Flight interneurons in the locust and the origin of insect wings.

Interneurons involved in the generation of motor activity for flight in the locust were found in the first three abdominal ganglia as well as in thoracic ganglia. The evidence that sets of homologous flight interneurons occur in abdominal and thoracic ganglia supports theories that insect wings originated from movable appendages which were serially distributed along the thorax and abdomen and which were under central nervous control.

Role of sensory feedback in the control of stance duration in walking cats.

The rate of stepping in the hind legs of chronic spinal and decerebrate cats adapts to the speed of the treadmill on which the animals walk. This adaptive behavior depends on sensory signals generated near the end of stance phase controlling the transition from stance to swing. Two sensory signals have been identified to have this role: one from afferents activated by hip extension, most likely arising from muscle spindles in hip flexor muscles, and the other from group Ib afferents from Golgi tendon organs in the ankle extensor muscles. The relative importance of these two signals in controlling the stance to swing transition differs in chronic spinal cats and in decerebrate cats. Activation of hip afferents is necessary for controlling the transition in chronic spinal cats but not in decerebrate cats, while reduction in activity in group Ib afferents from GTOs is the primary factor controlling the transition in decerebrate cats. Possible mechanisms for this difference are discussed. The extent to which these two sensory signals control the stance to swing transition in normal walking cats is unknown, but it is likely that both could play an important role when animals are walking in a variable environment.

Long-lasting memories of obstacles guide leg movements in the walking cat.

We examined the ways in which memories of previously seen obstacles can alter the stepping of walking cats. Cats were paused after the forelegs, but not the hindlegs, had stepped over an obstacle. Near the beginning of a variable delay period, the obstacle was lowered. On the subsequent step, the path of the hindlegs allowed us to make inferences about whether the memory of the obstacle was influencing leg movements. We present two main findings. First, the memory of the obstacle persisted for the duration that the animal straddled the original location of the obstacle. In one instance, this interval was 10 min. Second, this memory includes information regarding the size and position of the obstacle relative to the animal. This information is used to plan foot placement and to redirect the step in mid-swing to avoid the previous position of the obstacle.

Proprioceptive regulation of locomotion.

Recent investigations of proprioreceptors in the walking systems of cats, insects and crustaceans have identified reflex pathways that regulate the timing of the transition from stance to swing, and control the magnitude of ongoing motoneuronal activity. An important finding in the cat is that during locomotor activity, the influence of feedback from the Golgi tendon organs in extensor muscles onto extensor motoneurons is reversed from inhibition to excitation. The excitatory action of tendon organs during stance ensures that stance is maintained while extensor muscles are loaded, and may regulate the magnitude of extensor activity according to the load carried by the leg. Afferents from primary and secondary spindles in extensor and flexor muscles have also been found to influence the timing of the locomotor rhythm in a functionally relevant manner. Recent studies indicate that reflex reversals and the regulation of timing by multiple proprioceptive systems are also features of walking systems in arthropods.

Adaptive locomotor plasticity in chronic spinal cats after ankle extensors neurectomy.

After lateral gastrocnemius-soleus (LGS) nerve section in intact cats, a rapid locomotor compensation involving synergistic muscles occurs and is accompanied by spinal reflex changes. Only some of these changes are maintained after acute spinalization, indicating the involvement of descending pathways in functional recovery. Here, we address whether the development of these adaptive changes is dependent on descending pathways. The left LGS nerve was cut in three chronic spinal cats. Combined kinematics and electromyographic (EMG) recordings were obtained before and for 8 d after the neurectomy. An increased yield at the ankle was present early after neurectomy and, as in nonspinal cats, was gradually reduced within 8 d. Compensation involved transient changes in step cycle structure and a longer term increase in postcontact medial gastrocnemius (MG) EMG activity. Precontact MG EMG only increased in one of three cats. In a terminal experiment, the influence of group I afferents from MG and LGS on stance duration was measured in two cats. LGS effectiveness at increasing stance duration was largely decreased in both cats. MG effectiveness was only slightly changed: increased in one cat and decreased in another. In cat 3, the plantaris nerve was cut after LGS recovery. The recovery time courses from both neurectomies were similar (p > 0.8), suggesting that this spinal compensation is likely a generalizable adaptive strategy. From a functional perspective, the spinal cord therefore must be considered capable of adaptive locomotor plasticity after motor nerve lesions. This finding is of prime importance to the understanding of functional plasticity after spinal injury.

