Pathophysiology of Dyt1-Tor1a dystonia in mice is mediated by spinal neural circuit dysfunction.

Dystonia, a neurological disorder defined by abnormal postures and disorganized movements, is considered to be a neural circuit disorder with dysfunction arising within and between multiple brain regions. Given that spinal neural circuits constitute the final pathway for motor control, we sought to determine their contribution to this movement disorder. Focusing on the most common inherited form of dystonia in humans, DYT1-TOR1A, we generated a conditional knockout of the torsin family 1 member A (Tor1a) gene in the mouse spinal cord and dorsal root ganglia (DRG). We found that these mice recapitulated the phenotype of the human condition, developing early-onset generalized torsional dystonia. Motor signs emerged early in the mouse hindlimbs before spreading caudo-rostrally to affect the pelvis, trunk, and forelimbs throughout postnatal maturation. Physiologically, these mice bore the hallmark features of dystonia, including spontaneous contractions at rest and excessive and disorganized contractions, including cocontractions of antagonist muscle groups, during voluntary movements. Spontaneous activity, disorganized motor output, and impaired monosynaptic reflexes, all signs of human dystonia, were recorded from isolated mouse spinal cords from these conditional knockout mice. All components of the monosynaptic reflex arc were affected, including motor neurons. Given that confining the Tor1a conditional knockout to DRG did not lead to early-onset dystonia, we conclude that the pathophysiological substrate of this mouse model of dystonia lies in spinal neural circuits. Together, these data provide new insights into our current understanding of dystonia pathophysiology.

Electrical Properties of Adult Mammalian Motoneurons.

Motoneurons are the 'final common path' between the central nervous system (that intends, selects, commands, and organises movement) and muscles (that produce the behaviour). Motoneurons are not passive relays, but rather integrate synaptic activity to appropriately tune output (spike trains) and therefore the production of muscle force. In this chapter, we focus on studies of mammalian motoneurons, describing their heterogeneity whilst providing a brief historical account of motoneuron recording techniques. Next, we describe adult motoneurons in terms of their passive, transition, and active (repetitive firing) properties. We then discuss modulation of these properties by somatic (C-boutons) and dendritic (persistent inward currents) mechanisms. Finally, we briefly describe select studies of human motor unit physiology and relate them to findings from animal preparations discussed earlier in the chapter. This interphyletic approach to the study of motoneuron physiology is crucial to progress understanding of how these diverse neurons translate intention into behaviour.

C-bouton components on rat extensor digitorum longus motoneurons are resistant to chronic functional overload.

Mammalian motor systems adapt to the demands of their environment. For example, muscle fibre types change in response to increased load or endurance demands. However, for adaptations to be effective, motoneurons must adapt such that their properties match those of the innervated muscle fibres. We used a rat model of chronic functional overload to assess adaptations to both motoneuron size and a key modulatory synapse responsible for amplification of motor output, C-boutons. Overload of extensor digitorum longus (EDL) muscles was induced by removal of their synergists, tibialis anterior muscles. Following 21 days survival, EDL muscles showed an increase in fatigue resistance and a decrease in force output, indicating a shift to a slower phenotype. These changes were reflected by a decrease in motoneuron size. However, C-bouton complexes remained largely unaffected by overload. The C-boutons themselves, quantified by expression of vesicular acetylcholine transporter, were similar in size and density in the control and overload conditions. Expression of the post-synaptic voltage-gated potassium channel (KV 2.1) was also unchanged. Small conductance calcium-activated potassium channels (SK3) were expressed in most EDL motoneurons, despite this being an almost exclusively fast motor pool. Overload induced a decrease in the proportion of SK3+ cells, however, there was no change in density or size of clusters. We propose that reductions in motoneuron size may promote early recruitment of EDL motoneurons, but that C-bouton plasticity is not necessary to increase the force output required in response to muscle overload.

Spinal motoneuron firing properties mature from rostral to caudal during postnatal development of the mouse.

