Activity of Spinal Interneurons during Forward and Backward Locomotion.

Higher vertebrates are capable not only of forward but also backward and sideways locomotion. Also, single steps in different directions are generated for postural corrections. While the networks responsible for the control of forward walking (FW) have been studied in considerable detail, the networks controlling steps in other directions are mostly unknown. Here, to characterize the operation of the spinal locomotor network during FW and backward walking (BW), we recorded the activity of individual spinal interneurons from L4 to L6 during both FW and BW evoked by epidural stimulation (ES) of the spinal cord at L5-L6 in decerebrate cats of either sex. Three groups of neurons were revealed. Group 1 (45%) had a similar phase of modulation during both FW and BW. Group 2 (27%) changed the phase of modulation in the locomotor cycle depending on the direction of locomotion. Group 3 neurons were modulated during FW only (Group 3a, 21%) or during BW only (Group 3b, 7%). We suggest that Group 1 neurons belong to the network generating the vertical component of steps (the limb elevation and lowering) because it should operate similarly during locomotion in any direction, while Groups 2 and 3 neurons belong to the networks controlling the direction of stepping. Results of this study provide new insights into the organization of the spinal locomotor circuits, advance our understanding of ES therapeutic effects, and can potentially be used for the development of novel strategies for recuperation of impaired balance control, which requires the generation of corrective steps in different directions.SIGNIFICANCE STATEMENT Animals and humans can perform locomotion in different directions in relation to the body axis (forward, backward, sideways). While the networks that control forward walking have been studied in considerable detail, the networks controlling steps in other directions are unknown. Here, by recording the activity of the same spinal neurons during forward and backward walking, we revealed three groups of neurons forming, respectively, the network operating similarly during stepping in different directions, the network changing its operation with a change in the direction of stepping, and the network operating only during locomotion in a specific direction. These networks presumably control different aspects of the step. The obtained results provide new insights into the organization of the spinal locomotor networks.

Changes in operation of postural networks in rabbits with postural functions recovered after lateral hemisection of the spinal cord.

Acute lateral hemisection of the spinal cord (LHS) severely impairs postural functions, which recover over time. Here, to reveal changes in the operation of postural networks underlying the recovery, male rabbits with recovered postural functions after LHS at T12 (R-rabbits) were used. After decerebration, we characterized the responses of individual spinal interneurons from L5 along with hindlimb EMG responses to stimulation causing postural limb reflexes (PLRs) that substantially contribute to postural corrections in intact animals. The data were compared with those obtained in our previous studies of rabbits with the intact spinal cord and rabbits after acute LHS. Although, in R-rabbits, the EMG responses to postural disturbances both ipsilateral and contralateral to the LHS (ipsi-LHS and co-LHS) were only slightly distorted, PLRs on the co-LHS side (unaffected by acute LHS) were distorted substantially and PLRs on the ipsi-LHS side (abolished by acute LHS) were close to control. Thus, in R-rabbits, plastic changes develop in postural networks both affected and unaffected by acute LHS. PLRs on the ipsi-LHS side recover mainly as a result of changes at brainstem-cerebellum-spinal levels, whereas the forebrain is substantially involved in the generation of PLRs on the co-LHS side. We found that, in areas of grey matter in which the activity of spinal neurons of the postural network was significantly decreased after acute LHS, it recovered to the control level, whereas, in areas unaffected by acute LHS, it was significantly changed. These changes underlie the recovery and distortion of PLRs on the ipsi-LHS and co-LHS sides, respectively. KEY POINTS: After lateral hemisection of the spinal cord (LHS), postural functions recover over time. The underlying changes in the operation of postural networks are unknown. We compared the responses of individual spinal neurons and hindlimb muscles to stimulation causing postural limb reflexes (PLRs) in recovered LHS-rabbits with those obtained in rabbits with the intact spinal cord and rabbits after acute LHS. We demonstrated that changes underlying the recovery of postural functions take place not only in postural networks that are severely impaired, but also in those that are almost unaffected by acute LHS. PLRs on the LHS side recover mainly as a result of changes at brainstem-cerebellum-spinal levels, whereas the forebrain is substantially involved in the generation of PLRs contralateral to the LHS.