A new electrode configuration for recording electromyographic activity in behaving mice.

With the increasing use of normal and genetically modified mice in the field of motor physiology, there is a need for a simple and reliable technique for recording electromyographic (EMG) activity in behaving mice. Here, we describe a new method for the fabrication and implantation of fine EMG recording electrodes into multiple muscles of adult mice. This method minimizes surgical damage to the muscles and the connecting leads have only a modest influence on leg movements when electrodes are implanted into distal muscles. We demonstrate that excellent EMG recordings can be obtained during walking, swimming and scratching for the vastus lateralis, tibialis anterior and gastrocnemius muscles in normal adult mice. EMG recordings were also made in a mutant EphA4 mouse to demonstrate the utility of the method for examining motor patterns in genetically modified animals. We also developed a method for constructing highly reflective markers that could be viewed over a range of orientations to measure the kinematics of legs movements during stepping. A commercial motion analysis system was used to track six markers during walking and to synchronize video and EMG data during walking sequences.

Interneurons in the flight system of the locust: distribution, connections, and resetting properties.

The organization and functional properties of interneurons in the flight system of the locust, Locusta migratoria, were investigated by using intracellular recording and staining techniques. Interneurons were found to be distributed within the three thoracic and the first three abdominal ganglia, and they could be subdivided into three organizational categories: (1) members of one of two serially homologous groups controlling either the forewing or the hindwing, (2) unique individuals with no known homologues in other ganglia, and (3) members of a set of serial homologous in the metathoracic and first three abdominal ganglia. Interneurons in the last two categories influenced both forewing and hindwing motoneurons in a similar manner. Thus interneuronal organization is not characterized by two distinct homologous groups of interneurons for the separate control of forewing and hindwing motor activity. Flight interneurons may also form two separate functional categories: (1) those making short latency connections to motoneurons (premotor interneurons), and (2) those which reset the flight rhythm when depolarized by brief current pulses (pattern generator interneurons). None of the ten premotor interneurons we identified influenced the flight rhythm when depolarized and none of the three groups of pattern generator interneurons were found to form short latency connections with motoneurons. This separation of function may allow phase-shifts in motor output for flight control without changes in wingbeat frequency. Pattern generator interneurons influence motor output to both forewings and hindwings. Thus we conclude that the flight rhythm is generated in a distributed neuronal oscillator driving both the pairs of wings. The organization of flight interneurons is considerably more complex than predicted from existing models of the flight system, or anticipated from the relative simplicity of the motor output. Our finding of homologous sets of interneurons in the abdominal ganglia supports the notion that insect flight evoked from a behavior using appendages distributed along the thorax and the abdomen. Thus the organization of flight interneurons may reflect an interneuronal system which controlled the behavior from which flight evolved.

Heterogeneous properties of segmentally homologous interneurons in the ventral nerve cord of locusts.

