Closed-loop automated reaching apparatus (CLARA) for interrogating complex motor behaviors.

Objective.Closed-loop neuromodulation technology is a rapidly expanding category of therapeutics for a broad range of indications. Development of these innovative neurological devices requires high-throughput systems for closed-loop stimulation of model organisms, while monitoring physiological signals and complex, naturalistic behaviors. To address this need, we developed CLARA, a closed-loop automated reaching apparatus.Approach.Using breakthroughs in computer vision, CLARA integrates fully-automated, markerless kinematic tracking of multiple features to classify animal behavior and precisely deliver neural stimulation based on behavioral outcomes. CLARA is compatible with advanced neurophysiological tools, enabling the testing of neurostimulation devices and identification of novel neurological biomarkers.Results.The CLARA system tracks unconstrained skilled reach behavior in 3D at 150 Hz without physical markers. The system fully automates trial initiation and pellet delivery and is capable of accurately delivering stimulation in response to trial outcome with short latency. Kinematic data from the CLARA system provided novel insights into the dynamics of reach consistency over the course of learning, suggesting that learning selectively improves reach failures but does not alter the kinematics of successful reaches. Additionally, using the closed-loop capabilities of CLARA, we demonstrate that vagus nerve stimulation (VNS) improves skilled reach performance and increases reach trajectory consistency in healthy animals.Significance.The CLARA system is the first mouse behavior apparatus that uses markerless pose tracking to provide real-time closed-loop stimulation in response to the outcome of an unconstrained motor task. Additionally, we demonstrate that the CLARA system was essential for our investigating the role of closed-loop VNS stimulation on motor performance in healthy animals. This approach has high translational relevance for developing neurostimulation technology based on complex human behavior.

Development and plasticity of complex movement representations.

The mammalian motor cortex is topographically organized into representations of discrete body parts (motor maps). Studies in adult rats using long-duration intracortical microstimulation (LD-ICMS) reveal that forelimb motor cortex is functionally organized into several spatially distinct areas encoding complex, multijoint movement sequences: elevate, advance, grasp, and retract. The topographical arrangement of complex movements during development and the influence of skilled learning are unknown. Here, we determined the emergence and topography of complex forelimb movement representations in rats between postnatal days (PND) 13 and 60. We further investigated the expression of the maps for complex movements under conditions of reduced cortical inhibition and whether skilled forelimb motor training could alter their developing topography. We report that simple forelimb movements are first evoked at PND 25 and are confined to the caudal forelimb area (CFA), whereas complex movements first reliably appear at PND 30 and are observed in both the caudal and rostral forelimb areas (RFA). During development, the topography of complex movement representations undergoes reorganization with "grasp" and "elevate" movements predominantly observed in the RFA and all four complex movements observed in CFA. Under reduced cortical inhibition, simple and complex movements were first observed in the CFA on PND 15 and 20, respectively, and the topography is altered relative to a saline control. Further, skilled motor learning was associated with increases in "grasp" and "retract" representations specific to the trained limb. Our results demonstrate that early-life motor experience during development can modify the topography of complex forelimb movement representations.NEW & NOTEWORTHY The motor cortex is topographically organized into maps of different body parts. We used to think that the function of motor cortex was to drive individual muscles, but more recently we have learned that it is also organized to make complex movements. However, the development and plasticity of those complex movements is completely unknown. In this paper, the emergence and topography of complex movement representation, as well as their plasticity during development, is detailed.

Forelimb Cortical Stroke Reduces Precision of Motor Control in Mice.

Background and Objective. Rodent models of stroke impairment should capture translatable features of behavioral injury. This study characterized poststroke impairment of motor precision separately from strength in an automated behavioral assay. Methods. We measured skilled distal forelimb reach-and-grasp motions within a target force range requiring moderate-strength. We assessed whether deficits reflected an increase in errors on only one or both sides of the target force range after photothrombotic cortical stroke. Results. Pull accuracy was impaired for 6 weeks after stroke, with errors redistributing to both sides of the target range. No decrease in maximum force was measured. Conclusions. This automated reach task measures sustained loss of motor precision following cortical stroke in mice.

Low acetylcholine during early sleep is important for motor memory consolidation.

