Paw-Dragging: a Novel,Sensitive Analysis of the Mouse Cylinder Test

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore by rearing and touching the walls of the cylinder with their forelimb paws for postural support. Following ischemic injury to the forelimb sensorimotor cortex, rats rely more heavily on their unaffected forelimb paw for postural support resulting in fewer touches with their affected paw which is termed forelimb asymmetry. In contrast, focal ischemic damage in the mouse brain fails to result in comparable consistent deficits in forelimb asymmetry. While forelimb asymmetry deficits are infrequently observed, mice do demonstrate a novel behaviour post stroke termed “paw-dragging”. Paw-dragging is the tendency for a mouse to drag its affected paw along the cylinder wall rather than directly push off from the wall when dismounting from a rear to a four-legged stance. We have previously demonstrated that paw-dragging behaviour is highly sensitive to small cortical ischemic injuries to the forelimb motor cortex. Here we provide a detailed protocol for paw-dragging analysis. We define what a paw-drag is and demonstrate how to quantify paw-dragging behaviour. The cylinder test is a simple and inexpensive test to administer and does not require pre-training or food deprivation strategies. In using paw-dragging analysis with the cylinder test, it fills a niche for predicting cortical ischemic injuries such as photothrombosis and Endothelin-1 (ET-1)-induced ischemia – two models that are ever-increasing in popularity and produce smaller focal injuries than middle cerebral artery occlusion. Finally, measuring paw-dragging behaviour in the cylinder test will allow studies of functional recovery after cortical injury using a wide cohort of transgenic mouse strains where previous forelimb asymmetry analysis has failed to detect consistent deficits.     

Comparative study of the forelimbs of the semifossorial prairie dog, Cynomys gunnisoni, and the scansorial fox squirrel, Sciurus niger

A comparative study of the forelimbs of the semifossorial prairie dog, Cynomys gunnisoni , and the scansorial tree squirrel, Sciurus niger, was focused on the musculoskeletal design for digging in the former and climbing in the latter. Based on lever arm mechanics, it was expected that the forelimb of the prairie dog would show features appropriate to the production of relatively large forces and that of the fox squirrel to relatively great velocity. Force and lever arm measurements were made of select forelimb muscles at the shoulder, elbow, and wrist joints for a series of angles in both species. Contraction time and fatigue indexes were determined for the same forelimb muscles. Contrary to expectation, in the few cases in which significant (P less than .05) differences were found, the forces, lever arms, and torques (force times its lever arm) were greater in the smaller fox squirrel. The observed variation in the torques produced fits the demands on the forelimb during climbing and digging as estimated from films. Several forelimb muscles of the fox squirrel show significantly higher mean contraction times than do the homologous muscles of the prairie dog. There were no significant differences between the two species in the fatigability of the selected forelimb muscles, although the mean fatigue index was always higher (less fatigable muscle) in the prairie dog. Similarities in the forelimbs of these two sciurids suggest that only minor modifications may have been required of the ancestral forelimb in order for descendent forms to operate successfully as climbers and diggers .     

Comparison of electrical thresholds for evoking movements from sensori-motor areas of the cat cerebral cortex and its relation to motor training

Motor maps and electrical thresholds for evoking movements from motor areas of the cerebral cortex were evaluated in normal cats by using intracortical microstimulation techniques. Stainless steel chambers were implanted over craniotomies in adult cats trained to perform reaching and retrieval movements with their forelimbs. Prehensile motor training was continued and movement performance monitored for about 6–10 weeks during which the cortex was progressively explored with sharp tungsten electrodes inserted into cortical gyri (anterior and posterior sigmoid, and coronal) and the banks of sulci (cruciate, presylvian and coronal). Twice weekly, under light general anaesthesia, 3–4 tracks were made in either hemisphere till about 50 tracks were made in each hemisphere. Mean thresholds for evoking forelimb movements from different cytoarchitectonic areas (4γ, 4δ, 6aγ and 3a) were compared and no consistent or significant differences were observed between the different areas. In the animals (4/6) which used either forelimb to perform the tasks, there were no consistent differences in the mean thresholds for evoking forelimb movements from the two hemispheres. However, in 2 animals, which used their right forelimbs predominantly or exclusively to perform all the tasks, mean thresholds for evoking forelimb movements was significantly higher in areas 4γ and 6aγ of the left hemisphere (compared to the right); no consistent differences in the mean thresholds for evoking hindlimb or facial movements were observed between the two hemispheres. These findings suggest that ICMS thresholds for evoking forelimb movements may be similar in different sensorimotor areas of the cat cerebral cortex, and these thresholds could be influenced by motor training.     