The G, B1, and B2 neurons are three prominent interneurons located in adjacent segmental ganglia in the central nervous system of locusts. Previous studies on the adult nervous system have shown that each of these cells has its own distinctive morphology and responsiveness to auditory input. Previous studies on the embryonic nervous system have described the lineage and development of one of these cells, the G neuron, in the mesothoracic (T2) segment. In this paper it is shown that the G, B1, and B2 neurons are segmental homologues in that they arise from equivalent lineages during embryogenesis in the T2, T3, and A1 segments, respectively. Each cell arises (along with its identified sibling neuron) from the division of the second ganglion mother cell of neuroblast 7-4. The segment-specific morphology of the G homologues was determined in the T3 and A1 segments between 60-70% of embryonic development, and their identity was established as the adult B1 and B2 neurons by comparing the distinctive cell-specific features of their morphology between embryo and adult. Although all three neurons display striking morphological differences, they all share certain structural features in common, including the location of their primary axons and neurites in specific tracts in the neuropil. By recording intracellularly from the main neurites of the G, B1, and B2 neurons, clear differences were found in the synaptic inputs each of the neurons receives and the synaptic outputs each makes. For example, G and B2, but not B1, receive direct monosynaptic input from the descending contralateral movement detector (DCMD) interneurons and from auditory afferents; B1, but not B2, connects directly to G; and B2, but not B1 or G, connects directly to flight motoneurons. The main conclusion from these observations is that lineally equivalent neurons in different segments can develop similar primary structures but quite different secondary morphologies and synaptic connections. How these segment-specific differences arise during embryogenesis remains unknown.

Modification of reflexes in normal and abnormal movements.

The trajectories observed for the limb during human locomotion are determined by a mixture of influences, some arising from neural circuits entirely within the central nervous system and others arising from a variety of sensory receptors. Muscle reflexes are highly modulated during locomotion in an adaptive manner within each phase of the step cycle. Furthermore, the modulation can be modified quickly for different tasks such as standing, walking and running, probably by changes in presynaptic inhibition. This modulation is often lost or severely reduced in patients with spasticity after spinal cord or head injury. In normal subjects cutaneous reflexes can be completely reversed from exciting to inhibiting a muscle during each step cycle, particularly in muscles that normally show two bursts of activity per cycle (e.g., tibialis anterior). In some patients stimulation of a mixed nerve (e.g., common peroneal) can directly produce muscle contraction, generate a reflex response (flexor reflex) and transiently reduce spasticity in antagonist (extensor) muscles. Thus, simple systems employing stimulation can enhance gait to a certain extent in patients with incomplete injuries.

Enhancement and resetting of locomotor activity by muscle afferents.

The generation of the normal motor pattern for walking in mammals requires feedback from muscle proprioceptors. Two characteristics of the motor pattern particularly dependent on proprioceptive signals are (1) the magnitude of activity in knee and ankle extensor muscles and (2) the duration of extensor bursts during stance. Sensory regulation of these characteristics ensures that the level of activity in extensor muscles during stance is appropriate for the load carried by the leg and that the swing phase is not initiated when a leg is loaded. Many different groups of afferents from flexor and extensor muscles can influence the locomotor pattern. Most attention has focused on the action of group I afferents from ankle extensors. Electrical stimulation of these afferents during extension increases the duration and the magnitude of extensor activity. The prolongation of extensor activity depends in part on excitation of the extensor half-center by group Ib afferents from Golgi tendon organs. The enhancement of the magnitude of extensor bursts is produced primarily via disynaptic and polysynaptic pathways opened only during locomotion. The involvement of the proprioceptive signals in the generation of locomotor activity means that the gains in reflex pathways must be constantly calibrated according to the biomechanical properties of the locomotor system. Alteration of these properties by weakening ankle extensor muscles has recently been found to produce compensatory changes in proprioceptive influences on the locomotor pattern.

Intracellular recordings from interneurons and motoneurons in intact flying locusts.

