State-dependent long lasting modulation of leg motoneuron membrane potential during stick insect walking.

The basis of locomotion is the rhythmic activity of locomotor organs. During walking, rhythmic alternating bursts occur in leg motoneurons which result from the integration of signals from central pattern generators, sense organs and coordinating signals from neighboring segments. The rhythmic bursting pattern is shaped by rhythmic excitatory and inhibitory drive, as well as a long lasting (tonic) depolarizing modulation. This dissertation investigates the mechanisms that underlie the tonic modulation of neuronal activity, which is the basis of the rhythmic activity. A single-legged preparation of the stick insect was used, that allows the analysis of neuronal activity in mesothoracic motoneurons during front leg stepping, without the influence of local sense organs. Intracellular recordings of the membrane potential in flexor motoneurons ipsilateral and contralateral to the stepping front leg revealed a mean tonic membrane depolarization of 1.8 ±1.1 mV that could outlast the stepping sequence by several seconds. Furthermore, a phasic modulation of membrane potential occurred on top of the tonic depolarization, which was variably coupled to front leg steps. The tonic depolarization was associated with a decrease in input resistance, its amplitude depended on membrane potential and its mean reversal potential was found to be -41 mV. These properties of the tonic depolarization indicate that it is based on a nonselective cation conductance or a mixed inward and outward current through different channels. Furthermore, the tonic depolarization increased the excitability of the membrane to depolarizing input and was found to have long repolarization time constants (tau=800 ms). Pharmacological experiments were performed to identify the participating transmitter(s) and to test for a possible involvement of second messengers. Flexor motoneurons were recorded intracellularly while superfusing pharmacological agents, which were restricted to the mesothoracic ganglion. The muscarinic antagonist atropine decreased the tonic depolarization amplitude, thus indicating a role for acetylcholine in mediating the tonic depolarization via metabotropic receptors. Other transmitters/receptors might be involved too, as octopamine increased the tonic depolarization amplitude. This was supported by the reduction of the tonic depolarization amplitude by an octopaminergic antagonist (mianserin). Serotonin had an opposing effect on the tonic depolarization amplitude. It was shown that serotonin increased, but also decreased the tonic depolarization amplitude in different experiments. Several second messenger pathways might be involved in mediating the tonic depolarization, one of which seems to include calcium in the flexor motoneurons. Furthermore, the increase of the tonic depolarization amplitude by 8-Br-cAMP suggests a role for cAMP in mediating the tonic depolarization. An involvement of an IP3/DAG pathway was indicated by the increase in tonic depolarization in the presence of neomycin and U-73122. Additional experiments addressed the role of the brain (supraesophageal ganglion) in controlling neuronal activity in the mesothoracic ganglion induced by front leg stepping. Especially the relevance of the brain for generating the tonic depolarization was tested in lesion experiments. Brain removal decreased the tonic depolarization amplitude, thus indicating a role for descending pathways in influencing the tonic depolarization. Another preparation was used to investigate the neuronal activity in neck connectives during front leg stepping. The neuronal activity increased and was correlated to front leg stepping velocity. In brainless animals, however, the increased activity in neck connectives was diminished.