Zhao-Zhe Hao

Strong interactions between spinal cord networks for locomotion and scratching

Distinct rhythmic behaviors involving a common set of motoneurons and muscles can be generated by separate central nervous system (CNS) networks, a single network, or partly overlapping networks in invertebrates. Less is known for vertebrates. Simultaneous activation of two networks can reveal overlap or interactions between them. The turtle spinal cord contains networks that generate locomotion and three forms of scratching (rostral, pocket, and caudal), having different knee-hip synergies. Here, we report that in immobilized spinal turtles, simultaneous delivery of types of stimulation, which individually evoked forward swimming and one form of scratching, could 1) increase the rhythm frequency; 2) evoke switches, hybrids, and intermediate motor patterns; 3) recruit a swim motor pattern even when the swim stimulation was reduced to subthreshold intensity; and 4) disrupt rhythm generation entirely. The strength of swim stimulation could influence the result. Thus even pocket scratching and caudal scratching, which do not share a knee-hip synergy with forward swimming, can interact with swim stimulation to alter both rhythm and pattern generation. Model simulations were used to explore the compatibility of our experimental results with hypothetical network architectures for rhythm generation. Models could reproduce experimental observations only if they included interactions between neurons involved in swim and scratch rhythm generation, with maximal consistency between simulations and experiments attained using a model architecture in which certain neurons participated actively in both swim and scratch rhythmogenesis. Collectively, these findings suggest that the spinal cord networks that generate locomotion and scratching have important shared components or strong interactions between them.

Distributions of active spinal cord neurons during swimming and scratching motor patterns

The spinal cord can generate motor patterns underlying several kinds of limb movements. Many spinal interneurons are multifunctional, contributing to multiple limb movements, but others are specialized. It is unclear whether anatomical distributions of activated neurons differ for different limb movements. We examined distributions of activated neurons for locomotion and scratching using an activity-dependent dye. Adult turtles were stimulated to generate repeatedly forward swimming, rostral scratching, pocket scratching, or caudal scratching motor patterns, while sulforhodamine 101 was applied to the spinal cord. Sulforhodamine-labeled neurons were widely distributed rostrocaudally, dorsoventrally, and mediolaterally after each motor pattern, concentrated bilaterally in the deep dorsal horn, the lateral intermediate zone, and the dorsal to middle ventral horn. Labeled neurons were common in all hindlimb enlargement segments and the pre-enlargement segment following swimming and scratching, but a significantly higher percentage were in the rostral segments following swimming than rostral scratching. These findings suggest that largely the same spinal regions are activated during swimming and scratching, but there are some differences that may indicate locations of behaviorally specialized neurons. Finally, the substantial inter-animal variability following a single kind of motor pattern may indicate that essentially the same motor output is generated by anatomically variable networks.

Partly Shared Spinal Cord Networks for Locomotion and Scratching

Animals produce a variety of behaviors using a limited number of muscles and motor neurons. Rhythmic behaviors are often generated in basic form by networks of neurons within the central nervous system, or central pattern generators (CPGs). It is known from several invertebrates that different rhythmic behaviors involving the same muscles and motor neurons can be generated by a single CPG, multiple separate CPGs, or partly overlapping CPGs. Much less is known about how vertebrates generate multiple, rhythmic behaviors involving the same muscles. The spinal cord of limbed vertebrates contains CPGs for locomotion and multiple forms of scratching. We investigated the extent of sharing of CPGs for hind limb locomotion and for scratching. We used the spinal cord of adult red-eared turtles. Animals were immobilized to remove movement-related sensory feedback and were spinally transected to remove input from the brain. We took two approaches. First, we monitored individual spinal cord interneurons (i.e., neurons that are in between sensory neurons and motor neurons) during generation of each kind of rhythmic output of motor neurons (i.e., each motor pattern). Many spinal cord interneurons were rhythmically activated during the motor patterns for forward swimming and all three forms of scratching. Some of these scratch/swim interneurons had physiological and morphological properties consistent with their playing a role in the generation of motor patterns for all of these rhythmic behaviors. Other spinal cord interneurons, however, were rhythmically activated during scratching motor patterns but inhibited during swimming motor patterns. Thus, locomotion and scratching may be generated by partly shared spinal cord CPGs. Second, we delivered swim-evoking and scratch-evoking stimuli simultaneously and monitored the resulting motor patterns. Simultaneous stimulation could cause interactions of scratch inputs with subthreshold swim inputs to produce normal swimming, acceleration of the swimming rhythm, scratch-swim hybrid cycles, or complete cessation of the rhythm. The type of effect obtained depended on the level of swim-evoking stimulation. These effects suggest that swim-evoking and scratch-evoking inputs can interact strongly in the spinal cord to modify the rhythm and pattern of motor output. Collectively, the single-neuron recordings and the results of simultaneous stimulation suggest that important elements of the generation of rhythms and patterns are shared between locomotion and scratching in limbed vertebrates.

