Turgay Akay

Neuronal Matrix Metalloproteinase-9 Is a Determinant of Selective Neurodegeneration

Selective neuronal loss is the hallmark of neurodegenerative diseases. In patients with amyotrophic lateral sclerosis (ALS), most motor neurons die but those innervating extraocular, pelvic sphincter, and slow limb muscles exhibit selective resistance. We identified 18 genes that show >10-fold differential expression between resistant and vulnerable motor neurons. One of these, matrix metalloproteinase-9 (MMP-9), is expressed only by fast motor neurons, which are selectively vulnerable. In ALS model mice expressing mutant superoxide dismutase (SOD1), reduction of MMP-9 function using gene ablation, viral gene therapy, or pharmacological inhibition significantly delayed muscle denervation. In the presence of mutant SOD1, MMP-9 expressed by fast motor neurons themselves enhances activation of ER stress and is sufficient to trigger axonal die-back. These findings define MMP-9 as a candidate therapeutic target for ALS. The molecular basis of neuronal diversity thus provides significant insights into mechanisms of selective vulnerability to neurodegeneration.

Patterns of Spinal Sensory-Motor Connectivity Prescribed by a Dorsoventral Positional Template

Sensory-motor circuits in the spinal cord are constructed with a fine specificity that coordinates motor behavior, but the mechanisms that direct sensory connections with their motor neuron partners remain unclear. The dorsoventral settling position of motor pools in the spinal cord is known to match the distal-to-proximal position of their muscle targets in the limb, but the significance of invariant motor neuron positioning is unknown. An analysis of sensory-motor connectivity patterns in FoxP1 mutant mice, where motor neuron position has been scrambled, shows that the final pattern of sensory-motor connections is initiated by the projection of sensory axons to discrete dorsoventral domains of the spinal cord without regard for motor neuron subtype or, indeed, the presence of motor neurons. By implication, the clustering and dorsoventral settling position of motor neuron pools serve as a determinant of the pattern of sensory input specificity and thus motor coordination.

Degradation of mouse locomotor pattern in the absence of proprioceptive sensory feedback

Significance Terrestrial locomotion is thought to be generated by the actions of a circuit of interconnected interneurons (central pattern generator) in the spinal cord that drive the patterned activity of pools of motor neurons, causing sequential contraction of dozens of leg muscles. Sensory feedback exerts a strong modulatory influence on this pattern; nevertheless, it remains unclear whether sensory feedback also plays a role in the generation of the normal locomotor pattern. Through the use of a combination of electrophysiology, behavior, and mouse genetics, we provide evidence that the absence of proprioceptive sensory feedback degrades locomotor pattern, indicating that proprioceptive feedback is required for the construction of locomotor pattern. Mammalian locomotor programs are thought to be directed by the actions of spinal interneuron circuits collectively referred to as “central pattern generators.” The contribution of proprioceptive sensory feedback to the coordination of locomotor activity remains less clear. We have analyzed changes in mouse locomotor pattern under conditions in which proprioceptive feedback is attenuated genetically and biomechanically. We find that locomotor pattern degrades upon elimination of proprioceptive feedback from muscle spindles and Golgi tendon organs. The degradation of locomotor pattern is manifest as the loss of interjoint coordination and alternation of flexor and extensor muscles. Group Ia/II sensory feedback from muscle spindles has a predominant influence in patterning the activity of flexor muscles, whereas the redundant activities of group Ia/II and group Ib afferents appear to determine the pattern of extensor muscle firing. These findings establish a role for proprioceptive feedback in the control of fundamental aspects of mammalian locomotor behavior.

Quantification of gait parameters in freely walking wild type and sensory deprived Drosophila melanogaster.