A Novel Method for Assessing Proximal and Distal Forelimb Function in the Rat: the Irvine,Beatties and Bresnahan (IBB) Forelimb Scale

Several experimental models of cervical spinal cord injury (SCI) have been developed recently to assess the consequences of damage to this level of the spinal cord (Pearse et al., 2005, Gensel et al., 2006, Anderson et al., 2009), as the majority of human SCI occur here (Young, 2010; www.sci-info-pages.com). Behavioral deficits include loss of forelimb function due to damage to the white matter affecting both descending motor and ascending sensory systems, and to the gray matter containing the segmental circuitry for processing sensory input and motor output for the forelimb. Additionally, a key priority for human patients with cervical SCI is restoration of hand/arm function (Anderson, 2004). Thus, outcome measures that assess both proximal and distal forelimb function are needed. Although there are several behavioral assays that are sensitive to different aspects of forelimb recovery in experimental models of cervical SCI (Girgis et al., 2007, Gensel et al., 2006, Ballerman et al., 2001, Metz and Whishaw, 2000, Bertelli and Mira, 1993, Montoya et al., 1991, Whishaw and Pellis, 1990), few techniques provide detailed information on the recovery of fine motor control and digit movement.The current measurement technique, the Irvine, Beatties and Bresnahan forelimb scale (IBB), can detect recovery of both proximal and distal forelimb function including digit movements during a naturally occurring behavior that does not require extensive training or deprivation to enhance motivation. The IBB was generated by observing recovery after a unilateral C6 SCI, and involves video recording of animals eating two differently shaped cereals (spherical and doughnut) of a consistent size. These videos were then used to assess features of forelimb use, such as joint position, object support, digit movement and grasping technique.The IBB, like other forelimb behavioral tasks, shows a consistent pattern of recovery that is sensitive to injury severity. Furthermore, the IBB scale could be used to assess recovery following other types of injury that impact normal forelimb function.     

Comparison of electrical thresholds for evoking movements from sensori-motor areas of the cat cerebral cortex and its relation to motor training

Motor maps and electrical thresholds for evoking movements from motor areas of the cerebral cortex were evaluated in normal cats by using intracortical microstimulation techniques. Stainless steel chambers were implanted over craniotomies in adult cats trained to perform reaching and retrieval movements with their forelimbs. Prehensile motor training was continued and movement performance monitored for about 6-10 weeks during which the cortex was progressively explored with sharp tungsten electrodes inserted into cortical gyri (anterior and posterior sigmoid, and coronal) and the banks of sulci (cruciate, presylvian and coronal). Twice weekly, under light general anaesthesia, 3-4 tracks were made in either hemisphere till about 50 tracks were made in each hemisphere. Mean thresholds for evoking forelimb movements from different cytoarchitectonic areas (4gamma, 4delta, 6agamma and 3a) were compared and no consistent or significant differences were observed between the different areas. In the animals (4/6) which used either forelimb to perform the tasks, there were no consistent differences in the mean thresholds for evoking forelimb movements from the two hemispheres. However, in 2 animals, which used their right forelimbs predominantly or exclusively to perform all the tasks, mean thresholds for evoking forelimb movements was significantly higher in areas 4gamma and 6agamma of the left hemisphere (compared to the right); no consistent differences in the mean thresholds for evoking hindlimb or facial movements were observed between the two hemispheres. These findings suggest that ICMS thresholds for evoking forelimb movements may be similar in different sensorimotor areas of the cat cerebral cortex, and these thresholds could be influenced by motor training.     