KEY POINTS: Many mammals are born with immature motor systems that develop through a critical period of postnatal development. In rodents, postnatal maturation of movement occurs from rostral to caudal, correlating with maturation of descending supraspinal and local spinal circuits. We asked whether development of fundamental electrophysiological properties of spinal motoneurons follows the same rostro-caudal sequence. We show that in both regions, repetitive firing parameters increase and excitability decreases with development; however, these characteristics mature earlier in cervical motoneurons. We suggest that in addition to autonomous mechanisms, motoneuron development depends on activity resulting from their circuit milieu. ABSTRACT: Altricial mammals are born with immature nervous systems comprised of circuits that do not yet have the neuronal properties and connectivity required to produce future behaviours. During the critical period of postnatal development, neuronal properties are tuned to participate in functional circuits. In rodents, cervical motoneurons are born prior to lumbar motoneurons, and spinal cord development follows a sequential rostro-caudal pattern. Here we asked whether birth order is reflected in the postnatal development of electrophysiological properties. We show that motoneurons of both regions have similar properties at birth and follow the same developmental profile, with maximal firing increasing and excitability decreasing into the third postnatal week. However, these maturative processes occur in cervical motoneurons prior to lumbar motoneurons, correlating with the maturation of premotor descending and local spinal systems. These results suggest that motoneuron properties do not mature by cell autonomous mechanisms alone, but also depend on developing premotor circuits.

Simultaneous Assessment of Homonymous and Heteronymous Monosynaptic Reflex Excitability in the Adult Rat.

In order to successfully perform motor tasks such as locomotion, the central nervous system must coordinate contractions of antagonistic and synergistic muscles across multiple joints. This coordination is largely dependent upon the function of proprioceptive afferents (PAs), which make monosynaptic connections with homonymous motoneurons. Homonymous pathways have been well studied in both health and disease but their collateral fibers projecting to heteronymous, synergistic muscles receive relatively less attention. This is surprising given that PA collaterals have significant effects on the excitability of heteronymous motoneurons, and that their synaptic terminal density is activity dependent. It is likely that the relative lack of literature is due to the lack of a preparation which allows synergistic heteronymous pathways to be assessed in vivo. Here, we describe a method to simultaneously evoke homonymous and heteronymous (synergistic) monosynaptic reflexes (MSRs) and study their modulation by descending pathways in adult rats. Through stimulation of the medial plantar nerve, we were able to produce an H reflex in the intrinsic foot (IF) muscles of the hind paw with a latency of 10.52 ± 3.8 ms. Increasing the stimulus intensity evoked a robust signal with a monosynaptic latency (11.32 ± 0.35 ms), recorded in the ipsilateral gastrocnemius (Gs). Our subsequent analyses suggest that Gs motoneurons were activated via heteronymous afferent collaterals from the medial plantar nerve. These reflexes could be evoked bilaterally and were modulated by conditioning stimuli to the cortex (Cx) and reticular formation. Interestingly, cortical stimulation was equally efficient at modulating both ipsilateral and contralateral reflexes, indicating that cortical modulation of lumbar sensory afferents lacks the laterality demonstrated by studies of cortical muscle activation. This technique represents a novel, relatively simple way to assess heteronymous afferent pathways in normal motor control as well as in models of motor disorders where adaptive and maladaptive plasticity of PAs and descending systems affects functional outcomes.

Descending Systems Direct Development of Key Spinal Motor Circuits.