Coordinated walking in vertebrates and multi-legged invertebrates such as Drosophila melanogaster requires a complex neural network coupled to sensory feedback. An understanding of this network will benefit from systems such as Drosophila that have the ability to genetically manipulate neural activities. However, the flys small size makes it challenging to analyze walking in this system. In order to overcome this limitation, we developed an optical method coupled with high-speed imaging that allows the tracking and quantification of gait parameters in freely walking flies with high temporal and spatial resolution. Using this method, we present a comprehensive description of many locomotion parameters, such as gait, tarsal positioning, and intersegmental and left-right coordination for wild type fruit flies. Surprisingly, we find that inactivation of sensory neurons in the flys legs, to block proprioceptive feedback, led to deficient step precision, but interleg coordination and the ability to execute a tripod gait were unaffected. DOI: http://dx.doi.org/10.7554/eLife.00231.001

Segment specificity of load signal processing depends on walking direction in the stick insect leg muscle control system.

In terrestrial locomotion, sensory feedback from load sensors is important for altering ongoing motor output on a step-by-step basis. We investigated the influence of load signals from the leg on motoneuron pools of the thorax-coxa (ThC) joint in the stick insect walking system. Load sensors were stimulated during rhythmic, alternating activity in protractor coxae (ProCx) and retractor coxae (RetCx) motoneuron pools. Alternating activity in the segment of interest was induced by mechanical stimulation of the animal or pharmacological activation of the isolated thoracic ganglia. Load signals from the legs altered the timing of ThC motoneuron activity by resetting and entraining the activity of the central rhythm generating network of the ThC joint. In the front and middle legs, load signals induced or promoted RetCx activity and decreased or terminated ProCx activity. In the hindleg, reverse transitions were elicited, with increasing load terminating RetCx and initiating ProCx activity. Studies in semi-intact walking animals showed that the effect of load on the ThC-joint motoneurons depended on walking direction, with increased load promoting the functional stance phase motoneuron pool (in forward walking, RetCx activity; in backward walking, ProCx activity). Thus, we show that modifications of sensory feedback in a locomotor system are related to walking direction. In a final set of ablation experiments, we show that the load influence is mediated by the three groups of trochanteral campaniform sensilla.

Circuits for Grasping: Spinal dI3 Interneurons Mediate Cutaneous Control of Motor Behavior

Accurate motor performance depends on the integration in spinal microcircuits of sensory feedback information. Hand grasp is a skilled motor behavior known to require cutaneous sensory feedback, but spinal microcircuits that process and relay this feedback to the motor system have not been defined. We sought to define classes of spinal interneurons involved in the cutaneous control of hand grasp in mice and to show that dI3 interneurons, a class of dorsal spinal interneurons marked by the expression of Isl1, convey input from low threshold cutaneous afferents to motoneurons. Mice in which the output of dI3 interneurons has been inactivated exhibit deficits in motor tasks that rely on cutaneous afferent input. Most strikingly, the ability to maintain grip strength in response to increasing load is lost following genetic silencing of dI3 interneuron output. Thus, spinal microcircuits that integrate cutaneous feedback crucial for paw grip rely on the intermediary role of dI3 interneurons.

Neural control of unloaded leg posture and of leg swing in stick insect, cockroach, and mouse differs from that in larger animals.

Stick insect (Carausius morosus) leg muscles contract and relax slowly. Control of stick insect leg posture and movement could therefore differ from that in animals with faster muscles. Consistent with this possibility, stick insect legs maintained constant posture without leg motor nerve activity when the animals were rotated in air. That unloaded leg posture was an intrinsic property of the legs was confirmed by showing that isolated legs had constant, gravity-independent postures. Muscle ablation experiments, experiments showing that leg muscle passive forces were large compared with gravitational forces, and experiments showing that, at the rest postures, agonist and antagonist muscles generated equal forces indicated that these postures depended in part on leg muscles. Leg muscle recordings showed that stick insect swing motor neurons fired throughout the entirety of swing. To test whether these results were specific to stick insect, we repeated some of these experiments in cockroach (Periplaneta americana) and mouse. Isolated cockroach legs also had gravity-independent rest positions and mouse swing motor neurons also fired throughout the entirety of swing. These data differ from those in human and horse but not cat. These size-dependent variations in whether legs have constant, gravity-independent postures, in whether swing motor neurons fire throughout the entirety of swing, and calculations of how quickly passive muscle force would slow limb movement as limb size varies suggest that these differences may be caused by scaling. Limb size may thus be as great a determinant as phylogenetic position of unloaded limb motor control strategy.