The synaptic homeostasis theory of sleep proposes that low neurotransmitter activity in sleep optimizes memory consolidation. We tested this theory by asking whether increasing acetylcholine levels during early sleep would weaken motor memory consolidation. We trained separate groups of adult mice on the rotarod walking task and the single pellet reaching task, and after training, administered physostigmine, an acetylcholinesterase inhibitor, to increase cholinergic tone in subsequent sleep. Post-sleep testing showed that physostigmine impaired motor skill acquisition of both tasks. Home-cage video monitoring and electrophysiology revealed that physostigmine disrupted sleep structure, delayed non-rapid-eye-movement sleep onset, and reduced slow-wave power in the hippocampus and cortex. Additional experiments showed that: (1) the impaired performance associated with physostigmine was not due to its effects on sleep structure, as 1 h of sleep deprivation after training did not impair rotarod performance, (2) a reduction in cholinergic tone by inactivation of cholinergic neurons during early sleep did not affect rotarod performance, and (3) stimulating or blocking muscarinic and nicotinic acetylcholine receptors did not impair rotarod performance. Taken together, the experiments suggest that the increased slow wave activity and inactivation of both muscarinic and nicotinic receptors during early sleep due to reduced acetylcholine contribute to motor memory consolidation.

Ghrelin signalling within the rat nucleus accumbens and skilled reach foraging.

Motivation alters behaviour in a complex manner and nucleus accumbens (NAc) shell has been implied as a key structure regulating such behaviour. Recent studies show that acute ghrelin signalling enhances motivation when assessed in a simple motor task. The aim of the present study was to define the role of ghrelin signalling on motivation in a more complex motor behaviour. Rats were tested in the Montoya staircase, an animal model of skilled reach foraging assessed by the number of sucrose pellets consumed. Electrophysiological recordings were conducted to explore the neurophysiological correlates of ghrelin signalling. The initial electrophysiological results displayed that ex vivo administration of ghrelin increased NAc shell output in brain slices from drug- and training-naïve rats. In rats with an acquired skilled reach performance, acute as well as repeated treatment with a ghrelin receptor (GHSR-1 A) antagonist (JMV2959) decreased the number of sucrose pellets consumed. Moreover, infusion of JMV2959 into NAc shell reduced this consumption. Sub-chronic, during ten days, JMV2959 treatment during training on the Montoya staircase reduced the number of pellets consumed, whereas ghrelin improved this behaviour. In addition, field potential and whole cell recordings were conducted in NAc shell of rats that had been treated with ghrelin or GHSR-1 A antagonist during training on the Montoya staircase. Sub-chronic administration of ghrelin during motor-skill learning selectively increased the frequency of inhibitory transmission in the NAc shell, resulting in a net suppression of accumbal output. Collectively these data suggest that ghrelin signalling in NAc shell enhances skilled reached foraging tentatively by increasing the motivation.

Precision of Discrete and Rhythmic Forelimb Movements Requires a Distinct Neuronal Subpopulation in the Interposed Anterior Nucleus.

The deep cerebellar nuclei (DCN) represent output channels of the cerebellum, and they transmit integrated sensorimotor signals to modulate limb movements. But the functional relevance of identifiable neuronal subpopulations within the DCN remains unclear. Here, we examine a genetically tractable population of neurons in the mouse interposed anterior nucleus (IntA). We show that these neurons represent a subset of glutamatergic neurons in the IntA and constitute a specific element of an internal feedback circuit within the cerebellar cortex and cerebello-thalamo-cortical pathway associated with limb control. Ablation and optogenetic stimulation of these neurons disrupt efficacy of skilled reach and locomotor movement and reveal that they control positioning and timing of the forelimb and hindlimb. Together, our findings uncover the function of a distinct neuronal subpopulation in the deep cerebellum and delineate the anatomical substrates and kinematic parameters through which it modulates precision of discrete and rhythmic limb movements.

Developmental abnormality contributes to cortex-dependent motor impairments and higher intracortical current requirement in the reeler homozygous mutants.