A preparation is described in which it is possible to record intracellularly from identified motoneurons and interneurons during flight in intact tethered locusts. Intracellular recordings could be obtained from either the large somata of the motoneurons or from the neuropil processes of interneurons and motoneurons in either the meso- or metathoracic ganglion. Intracellular recordings from motoneuron somata were sufficiently stable to allow surgical manipulations, e.g. deafferentation, of the nervous system without the loss of penetration. Recordings from neuropil processes required stabilizing the ganglion by sandwiching it between two supports. The preparation is suitable for the cellular analysis of many aspects of motor patterning in the flight system of the locust. So far we have used it to make a direct comparison between the patterns of synaptic activity in intact and deafferented animals. This comparison shows that sensory feedback from wing proprioceptors strongly influences the activity in elevator motoneurons and flight interneurons. From these and other observations we conclude that proprioceptive feedback is essential for generating important features of the intact motor pattern and that wing receptors are integral elements of the intact flight pattern generator.

The use of naloxone to facilitate the generation of the locomotor rhythm in spinal cats.

The locomotor rhythm evoked by perineal stimulation in clonidine-treated acute and chronic spinal cats can be produced more easily when the opioid receptor antagonist naloxone is also administered. Naloxone increases the frequency of the locomotor rhythm and decreases the intensity of skin stimulation required for evoking the rhythm. A useful property of naloxone is that it can restore the locomotor rhythm when the rhythm wanes, thus prolonging the time period over which locomotor activity can be generated. Administration of naloxone without clonidine does not enable the locomotor rhythm to be generated by skin stimulation, but it does reduce the concentration of clonidine required for the expression of a robust rhythm and may increase the chance of a successful preparation. We conclude that naloxone is a useful pharmacological tool for studies on the locomotor pattern generator.

Octopamine induces bursting and plateau potentials in insect neurones.

The membrane properties of some inter- and motoneurones in the respiratory and flight systems of the locust Locusta migratoria were characterized during octopamine perfusion by means of intracellular recording techniques. Octopamine induced active membrane properties in these neurones. Plateau-potentials were evoked by brief current pulses or synaptic input in 3 of the identified neurones and endogenous bursting was evoked by prolonged current pulses in one identified interneurone. Hyperpolarizing pulses injected into these neurones either prematurely terminated or suppressed these responses, indicating that these potentials are due to active membrane properties intrinsic to these neurones. Such intrinsic membrane properties have not been described in insects before. Further investigations are necessary to examine whether these properties may play an important role in the generation of rhythmic motor patterns as has previously been demonstrated in many other vertebrate and invertebrate motor systems.

Use-dependent gain change in the reflex contribution to extensor activity in walking cats.

Denervation of the synergists of the medial gastrocnemius (MG) muscle in the cat hind leg results in a progressive increase in the magnitude of burst activity in the MG muscle during walking. The increase in burst magnitude is associated with an increase in the slope of the relationship between the magnitude of individual MG bursts and the amplitude of ankle flexion during stance. This finding is consistent with the hypothesis that the increase in MG burst magnitude is due to an increase in gain of reflex pathways reinforcing the activation of MG. The increase in slope is use-dependent since it was not observed when the leg was released from a cast that immobilized the leg for 6 days.

Inhibition of flexor burst generation by loading ankle extensor muscles in walking cats.

The role of proprioceptive input from the ankle extensor triceps surae in the control of walking was examined in premammillary cats walking on a treadmill. The left hindlimb was rigidly fixed in one position after denervating almost all the leg muscles except the ankle extensor (triceps surae) and ankle flexor (tibialis anterior). Rhythmic alternating contractions of the isolated ankle flexor and extensor occurred in the fixed hindleg during periods of walking in the other three intact limbs. These rhythmic contractions disappeared when the isolated triceps surae was stretched so as to increase the force of the active contractions to beyond 4 kg. With maintained stretch the periodic contractions in the ankle flexor and extensor returned only after the force in the stretched triceps surae gradually decreased and fell below approximately 4 kg. Isometric contractions of the triceps surae produced either by stimulation of ventral root S1 or by large amplitude vibrations also led to the sudden disappearance of ankle flexor bursts. Inhibition of the locomotory rhythm could also be produced in all muscles of a single intact hindleg by clamping the ankle joint in a flexed position so as to stretch the ankle extensor. In all these cases, an increased rate of stepping of the contralateral hindleg was associated with the inhibition of the rhythmic locomotory activity. It is suggested that triceps surae proprioceptors signalling the presence of loading of the hindlimb extensor muscles inhibit the central generation of hindlimb flexion. During normal walking this mechanism could be of major importance during stance to prevent the initiation of the swing phase of a time when hindlimb extension is fully needed to support the weight of the animal. Thus a necessary, but not always sufficient, condition for the initiation of swing may be an unloading of leg extensor muscles.