Differential Contribution of V0 Interneurons to Execution of Rhythmic and Nonrhythmic Motor Behaviors.

Locomotion, scratching, and stabilization of the body orientation in space are basic motor functions which are critically important for animal survival. Their execution requires coordinated activity of muscles located in the left and right halves of the body. Commissural interneurons (CINs) are critical elements of the neuronal networks underlying the left-right motor coordination. V0 interneurons (characterized by the early expression of the transcription factor Dbx1) contain a major class of CINs in the spinal cord (excitatory, V0V; inhibitory, V0D), and a small subpopulation of excitatory ipsilaterally projecting interneurons. The role of V0 CINs in left-right coordination during forward locomotion was demonstrated earlier. Here, to reveal the role of glutamatergic V0 and other V0 subpopulations in control of backward locomotion, scratching, righting behavior, and postural corrections, kinematics of these movements performed by wild-type mice and knock-out mice with glutamatergic V0 or all V0 interneurons ablated were compared. Our results suggest that the functional effect of excitatory V0 neurons during backward locomotion and scratching is inhibitory, and that the execution of scratching involves active inhibition of the contralateral scratching central pattern generator mediated by excitatory V0 neurons. By contrast, other V0 subpopulations are elements of spinal networks generating postural corrections. Finally, all V0 subpopulations contribute to the generation of righting behavior. We found that different V0 subpopulations determine left-right coordination in the anterior and posterior parts of the body during a particular behavior. Our study shows a differential contribution of V0 subpopulations to diverse motor acts that provides new insight to organization of motor circuits.SIGNIFICANCE STATEMENT Commissural interneurons with their axons crossing the midline of the nervous system are critical elements of the neuronal networks underlying the left-right motor coordination. For the majority of motor behaviors, the neuronal mechanisms underlying left-right coordination are unknown. Here, we demonstrate the functional role of excitatory V0 neurons and other subpopulations of V0 interneurons in control of a number of basic motor behaviors-backward locomotion, scratching, righting behavior, and postural corrections-which are critically important for animal survival. We have shown that different subpopulations of V0 neurons determine left-right coordination in the context of different behaviors as well as in the anterior and posterior parts of the body during a particular behavior.

Comparison of operation of spinal locomotor networks activated by supraspinal commands and by epidural stimulation of the spinal cord in cats.

KEY POINTS: Epidural electrical stimulation (ES) of the spinal cord restores/improves locomotion in patients. ES-evoked locomotor movements differ to some extent from the normal ones. Operation of the locomotor network during ES is unknown. We compared the activity of individual spinal neurons during locomotion initiated by signals from the brainstem and by ES. We demonstrated that the spinal network generating locomotion under each of the two conditions is formed by the same neurons. A part of this network operates similarly under the two conditions, suggesting that it is essential for generation of locomotion under both conditions. Another part of this network operates differently under the two conditions, suggesting that it is responsible for differences in the movement kinematics observed under the two conditions. ABSTRACT: Locomotion is a vital motor function for both animals and humans. Epidural electrical stimulation (ES) of the spinal cord is used to restore/improve locomotor movements in patients. However, operation of locomotor networks during ES has never been studied. Here we compared the activity of individual spinal neurons recorded in decerebrate cats of either sex during locomotion initiated by supraspinal commands (caused by stimulation of the mesencephalic locomotor region, MLR) and by ES. We found that under both conditions, the same neurons had modulation of their activity related to the locomotor rhythm, suggesting that the network generating locomotion under the two conditions is formed by the same neurons. About 40% of these neurons had stable modulation (i.e. small dispersion of their activity phase in sequential cycles), as well as a similar phase and shape of activity burst in MLR- and ES-evoked locomotor cycles. We suggest that these neurons form a part of the locomotor network that operates similarly under the two conditions, and are critical for generation of locomotion. About 23% of the modulated neurons had stable modulation only during MLR-evoked locomotion. We suggest that these neurons are responsible for some differences in kinematics of MLR- and ES-evoked locomotor movements. Finally, 25% of the modulated neurons had unstable modulation during both MLR- and ES-evoked locomotion. One can assume that these neurons contribute to maintenance of the excitability level of locomotor networks necessary for generation of stepping, or belong to postural networks, activated simultaneously with locomotor networks by both MLR stimulation and ES.

