotonin reuptake inhibitors (SSRIs) and other antidepressants are often prescribed to amyotrophic lateral sclerosis (ALS) patients; however, the impact of these prescriptions on ALS disease progression has not been systematically tested. To determine whether SSRIs impact disease progression, fluoxetine (Prozac, 5 or 10 mg/kg) was administered to mutant superoxide dismutase 1 (SOD1) mice during one of three age ranges: neonatal [postnatal day (P)5-11], adult presymptomatic (P30 to end stage), and adult symptomatic (P70 to end stage). Long-term adult fluoxetine treatment (started at either P30 or P70 and continuing until end stage) had no significant effect on disease progression. In contrast, neonatal fluoxetine treatment (P5-11) had two effects. First, all animals (mutant SOD1(G93A) and control: nontransgenic and SOD1(WT)) receiving the highest dose (10 mg/kg) had a sustained decrease in weight from P30 onward. Second, the high-dose SOD1(G93A) mice reached end stage ∼8 days (∼6% decrease in life span) sooner than vehicle and low-dose animals because of an increased rate of motor impairment. Fluoxetine increases synaptic serotonin (5-HT) levels, which is known to increase spinal motoneuron excitability. We confirmed that 5-HT increases spinal motoneuron excitability during this neonatal time period and therefore hypothesized that antagonizing 5-HT receptors during the same time period would improve disease outcome. However, cyproheptadine (1 or 5 mg/kg), a 5-HT receptor antagonist, had no effect on disease progression. These results show that a brief period of antidepressant treatment during a critical time window (the transition from neonatal to juvenile states) can be detrimental in ALS mouse models.

Comparison of dendritic calcium transients in juvenile wild type and SOD1G93A mouse lumbar motoneurons

Previous studies of spinal motoneurons in the SOD1 mouse model of amyotrophic lateral sclerosis have shown alterations long before disease onset, including increased dendritic branching, increased persistent Na+ and Ca2+ currents, and impaired axonal transport. In this study dendritic Ca2+ entry was investigated using two photon excitation fluorescence microscopy and whole-cell patch-clamp of juvenile (P4-11) motoneurons. Neurons were filled with both Ca2+ Green-1 and Texas Red dextrans, and line scans performed throughout. Steps were taken to account for different sources of variability, including (1) dye filling and laser penetration, (2) dendritic anatomy, and (3) the time elapsed from the start of recording. First, Ca2+ Green-1 fluorescence was normalized by Texas Red; next, neurons were reconstructed so anatomy could be evaluated; finally, time was recorded. Customized software detected the largest Ca2+ transients (area under the curve) from each line scan and matched it with parameters above. Overall, larger dendritic diameter and shorter path distance from the soma were significant predictors of larger transients, while time was not significant up to 2 h (data thereafter was dropped). However, Ca2+ transients showed additional variability. Controlling for previous factors, significant variation was found between Ca2+ signals from different processes of the same neuron in 3/7 neurons. This could reflect differential expression of Ca2+ channels, local neuromodulation or other variations. Finally, Ca2+ transients in SOD1G93A motoneurons were significantly smaller than in non-transgenic motoneurons. In conclusion, motoneuron processes show highly variable Ca2+ transients, but these transients are smaller overall in SOD1G93A motoneurons.

Spinal cord injury in hypertonic newborns after antenatal hypoxia-ischemia in a rabbit model of cerebral palsy

ABSTRACT While antenatal hypoxia‐ischemia (H‐I) is a well‐established cause of brain injury, the effects of H‐I on the spinal cord remain undefined. This study examined whether hypertonia in rabbits was accompanied by changes in spinal architecture. Rabbit dams underwent global fetal H‐I at embryonic day 25 for 40 min. High resolution diffusion tensor imaging was performed on fixed neonatal CNS. Fractional anisotropy (FA) and regional volumetric measurements were compared between kits with and without hypertonia after H‐I and sham controls using Tract Based Spatial Statistics. Hypertonic kits showed evidence of damage from hypoxia not only in the brain, but in spinal cord as well. Hypertonic kits showed reduced FA and thickness in corticospinal tracts, external capsule, fimbria, and in white and gray matter of both cervical and lumbar spinal cord. Dorsal white matter of the spinal cord was the exception, where there was thickening and increased FA in hypertonic kits. Direct damage to the spinal cord was demonstrated in a subset of dams imaged during H‐I with a 3 T magnetic resonance scanner, where apparent diffusion coefficient in fetal spinal cords acutely decreased during hypoxia. Hypertonic kits showed subsequent decreases in lumbar motoneuron counts and extensive TUNEL‐ and Fluoro‐Jade C‐positive labeling was present in the spinal cord 48 h after H‐I, demonstrating spinal neurodegeneration. We speculate that global H‐I causes significant loss of both spinal white and gray matter in hypertonic newborns due to direct H‐I injury to the spinal cord as well as due to upstream brain injury and consequent loss of descending projections. HIGHLIGHTSMotor deficits caused by antenatal hypoxia typical of cerebral palsy are often attributed solely to brain injuries.Evidence here shows an acute drop in apparent diffusion coefficient in the fetal rabbit spinal cord, similar to that experienced in the brain.Extensive neurodegeneration was apparent in the spinal cord, resulting in smaller size of most spinal regions.Extent of brain injuries corresponded to loss of spinal white and gray matter, indicating neural damage throughout CNS.Thus motor deficits likely arise from the concurrent injuries in the developing brain and spinal cord.