Effects of extensor muscle afferents on the timing of locomotor activity during walking in adult rats.

The influence of hind leg extensor muscle afferents on the timing of locomotor phase transitions was examined in adult, decerebrate rats, walking on a treadwheel. Walking occurred either spontaneously or was induced by stimulation of the mesencephalic locomotor region. Large diameter muscle afferents innervating the lateral or medial gastrocnemius were electrically stimulated during walking. A stimulus was delivered either at the onset of extensor muscle activity, or randomly during the step cycle. Stimulation with a train duration of 300 ms at the onset of extension increased the duration of the extensor bursts. The subsequent flexion phase was delayed. Stimulation with a shorter stimulus train (150 ms) early in extension had little effect on the extension phase duration. However when delivered at the end of extension the same stimulus significantly increased the duration of the extension phase and decreased the duration of the following flexion phase. Stimulating near the end of the flexion phase delayed onset and decreased duration of the subsequent extension phase. The effects of stimulating extensor afferents during the extension phase were weaker but qualitatively similar, to those in cats, suggesting similar mechanisms. The results of this study also show major differences in the integration of extensor muscle afferents between adult and neonatal rats.

Modification of group I field potentials in the intermediate nucleus of the cat spinal cord after chronic axotomy of an extensor nerve.

In walking decerebrate cats the influence of group I afferents from the medial gastrocnemius (MG) and the lateral gastrocnemius/soleus (LGS) muscles on stance phase duration is altered after axotomy of the LGS nerve [Whelan, P.J., Hiebert, G.W. and Pearson, K.G., J. Neurophysiol., 74 (1995) 2782-2787]. We examined whether a site for this plasticity is the connection from group I afferents onto spinal interneurons. Group I field potentials from MG, LGS and plantaris (PL) muscle afferents were recorded in the intermediate nucleus of the L6/L7 segments. Within 5 days following the transection of the LGS nerve in one hind leg, the field potential amplitudes from LGS afferents were decreased, from MG increased and those from PL were unchanged. The changes in the field potentials parallel modifications in the influence of group I afferents from the MG and LGS muscles on stance phase duration during walking.

From cat to man: basic aspects of locomotion relevant to motor rehabilitation of SCI.

On the assumption that locomotion is partly produced by a central pattern generator (CPG) in the spinal cord of both cat and man, it is essential to learn more about how such a CPG is controlled by sensory input produced during gait. For the cat there is evidence that load receptor input both from extensor muscles and from cutaneous receptors from the foot, is able to reinforce the ongoing extensor activity in the stance phase and delay the ensuing swing phase. Original data on electrical stimulation of nerves in walking premammillary cats with one hindlimb fixed, support the notion that this type of load afferent input acts directly on the CPG. A second potential source of sensory input on the CPG is derived from sensory signals related to hip position. One would therefore expect that hip position is a more tightly controlled variable than the position of other joints. This was investigated by measuring these angles under conditions of constrained gait (crouch). It was found that cats indeed maintained the maximum excursions of hip flexion and extension within stricter limits than the corresponding angles at other joints. Finally, experiments on hip joint denervation show that there is very little effect on step cycle parameters, thereby supporting the idea that the important hip signal is unlikely to be derived from hip joint afferents. It is suggested that procedures aimed at activating the locomotor CPG in SCI patients could benefit from the use of periodic stimulation of ankle muscle load afferents and hip flexor stretch receptors.