Putative spinal interneurons mediating postural limb reflexes provide a basis for postural control in different planes.

The dorsal-side-up trunk orientation in standing quadrupeds is maintained by the postural system driven mainly by somatosensory inputs from the limbs. Postural limb reflexes (PLRs) represent a substantial component of this system. Earlier we described spinal neurons presumably contributing to the generation of PLRs. The first aim of the present study was to reveal trends in the distribution of neurons with different parameters of PLR-related activity across the gray matter of the spinal cord. The second aim was to estimate the contribution of PLR-related neurons with different patterns of convergence of sensory inputs from the limbs to stabilization of body orientation in different planes. For this purpose, the head and vertebral column of the decerebrate rabbit were fixed and the hindlimbs were positioned on a platform. Activity of individual neurons from L5 to L6 was recorded during PLRs evoked by lateral tilts of the platform. In addition, the neurons were tested by tilts of the platform under only the ipsilateral or only the contralateral limb, as well as during in-phase tilts of the platforms under both limbs. We found that, across the spinal gray matter, strength of PLR-related neuronal activity and sensory input from the ipsilateral limb decreased in the dorsoventral direction, while strength of the input from the contralateral limb increased. A near linear summation of tilt-related sensory inputs from different limbs was found. Functional roles were proposed for individual neurons. The obtained data present the first characterization of posture-related spinal neurons, forming a basis for studies of postural networks impaired by injury.

Effects of reversible spinalization on individual spinal neurons.

Postural limb reflexes (PLRs) represent a substantial component of the postural system responsible for stabilization of dorsal-side-up trunk orientation in quadrupeds. Spinalization causes spinal shock, that is a dramatic reduction of extensor tone and spinal reflexes, including PLRs. The goal of our study was to determine changes in activity of spinal interneurons, in particular those mediating PLRs, that is caused by spinalization. For this purpose, in decerebrate rabbits, activity of individual interneurons from L5 was recorded during stimulation causing PLRs under two conditions: (1) when neurons received supraspinal influences and (2) when these influences were temporarily abolished by a cold block of spike propagation in spinal pathways at T12 ("reversible spinalization"; RS). The effect of RS, that is a dramatic reduction of PLRs, was similar to the effect of surgical spinalization. In the examined population of interneurons (n = 199), activity of 84% of them correlated with PLRs, suggesting that they contribute to PLR generation. RS affected differently individual neurons: the mean frequency decreased in 67% of neurons, increased in 15%, and did not change in 18%. Neurons with different RS effects were differently distributed across the spinal cord: 80% of inactivated neurons were located in the intermediate area and ventral horn, whereas 50% of nonaffected neurons were located in the dorsal horn. We found a group of neurons that were coactivated with extensors during PLRs before RS and exhibited a dramatic (>80%) decrease in their activity during RS. We suggest that these neurons are responsible for reduction of extensor tone and postural reflexes during spinal shock.

Spinal and supraspinal control of the direction of stepping during locomotion.