Nonoverlapping Thalamocortical Connections to Normal and Deprived Primary Somatosensory Cortex for Similar Forelimb Receptive Fields in Chronic Spinal Cats

The fluorescent dye retrograde tracing technique, using fast blue in combination with fluorogold, was used to examine thalamocortical projections from the ventrobasal complex to primary somatosensory cortex in chronic spinal cats that sustained T12 cord transection at 2 weeks of age. Following cord transection at this age, it has been shown that forelimb afferents can excite the deprived hindlimb projection zone, in addition to the region of somatosensory cortex that they normally occupy (McKinley et al, 1987). These two regions of cortex are separated by over 10 mm, thus facilitating the determination of whether the forelimb representation in “hindlimb cortex” is derived from the sector of the ventrobasal complex of the thalamus representing the forelimb, hindlimb, or both. Injections of the two dyes into separate regions of the cortex that were excited by the same peripheral forelimb receptive fields produced single labeling of two nonoverlapping clusters of thalamic neurons. This finding suggests that the projections for these two areas are independent and distinct, and indicates that altered thalamocortical projections do not contribute the critical component underlying reorganizational changes observed at the cortical level after spinal cord transection. It is hypothesized that the degree of reorganization required to achieve the magnitude of change observed in the cortex must occur below the level of the thalamocortical relay.     

Role of different projection areas of the motor cortex in reorganization of the innate head-forelimb coordination in dogs

Dogs were trained to perform the forelimb tonic flexion in order to lift a cup with meat from a bottom of the foodwell and hold it during eating with the head bent down to the cup. It is known that conditioning of the instrumental reaction is based on reorganization of the innate head-forelimb coordination into the opposite one. In untrained dogs, the forelimb flexion is accompanied by the anticipatory lifting of the head bent down to the foodwell. The following lowering of the head leads to an extension of the flexed forelimb. Tonic forelimb flexion is possible if the head is in the up position. Simultaneous holding of the flexed forelimb and lowered head providing food reinforcement is achieved only by learning. It was shown earlier that the lesion of the motor cortex contralateral to the "working" forelimb led to a prolonged disturbance of the elaborated coordination and reappearance of the innate coordination. In the present work we studied the influence of local lesions of the projection areas in the motor cortex, such as a "working" forelimb area, bilateral representation of the neck, and the medial part of the motor cortex, on the learned instrumental feeding reaction. It was found that only the lesion of the forelimb but not neck projection led to a disturbance of the learned head-forelimb movement coordination.     

Directional asymmetry in the forelimb of Macaca mulatta

Asymmetry was investigated in the forelimbs of 150 rhesus monkey (Macaca mulatta) skeletons using measurements of right and left humerii, radii, ulnae, second metacarpals, and femora. Seven of the ten forelimb dimensions were larger on the right than on the left side. Paired t-tests revealed that the mean of the right side was significantly larger than that for the left for two measurements of the ulna and two of the humerus. No measurement was significantly larger on the left than on the right side. These results indicate a small but significant asymmetry in the forelimb bones of rhesus monkeys and, as is the case for humans, the direction of asymmetry favors the right side. Our findings are consistent with an interpretation of hypertrophy of certain muscles and opens the question of whether rhesus monkeys preferentially use their right forelimbs for manipulative tasks that require manual dexterity, as is the case for humans. These forelimb skeletal asymmetries are discussed in light of the recent literature on cortical asymmetry and handedness in nonhuman primates.     

Is it possible to transfer learned coordination of head and forepaw movements in dogs

Dogs were trained for tonic forelimb flexion fixed to a lever in order to hold a cup with meat during eating, when the head was bent down to a foodwell. Before learning, the forelimb flexion is accompanied by the anticipatory lifting of the head bent down to the foodwell; following lowering of the head leads to an extension of the flexed forelimb. Simultaneous holding of the flexed forelimb and lowered head is achieved by learning. During the original learning, the innate head-forelimb coordination was rearranged into the opposite one. After the initial instrumental learning, the "working" forelimb was changed to test whether a transfer of the learned head-forelimb coordination would occur. It was shown that the execution of the instrumental reaction by the untrained forelimb was impossible, because the innate coordination between the head and this forelimb persisted. It could also be rearranged by learning. The involvement of the motor cortex in the unilateral rearrangement of the innate head-forelimb movement coordination is discussed.     