The formation of mature spinal motor circuits is dependent on both activity-dependent and independent mechanisms during postnatal development. During this time, reorganization and refinement of spinal sensorimotor circuits occurs as supraspinal projections are integrated. However, specific features of postnatal spinal circuit development remain poorly understood. This study provides the first detailed characterization of rat spinal sensorimotor circuit development in the presence and absence of descending systems. We show that the development of proprioceptive afferent input to motoneurons (MNs) and Renshaw cells (RCs) is disrupted by thoracic spinal cord transection at postnatal day 5 (P5TX). P5TX also led to malformation of GABApre neuron axo-axonic contacts on Ia afferents and of the recurrent inhibitory circuit between MNs and RCs. Using a novel in situ perfused preparation for studying motor control, we show that malformation of these spinal circuits leads to hyperexcitability of the monosynaptic reflex. Our results demonstrate that removing descending input severely disrupts the development of spinal circuits and identifies key mechanisms contributing to motor dysfunction in conditions such as cerebral palsy and spinal cord injury.SIGNIFICANCE STATEMENT Acquisition of mature behavior during postnatal development correlates with the arrival and maturation of supraspinal projections to the spinal cord. However, we know little about the role that descending systems play in the maturation of spinal circuits. Here, we characterize postnatal development of key spinal microcircuits in the presence and absence of descending systems. We show that formation of these circuits is abnormal after early (postnatal day 5) removal of descending systems, inducing hyperexcitability of the monosynaptic reflex. The study is a detailed characterization of spinal circuit development elucidating how these mechanisms contribute to motor dysfunction in conditions such as cerebral palsy and spinal cord injury. Understanding these circuits is crucial to developing new therapeutics and improving existing ones in such conditions.

Sequential therapy of anti-Nogo-A antibody treatment and treadmill training leads to cumulative improvements after spinal cord injury in rats.

Intense training is the most clinically successful treatment modality following incomplete spinal cord injuries (SCIs). With the advent of plasticity enhancing treatments, understanding how treatments might interact when delivered in combination becomes critical. Here, we investigated a rational approach to sequentially combine treadmill locomotor training with antibody mediated suppression of the fiber growth inhibitory protein Nogo-A. Following a large but incomplete thoracic lesion, rats were immediately treated with either anti-Nogo-A or control antibody (2weeks) and then either left untrained or step-trained starting 3weeks after injury for 8weeks. It was found that sequentially combined therapy improved step consistency and reduced toe dragging and climbing errors, as seen with training and anti-Nogo-A individually. Animals with sequential therapy also adopted a more parallel paw position during bipedal walking and showed greater overall quadrupedal locomotor recovery than individual treatments. Histologically, sequential therapy induced the greatest corticospinal tract sprouting caudally into the lumbar region and increased the number of serotonergic synapses onto lumbar motoneurons. Increased primary afferent sprouting and synapse formation onto lumbar motoneurons observed with anti-Nogo-A antibody were reduced by training. Animals with sequential therapy also showed the highest reduction of lumbar interneuronal activity associated with walking (c-fos expression). No treatment effects for thermal nociception, mechanical allodynia, or lesion volume were observed. The results demonstrate that sequential administration of anti-Nogo-A antibody followed in time with intensive locomotor training leads to superior recovery of lost locomotor functions, which is probably mediated by changes in the interaction between descending sprouting and local segmental networks after SCI.

Differential regulation of perineuronal nets in the brain and spinal cord with exercise training.

Perineuronal nets (PNNs) are lattice like structures which encapsulate the cell body and proximal dendrites of many neurons and are thought to be involved in regulating synaptic plasticity. It is believed that exercise can enhance the plasticity of the Central Nervous System (CNS) in healthy and dysfunctional states by shifting the balance between plasticity promoting and plasticity inhibiting factors in favor of the former. Recent work has focused on exercise effects on trophic factors but its effect on other plasticity regulators is poorly understood. In the present study we investigated how exercise regulates PNN expression in the lumbar spinal cord and areas of the brain associated with motor control and learning and memory. Adult, female Sprague-Dawley rats with free access to a running wheel for 6 weeks had significantly increased PNN expression in the spinal cord compared to sedentary rats (PNN thickness around motoneurons, exercise=15.75±0.63µm, sedentary=7.98±1.29µm, p<0.01). Conversely, in areas of the brain associated with learning and memory there was a significant reduction in perineuronal net expression (number of neurons with PNN in hippocampus CA1-exercise 21±0.56 and sedentary 24±0.34, p<0.01, thickness-exercised=2.37±0.13µm, sedentary=4.27±0.21µm; p<0.01). Our results suggest that in response to exercise, PNNs are differentially regulated in select regions of the CNS, with a general decreased expression in the brain and increased expression in the lumbar spinal cord. This differential expression may indicate different regulatory mechanisms associated with plasticity in the brain compared to the spinal cord.