This project is designed to provide information about the organization of neuronal systems in the mammalian spinal cord that are involved in the neural control of movement. In the past, we investigated this problem using the in vivo cat spinal cord but current work uses the isolated brain stem and spinal cord of neonatal mice or amphibians studied in vitro. During FY 2000, we continued a study of low-frequency synaptic depression of monosynaptic excitatory postsynaptic potentials (EPSPs) produced in lumbosacral spinal motoneurons by short trains (10 pulses) of stimuli to dorsal roots at 0.0125 - 8.0 Hz in 2-4 day old (P2-4) mice. The NMDA receptor blocker, AP5, was added to the bathing solution to suppress spontaneous and stimulus-evoked background discharges, which distorted the data obtained in earlier experiments. At frequencies greater than 0.5 Hz the second response (R2) was larger than R2 at lower frequencies, while the average of the last three responses (Tail) were monotonically smaller as stimulus frequency increased. All responses were normalized by that of the first response (R1) to permit comparisons. Lowering external Ca concentration from 2.0 mM to 0.8 mM without changing external Mg reduced average R1 amplitudes and R2 depression, with little change in Tail depression. Conversely, increasing external Ca to 4.0 mM increased average R1 amplitude and R2 depression, but again did not change Tail depression. Increasing bath temperature from 24 to 32 deg. C produced little change in R1 amplitudes but reduced the depression of all responses at most frequencies. Based on these data, we developed an empirical model that assumes: 1) depletion of two presynaptic compartments (N and S) that are renewed by independent processes with exponential time constants; 2) a rapidly decaying facilitation of transmitter release probability, and; 3) a more slowly decaying augmentation of the rate of renewal of the N compartment. This model gave satisfactory fits to the data from all external Ca conditions and implied that the release fraction and the incremental weights of the facilitation and augmentation processes were all changed in parallel with Ca concentration, without change in time constants. In contrast, model fits to the 32 deg. C data implied that the process time constants all decreased by 45 to 50% while the presumably calcium-related weighting factors were unchanged. A full report has been submitted. The work is continuing with a similar examination of the system in P10-12 animals and an exploration of the effect of metabotropic glutamate receptor agonists and antagonists on synaptic depression in both age ranges. This work will enable us to develop a profile of synaptic development during early postnatal life. During FY2000 we began a new project designed to elucidate spinal cord mechanisms that control rhythmic walking movements in a relatively primitive amphibian, Necturus. We have successfully developed an animal husbandry system for maintaining these animals and for recording locomotor-like activity from the isolated spinal cord in vitro. Preliminary studies are underway to examine the influence of lesions on the quality of rhythmic stepping, in order to define the optimum preparation to use for intra- and extracellular recording studies of the spinal circuits that produce locomotion. The Necturus model is of the spinal central pattern generator for locomotion is a useful bridge between studies of this system in primitive fish and in the much more complex spinal cord of mammals.
