Proposal #0950370 Selection and generation of limb movements by a combination of multifunctional and specialized spinal interneurons.
Animals and people must constantly decide how to move their limbs. They often do so without conscious awareness. Their nervous systems are responsible for making and implementing decisions about limb movements that are appropriate for each situation. The turtle spinal cord is an excellent model system to investigate how this is done. The turtle spinal cord can appropriately produce several types of swimming, scratching, and limb withdrawal movements, even without input from the brain, essentially using programs that are stored within networks of spinal cord interneurons. (Interneurons are the nerve cells in between sensory neurons and motor neurons; motor neurons excite muscles to contract.) Turtles are also easier to study physiologically than mammals because their tissues remain healthy in physiologically challenging conditions. This project will use electrophysiological, anatomical, immunocytochemical, and pharmacological methods to study two types of spinal cord interneurons previously identified by this group of researchers. Interneurons of one type, called transverse interneurons, are active during several kinds of limb movements, and thus are multifunctional. Interneurons of the other type, called scratch-specialized interneurons, are activated during scratching but are often inhibited during swimming. For each type of interneuron, this project will determine which neurotransmitter(s) the interneurons use, and therefore whether they are excitatory or inhibitory; the project will also determine which neurotransmitter inhibits scratch-specialized interneurons during swimming. Finally, the project will determine whether interneurons of each type directly contact motor neurons. This project will thus test specific hypotheses about how neuronal networks combine multifunctional and specialized interneurons to select and generate limb movements. The project may reveal mechanisms that nervous systems use to make and implement movement decisions, not only in turtles but in vertebrates generally. This project will also provide training to two graduate students and to undergraduates in electrophysiological, anatomical, pharmacological, and general scientific approaches.
The major intellectual goal of this project was to reveal central nervous system mechanisms that generate an appropriate movement for each circumstance an animal faces. More specifically, we addressed whether the spinal cord uses one network of nerve cells to generate several types of leg movements or instead uses a separate network for each type of movement. This goal is fundamental to understanding how animals interact successfully with their environments. An increased understanding of how the spinal cord generates leg movements may also lead to improvements for human patients with spinal cord injuries. To achieve this goal, this project took advantage of a particularly tractable vertebrate model system, the turtle spinal cord. The turtle spinal cord can generate a variety of hind leg movements appropriately, even without input from the brain and sensory feedback from the legs. In addition, turtles, being diving animals, have evolved cellular and molecular mechanisms that keep their tissues healthy under conditions that reduce their oxygen supply. This makes turtles especially favorable for a variety of physiological experiments. In this project, we focused on multiple types of rhythmic leg movements: forward swimming and three forms of scratching. We used a chemical that blocks signals between the spinal cord nerve cells that activate muscles (i.e., motor neurons) and muscles. This kept the spinal cord stable and allowed us to study its output in the absence of sensory feedback from moving legs. We could then determine which kind of movement output the spinal cord was generating at each moment by monitoring electrical signals in motor nerves that normally activate muscles. In one type of experiment, we repeatedly triggered swimming outputs in some animals and scratching outputs in other animals and then looked at the distribution of activated spinal cord nerve cells in each group, using an activity-dependent fluorescent dye. These experiments showed that the distributions of active nerve cells largely (but not entirely) overlapped in the spinal cord for swimming and scratching. In a second type of experiment, we cut away segments of the spinal cord, one by one, and assessed the effect of each cut on swimming and scratching. These experiments demonstrated that largely the same spinal cord segments generate the rhythms of swimming and scratching. However, these cuts often caused deletion of the limb extension portions of the spinal cord outputs for scratching, but not for swimming, demonstrating a key difference in the nerve cell networks for these two types of movement. In a third type of experiment, we applied stimulation that triggers swimming and stimulation that triggers scratching at the same time. If separate networks of spinal cord nerve cells generate these two rhythmic outputs, then we would expect the output to reflect the addition of the two rhythmic signals. But this never happened. Instead, a variety of other effects occurred that demonstrated coordination between swimming and scratching. These effects included generation of a single, coordinated, swim-like rhythm that was faster than either the swim-alone or the scratch-alone rhythm, as well as switches between swim and scratch outputs on a cycle-by-cycle basis. This shows that the swim and scratch networks are largely the same or interact strongly. To assess where such interactions occur, in a fourth type of experiment, we monitored the electrical signals in individual motor neurons by poking each with a sharp, glass micropipette. These experiments showed that motor neurons receive a single, rhythmic input during swim/scratch dual stimulation. This showed that any interactions between swim and scratch nerve cell networks occur earlier than motor neurons in the spinal cord pathways (i.e., in interneurons). We can similarly poke micropipettes into individual spinal cord interneurons while triggering different spinal cord outputs. After assessing whether each interneuron is activated during swimming, scratching, or both, we can inject the interneuron with a dye, which allows us to later see the anatomy of the interneuron and in some cases which neurotransmitter it used, from which we can infer whether its signals are excitatory or inhibitory. We previously found that many spinal cord interneurons contribute to multiple types of leg movements and thus are multifunctional, but other types are specialized for scratching and are inhibited during swimming. We are currently investigating whether the multifunctional and scratch-specialized interneurons are each excitatory or inhibitory and whether they connect directly to motor neurons. This information will help us understand how a combination of multifunctional and specialized spinal cord neurons generates the right movement at the right time. The major broader impacts achieved by this project were to disseminate our results within the scientific community, to train undergraduates and graduate students for careers in biology, and to lead hands-on brain activities at several annual events at Oklahoma museums (each of which attracts hundreds of visitors) to educate the general public about the functions of the nervous system.