Most bipeds and quadrupeds, in addition to forward walking, are also capable of backward and sideward walking. The direction of walking is determined by the direction of stepping movements of individual limbs in relation to the front-to-rear body axis. Our goal was to assess the functional organization of the system controlling the direction of stepping. Experiments were performed on decerebrate cats walking on the treadmill with their hindlimbs, whereas the head and trunk were rigidly fixed. Different directions of the treadmill motion relative to the body axis were used (0, ± 45, ± 90, and 180°). For each direction, we compared locomotion evoked from the brainstem (by stimulation of the mesencephalic locomotor region, MLR) with locomotion evoked by epidural stimulation of the spinal cord (SC). It was found that SC stimulation evoked well coordinated stepping movements at different treadmill directions. The direction of steps was opposite to the treadmill motion, suggesting that this direction was determined by sensory input from the limb during stance. Thus, SC stimulation activates limb controllers, which are able to generate stepping movements in different directions. By contrast, MLR stimulation evoked well coordinated stepping movements only if the treadmill was moving in the front-to-rear direction. One can conclude that supraspinal commands (caused by MLR stimulation) select one of the numerous forms of operation of the spinal limb controllers, namely, the forward walking. The MLR can thus be considered as a command center for forward locomotion, which is the main form of progression in bipeds and quadrupeds.

Changes in Activity of Spinal Postural Networks at Different Time Points After Spinalization.

Postural limb reflexes (PLRs) are an essential component of postural corrections. Spinalization leads to disappearance of postural functions (including PLRs). After spinalization, spastic, incorrectly phased motor responses to postural perturbations containing oscillatory EMG bursting gradually develop, suggesting plastic changes in the spinal postural networks. Here, to reveal these plastic changes, rabbits at 3, 7, and 30 days after spinalization at T12 were decerebrated, and responses of spinal interneurons from L5 along with hindlimb muscles EMG responses to postural sensory stimuli, causing PLRs in subjects with intact spinal cord (control), were characterized. Like in control and after acute spinalization, at each of three studied time points after spinalization, neurons responding to postural sensory stimuli were found. Proportion of such neurons during 1st month after spinalization did not reach the control level, and was similar to that observed after acute spinalization. In contrast, their activity (which was significantly decreased after acute spinalization) reached the control value at 3 days after spinalization and remained close to this level during the following month. However, the processing of postural sensory signals, which was severely distorted after acute spinalization, did not recover by 30 days after injury. In addition, we found a significant enhancement of the oscillatory activity in a proportion of the examined neurons, which could contribute to generation of oscillatory EMG bursting. Motor responses to postural stimuli (which were almost absent after acute spinalization) re-appeared at 3 days after spinalization, although they were very weak, irregular, and a half of them was incorrectly phased in relation to postural stimuli. Proportion of correct and incorrect motor responses remained almost the same during the following month, but their amplitude gradually increased. Thus, spinalization triggers two processes of plastic changes in the spinal postural networks: rapid (taking days) restoration of normal activity level in spinal interneurons, and slow (taking months) recovery of motoneuronal excitability. Most likely, recovery of interneuronal activity underlies re-appearance of motor responses to postural stimuli. However, absence of recovery of normal processing of postural sensory signals and enhancement of oscillatory activity of neurons result in abnormal PLRs and loss of postural functions.

Effects of acute spinalization on neurons of postural networks.

Postural limb reflexes (PLRs) represent a substantial component of postural corrections. Spinalization results in loss of postural functions, including disappearance of PLRs. The aim of the present study was to characterize the effects of acute spinalization on two populations of spinal neurons (F and E) mediating PLRs, which we characterized previously. For this purpose, in decerebrate rabbits spinalized at T12, responses of interneurons from L5 to stimulation causing PLRs before spinalization, were recorded. The results were compared to control data obtained in our previous study. We found that spinalization affected the distribution of F- and E-neurons across the spinal grey matter, caused a significant decrease in their activity, as well as disturbances in processing of posture-related sensory inputs. A two-fold decrease in the proportion of F-neurons in the intermediate grey matter was observed. Location of populations of F- and E-neurons exhibiting significant decrease in their activity was determined. A dramatic decrease of the efficacy of sensory input from the ipsilateral limb to F-neurons, and from the contralateral limb to E-neurons was found. These changes in operation of postural networks underlie the loss of postural control after spinalization, and represent a starting point for the development of spasticity.