The motor deficit of the reeler mutants has largely been considered cerebellum related, and the developmental consequences of the cortex on reeler motor behavior have not been examined. We herein showed that there is a behavioral consequence to reeler mutation in models examined at cortex-dependent bimanual tasks that require forepaw dexterity. Using intracortical microstimulation, we found the forelimb representation in the motor cortex was significantly reduced in the reeler. The reeler cortex required a significantly higher current to evoke skeletal muscle movements, suggesting the cortical trans-synaptic propagation is disrupted. When the higher current was applied, the reeler motor representation was found preserved. To elucidate the influence of cerebellum atrophy and ataxia on the obtained results, the behavioral and neurophysiological findings in reeler mice were reproduced using the Disabled-1 (Dab1) cKO mice, in which the Reelin-Dab1 signal deficiency is confined to the cerebral cortex. The Dab1 cKO mice were further assessed at the single-pellet reach and retrieval task, displaying a lower number of successfully retrieved pellets. It suggests the abnormality confined to the cortex still reduced the dexterous motor performance. Although possible muscular dysfunction was reported in REELIN-deficient humans, the function of the reeler forelimb muscle examined by electromyography, morphology of neuromuscular junction and the expression level of choline acetyltransferase were normal. Our results suggest that the mammalian laminar structure is necessary for the forepaw skill performance and for trans-synaptic efficacy in the cortical output.

Manganese-Enhanced Magnetic Resonance Imaging and Studies of Rat Behavior: Transient Motor Deficit in Skilled Reaching, Rears, and Activity in Rats After a Single Dose of MnCl2.

Manganese-enhanced magnetic resonance imaging (MEMRI) has been suggested to be a useful tool to visualize and map behavior-relevant neural populations at large scale in freely behaving rodents. A primary concern in MEMRI applications is Mn2+ toxicity. Although a few studies have specifically examined toxicity on gross motor behavior, Mn2+ toxicity on skilled motor behavior was not explored. Thus, the objective of this study was to combine manganese as a functional contrast agent with comprehensive behavior evaluation. We evaluated Mn2+ effect on skilled reach-to-eat action, locomotion, and balance using a single pellet reaching task, activity cage, and cylinder test, respectively. The tests used are sensitive to the pathophysiology of many neurological and neurodegenerative disorders of the motor system. The behavioral testing was done in combination with a moderate dose of manganese. Behavior was studied before and after a single, intravenous infusion of MnCl2 (48 mg/kg). The rats were imaged at 1, 3, 5, 7, and 14 days following infusion. The results show that MnCl2 infusion resulted in detectable abnormalities in skilled reaching, locomotion, and balance that recovered within 3 days compared with the infusion of saline. Because some tests and behavioral measures could not detect motor abnormalities of skilled movements, comprehensive evaluation of motor behavior is critical in assessing the effects of MnCl2. The relaxation mapping results suggest that the transport of Mn2+ into the brain is through the choroid plexus-cerebrospinal fluid system with the primary entry point and highest relaxation rates found in the pituitary gland. Relaxation rates in the pituitary gland correlated with measures of motor skill, suggesting that altered motor ability is related to the level of Mn circulating in the brain. Thus, combined MEMRI and behavioral studies that both achieve adequate image enhancement and are also free of motor skills deficits are difficult to achieve using a single systemic dose of MnCl2.

Effects of skilled reach training with affected forelimb and treadmill exercise on the expression of neurotrophic factor following ischemia-induced brain injury in rats.

[Purpose] The aim of the present study was to investigate effects of skilled reach training with affected forelimb and treadmill exercise on the expression of neurotrophic factor following ischemia-induced brain injury in rats. [Subjects and Methods] Thirty male Sprague-Dawley rats were divided into 3 groups randomly: namely, the control sacrified 2 weeks after surgery, skilled reach training with forepaw contralateral to brain injury for 2 weeks, and treadmill exercise for 2 weeks. Transient focal cerebral ischemia was induced by intraluminal occlusion of the left middle cerebral artery. After that, skilled reach training and treadmill exercise were conducted. Western blot analysis was performed to investigate expressions of neurotrophic factors. [Results] There were significant differences in brain-derived neurotrophic factor and nerve growth factor expression between the control group and the experimental group. There were no significant differences in brain-derived neurotrophic factor and nerve growth factor expression between the skilled reach training group and the treadmill exercise group. [Conclusion] Skilled reach training and treadmill exercise can affect the expression of neurotrophic factors.

Behavioral characterization of female zinc transporter 3 (ZnT3) knockout mice.