Spinal 5-HT7 receptors are critical for alternating activity during locomotion: in vitro neonatal and in vivo adult studies using 5-HT7 receptor knockout mice.

5-HT7 receptors have been implicated in the control of locomotion. Here we use 5-HT7 receptor knockout mice to rigorously test whether 5-HT acts at the 5-HT7 receptor to control locomotor-like activity in the neonatal mouse spinal cord in vitro and voluntary locomotion in adult mice. We found that 5-HT applied onto in vitro spinal cords of 5-HT7+/+ mice produced locomotor-like activity that was disrupted and subsequently blocked by the 5-HT7 receptor antagonist SB-269970. In spinal cords isolated from 5-HT7-/- mice, 5-HT produced either uncoordinated rhythmic activity or resulted in synchronous discharges of the ventral roots. SB-269970 had no effect on 5-HT-induced rhythmic activity in the 5-HT7-/- mice. In adult in vivo experiments, SB-269970 applied directly to the spinal cord consistently disrupted locomotion and produced prolonged-extension of the hindlimbs in 5-HT7+/+ but not 5-HT7-/- mice. Disrupted EMG activity produced by SB-269970 in vivo was similar to the uncoordinated rhythmic activity produced by the drug in vitro. Moreover, 5-HT7-/- mice displayed greater maximal extension at the hip and ankle joints than 5-HT7+/+ animals during voluntary locomotion. These results suggest that spinal 5-HT7 receptors are required for the production and coordination of 5-HT-induced locomotor-like activity in the neonatal mouse and are important for the coordination of voluntary locomotion in adult mice. We conclude that spinal 5-HT7 receptors are critical for alternating activity during locomotion.

Quantification of gait parameters in freely walking rodents

BackgroundQualitative and quantitative measurements of motor performance are essential for characterizing perturbations of motor systems. Although several methods exist for analyzing specific motor tasks, few behavioral assays are readily available to researchers that provide a complete set of kinematic parameters in rodents.ResultsHere we present MouseWalker, an integrated hardware and software system that provides a comprehensive and quantitative description of kinematic features in freely walking rodents. Footprints are visualized with high spatial and temporal resolution by a non-invasive optical touch sensor coupled to high-speed imaging. A freely available and open-source software package tracks footprints and body features to generate a comprehensive description of many locomotion features, including static parameters such as footprint position and stance patterns and dynamic parameters, such as step and swing cycle duration, and inter-leg coordination. Using this method, we describe walking by wild-type mice including several previously undescribed parameters. For example, we demonstrate that footprint touchdown occurs instantaneously by the entire paw with no obvious rostral–caudal or lateral–medial bias.ConclusionsThe readily available MouseWalker system and the large set of readouts it generates greatly increases the currently available toolkit for the analysis of wild type and aberrant locomotion in rodents.

Spinal microcircuits comprising dI3 interneurons are necessary for motor functional recovery following spinal cord transection

The spinal cord has the capacity to coordinate motor activities such as locomotion. Following spinal transection, functional activity can be regained, to a degree, following motor training. To identify microcircuits involved in this recovery, we studied a population of mouse spinal interneurons known to receive direct afferent inputs and project to intermediate and ventral regions of the spinal cord. We demonstrate that while dI3 interneurons are not necessary for normal locomotor activity, locomotor circuits rhythmically inhibit them and dI3 interneurons can activate these circuits. Removing dI3 interneurons from spinal microcircuits by eliminating their synaptic transmission left locomotion more or less unchanged, but abolished functional recovery, indicating that dI3 interneurons are a necessary cellular substrate for motor system plasticity following transection. We suggest that dI3 interneurons compare inputs from locomotor circuits with sensory afferent inputs to compute sensory prediction errors that then modify locomotor circuits to effect motor recovery. DOI: http://dx.doi.org/10.7554/eLife.21715.001