Forearm elongation in gibbons: Hypothesis and preliminary results

The characteristic long forelimbs of gibbons are a consequence of elongation of all segments (hand, forearm, and arm) but especially the forearm. While the hand is limited in size by its prehensile role, both the arm and the forearm do not seem to be directly limited in a similar manner. Why is the elongation concentrated specifically in the forearm? One hypothesis, originally applied to cursorial ungulates, relates to the need to enhance angular velocity during the swing phase at a minimum cost by distributing the heaviest forelimb loads proximally near the shoulder joint. This appears to be a plausible explanation despite complicating factors associated with competing functions of the forelimb during the support phase of arm-swinging and the fact that the animal behaviorally adjusts inertial properties of its forelimb by flexing it while reaching for the support. In order to test this hypothesis, the forelimb was modeled as a series of uniform cylinders from which the radii of gyration (k) were calculated. After converting into relative values (%k), they were compared (1) interspecifically, among two hylobatids, an orangutan, common chimpanzee, and gorilla, and (2) hypothetically, among imaginary gibbons with brachial indices extending beyond the range found in hylobatids. The results of both tests were equivocal, suggesting that forelimb mass distribution may not be the dominant factor influencing elongation of the forearms in gibbons. Even though gibbons are unique in their great dependence upon arm-swinging, efficient generation of high angular velocities during the swing phase does not appear to be the overriding function beyond others related to support, propulsion, and prehension.     

Changes in electrical thresholds for evoking movements from the cat cerebral cortex following lesions of the sensori-motor area

We evaluated motor maps in the cerebral cortex and motor performance in cats before and after lesions of the forelimb representation in the primary motor area. After the lesion there was a reduction in the use of the affected forelimb and loss of accuracy in prehension tasks using the forelimb; some recovery occurred during the mapping study. Electrode tracts and lesion sites were located in cytoarchitectonically identified cortical areas 4γ, 4δ, 6aα, 6aγ, 3a. The lesions were mainly in area 4γ. In the lesioned hemisphere there were many points around the lesion site (in areas 4γ and 3a) from which movements could not be evoked. In some areas distant from the lesion site (e.g. area 6aγ) the mean thresholds for evoking forelimb movements were significantly elevated. Mean thresholds for evoking hindlimb and facial movements were not different from before. In the contralateral hemisphere mean thresholds for evoking forelimb, but not hindlimb or facial movements, were significantly elevated in several sensorimotor areas (area 4γ, 6aγ and 3a). Mean thresholds for evoking forelimb movements appeared to progressively increase during the time of study. Minimal currents required to evoke forelimb movements from the cerebral cortex increase (possibly progressively) following a lesion of the forelimb representation in the primary motor area, affecting many interconnected motor areas in the hemispheres ipsilateral and contralateral to the lesioned site. This increase in thresholds may play a role in the changes in cortical control of the affected and contralateral limbs following brain lesions and explain the increased sense of effort required to produce movements.     

Changes in electrical thresholds for evoking movements from the cat cerebral cortex following lesions of the sensori-motor area

We evaluated motor maps in the cerebral cortex and motor performance in cats before and after lesions of the forelimb representation in the primary motor area. After the lesion there was a reduction in the use of the affected forelimb and loss of accuracy in prehension tasks using the forelimb; some recovery occurred during the mapping study. Electrode tracts and lesion sites were located in cytoarchitectonically identified cortical areas 4gamma, 4delta, 6aalpha, 6agamma, 3a. The lesions were mainly in area 4gamma. In the lesioned hemisphere there were many points around the lesion site (in areas 4gamma and 3a) from which movements could not be evoked. In some areas distant from the lesion site (e.g. area 6agamma) the mean thresholds for evoking forelimb movements were significantly elevated. Mean thresholds for evoking hindlimb and facial movements were not different from before. In the contralateral hemisphere mean thresholds for evoking forelimb, but not hindlimb or facial movements, were significantly elevated in several sensorimotor areas (area 4gamma, 6agamma and 3a). Mean thresholds for evoking forelimb movements appeared to progressively increase during the time of study. Minimal currents required to evoke forelimb movements from the cerebral cortex increase (possibly progressively) following a lesion of the forelimb representation in the primary motor area, affecting many interconnected motor areas in the hemispheres ipsilateral and contralateral to the lesioned site. This increase in thresholds may play a role in the changes in cortical control of the affected and contralateral limbs following brain lesions and explain the increased sense of effort required to produce movements.     