Zinc is an important element in all cells of the body, having structural, enzymatic, and regulatory functions. In some neurons, zinc is loaded into synaptic vesicles by zinc transporter 3 (ZnT3) and released into the synaptic cleft, where it can modulate neuronal function. ZnT3 knockout (KO) mice lack ZnT3 and thus lack synaptic zinc. Previous studies have examined the behavioral phenotype of ZnT3 KO mice, mostly using mixed-sex or male-only groups. In the present study we focused specifically on the behavior of female ZnT3 KO mice (2-3 months old). An extensive battery of tests was administered to assess sensorimotor and cognitive behaviours, as well as to examine for a possible schizophrenia-like phenotype. ZnT3 KO mice performed similarly to wild type controls in the majority of tests. However, they were less accurate in the skilled reach task, suggesting impaired skilled motor learning, and faster to descend a vertical pole. ZnT3 KO mice were also slower in the open field and made fewer chamber entries in the social preference test, suggesting decreased exploratory locomotion. No differences were observed in the Morris water task or fear conditioning test. This is the first study to show a behavioural phenotype specifically for female ZnT3 KO mice. Comparing our results to previous studies, it appears that there may be sex-specific effects of eliminating ZnT3. Female ZnT3 KO mice exhibit abnormalities in locomotion and at skilled motor learning, but we were unable to detect spatial or fear learning deficits previously described in male ZnT3 KO mice.

Corticospinal circuit plasticity in motor rehabilitation from spinal cord injury.

Restoring corticospinal function after spinal cord injury is a significant challenge as the corticospinal tract elicits no substantive, spontaneous regeneration, and its interruption leaves a permanent deficit. The corticospinal circuit serves multiple motor and sensory functions within the mammalian nervous system as the direct link between isocortex and spinal cord. Maturation of the corticospinal circuit involves the refinement of projections within the spinal cord and a subsequent refinement of motor maps within the cortex. The plasticity of these cortical motor maps mirrors the acquisition of skilled motor learning, and both the maps and motor skills are disrupted following injury to the corticospinal tract. The motor cortex exhibits the capacity to incorporate changes in corticospinal projections induced by both spontaneous and therapeutic-mediated plasticity of corticospinal axons through appropriate rehabilitation. An understanding of the mechanisms of corticospinal plasticity in motor learning will undoubtedly help inform strategies to improve motor rehabilitation after spinal cord injury.

Effects of acute sleep deprivation on motor and reversal learning in mice.

Sleep supports the formation of a variety of declarative and non-declarative memories, and sleep deprivation often impairs these types of memories. In human subjects, natural sleep either during a nap or overnight leads to long-lasting improvements in visuomotor and fine motor tasks, but rodent models recapitulating these findings have been scarce. Here we present evidence that 5h of acute sleep deprivation impairs mouse skilled reach learning compared to a matched period of ad libitum sleep. In sleeping mice, the duration of total sleep time during the 5h of sleep opportunity or during the first bout of sleep did not correlate with ultimate gain in motor performance. In addition, we observed that reversal learning during the skilled reaching task was also affected by sleep deprivation. Consistent with this observation, 5h of sleep deprivation also impaired reversal learning in the water-based Y-maze. In conclusion, acute sleep deprivation negatively impacts subsequent motor and reversal learning and memory.

Skilled reach training influences brain recovery following intracerebral hemorrhage in rats.

[Purpose] The present study investigated how skilled reach training influences functional and neurological brain recovery via a rat model with intracerebral hemorrhage. [Subjects] Thirty rats with intracerebral hemorrhage were divided into 2 groups randomly: the control group (CON) that did not receive any treatment, and the experimental group (SRT) that received skilled reach training. [Methods] The experimental group was trained through skilled reaching training with the affected upper limb in 15-minute sessions administered 6 days per week for 4 weeks. [Results] In the behavioral test, the results showed that motor function was significantly improved in the skilled reach training group compared with the control group. In the neurological teat, the expression level of brain-derived neurotrophic factor (BDNF) was significantly increased in the skilled reach training group compared with the control group. [Conclusion] Skilled reach training is able to facilitate both the expression of neurotrophic factor in the motor cortex and motor function recovery following intracerebral hemorrhage.