Rostral spinal cord segments are sufficient to generate a rhythm for both locomotion and scratching but affect their hip extensor phases differently

Rostral segments of the spinal cord hindlimb enlargement are more important than caudal segments for generating locomotion and scratching rhythms in limbed vertebrates, but the adequacy of rostral segments has not been directly compared between locomotion and scratching. We separated caudal segments from immobilized low-spinal turtles by sequential spinal cord transections. After separation of the caudal four segments of the five-segment hindlimb enlargement, the remaining enlargement segment and five preenlargement segments still produced rhythms for forward swimming and both rostral and pocket scratching. The swimming rhythm frequency was usually maintained. Some animals continued to generate swimming and scratching rhythms even with no enlargement segments remaining, using only preenlargement segments. The preenlargement segments and rostral-most enlargement segment were also sufficient to maintain hip flexor (HF) motoneuron quiescence between HF bursts [which normally occurs during each hip extensor (HE) phase] during swimming. In contrast, the HF-quiescent phase was increasingly absent (i.e., HE-phase deletions) during rostral and pocket scratching. Moreover, respiratory motoneurons that normally burst during HE bursts continued to burst during the HF quiescence of swimming even with the caudal segments separated. Thus the same segments are sufficient to generate the basic rhythms for both locomotion and scratching. These segments are also sufficient to produce a reliable HE phase during locomotion but not during rostral or pocket scratching. We hypothesize that the rostral HE-phase interneurons that rhythmically inhibit HF motoneurons and interneurons are sufficient to generate HF quiescence during HE-biased swimming but not during the more HF-biased rostral and pocket scratching.

Shared Components of Rhythm Generation for Locomotion and Scratching Exist Prior to Motoneurons

Does the spinal cord use a single network to generate locomotor and scratching rhythms or two separate networks? Previous research showed that simultaneous swim and scratch stimulation (“dual stimulation”) in immobilized, spinal turtles evokes a single rhythm in hindlimb motor nerves with a frequency often greater than during swim stimulation alone or scratch stimulation alone. This suggests that the signals that trigger swimming and scratching converge and are integrated within the spinal cord. However, these results could not determine whether the integration occurs in motoneurons themselves or earlier, in spinal interneurons. Here, we recorded intracellularly from hindlimb motoneurons during dual stimulation. Motoneuron membrane potentials displayed regular oscillations at a higher frequency during dual stimulation than during swim or scratch stimulation alone. In contrast, arithmetic addition of the oscillations during swimming alone and scratching alone with various delays always generated irregular oscillations. Also, the standard deviation of the phase-normalized membrane potential during dual stimulation was similar to those during swimming or scratching alone. In contrast, the standard deviation was greater when pooling cycles of swimming alone and scratching alone for two of the three forms of scratching. This shows that dual stimulation generates a single rhythm prior to motoneurons. Thus, either swimming and scratching largely share a rhythm generator or the two rhythms are integrated into one rhythm by strong interactions among interneurons.