Differential expression of choline kinase isoforms in skeletal muscle explains the phenotypic variability in the rostrocaudal muscular dystrophy mouse

Choline kinase in mammals is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneous genomic deletion in murine Chkb results in neonatal forelimb bone deformity and hindlimb muscular dystrophy. Surprisingly, muscular dystrophy isn't significantly developed in the forelimb. We have investigated the mechanism by which a lack of choline kinase β, encoded by Chkb, results in minimal muscular dystrophy in forelimbs. We have found that choline kinase β is the major isoform in hindlimb muscle and contributes more to choline kinase activity, while choline kinase α is predominant in forelimb muscle and contributes more to choline kinase activity. Although choline kinase activity is decreased in forelimb muscles of Chkb^−/− mice, the activity of CTP:phosphocholine cytidylyltransferase is increased, resulting in enhanced phosphatidylcholine biosynthesis. The activity of phosphatidylcholine phospholipase C is up-regulated while the activity of phospholipase A₂ in forelimb muscle is not altered. Regeneration of forelimb muscles of Chkb^−/− mice is normal when challenged with cardiotoxin. In contrast to hindlimb muscle, mega-mitochondria are not significantly formed in forelimb muscle of Chkb^−/− mice. We conclude that the relative lack of muscle degeneration in forelimbs of Chkb^−/− mice is due to abundant choline kinase α and the stable homeostasis of phosphatidylcholine.     

A gradient of homeodomain protein in developing forelimbs of Xenopus and mouse embryos

The expression of the homeodomain protein XIHbox 1 in developing Xenopus limbs was analyzed using specific antibodies. In the forelimb bud mesoderm XIHbox 1 shows a clear antero-posterior gradient that is strongest in the anterior and proximal region of the forelimb. Hindlimb bud mesoderm is devoid of XIHbox 1, indicating an early molecular difference between arm and leg. The innermost ectodermal cell layer is positive throughout the forelimb and hindlimb bud ectoderm, but no other areas of the skin. Similar results are obtained in developing mouse limbs, suggesting that XIHbox 1 participates in forelimb development in a variety of tetrapods. In early tadpoles analyzed at stages preceding limb bud formation, the lateral plate mesoderm is positive in the region corresponding to the earliest "field" of forelimb information, but not in the hindlimb field. These results suggest a molecular link between morphogenetic fields, gradients, and homeobox genes in vertebrate development.     

A biomechanical perspective on the use of forelimb length as a measure of sexual selection in frogs

Differences in forelimb length between male and female frogs and between amplexing and non-amplexing males have been interpreted to be the results of sexual selection on forelimb length. The causal feature of the forelimb that has been posited to cause such selection is the observation that non-amplexing males attempt to disrupt breeding by prying amplexing males from females. A biomechanical model of forelimb function suggests that total length per se may not be the most appropriate measure to use. There are more functionally significant aspects of forelimb morphology, such as lever arm lengths, that should influence amplexing ability and may make measures of overall forelimb length misleading. This example highlights the relevance of functional analysis to current questions in evolutionary biology that rely on postulated roles for morphological structures under selection.     

Restraint of Fgf8 signaling by retinoic acid signaling is required for proper heart and forelimb formation

Cardiomelic or heart–hand syndromes include congenital defects affecting both the forelimb and heart, suggesting a hypothesis where similar signals may coordinate their development. In support of this hypothesis, we have recently defined a mechanism by which retinoic acid (RA) signaling acts on the forelimb progenitors to indirectly restrict cardiac cell number. However, we still do not have a complete understanding of the mechanisms downstream of RA signaling that allow for the coordinated development of these structures. Here, we test the hypothesis that appropriate Fgf signaling in the cardiac progenitor field downstream of RA signaling is required for the coordinated development of the heart and forelimb. Consistent with this hypothesis, we find that increasing Fgf signaling can autonomously increase cardiac cell number and non-autonomously inhibit forelimb formation over the same time period that embryos are sensitive to loss of RA signaling. Furthermore, we find that Fgf8a, which is expressed in the cardiac progenitors, is expanded into the posterior in RA signaling-deficient zebrafish embryos. Reducing Fgf8a function in RA signaling-deficient embryos is able to rescue both heart and forelimb development. Together, these results are the first to directly support the hypothesis that RA signaling is required shortly after gastrulation in the forelimb field to temper Fgf8a signaling in the cardiac field, thus coordinating the development of the heart and forelimb.