Calcium dynamics and compartmentalization in leech neurons

The aim of this project was to study how and where action potentials arise and propagate in the arborizations of identified neurons in the central nervous system of the leech. A major aim was to assess whether the entry of calcium is localized to distinct regions of the cells and to determine whether there are significant differences in calcium channel distribution between different types of neurons. A combination of electrophysiological techniques, optical recording and image analysis was used to approach these problems. I developed an experimental set-up for optical recordings of calcium transients by a fast CCD-camera. By use of calcium sensitive dyes I analysed in detail optical responses to electrical stimulation of neurons and the density of calcium channels, spatially and temporarily, in different neural cell types, including mechanosensory neurons and motoneurons. Fluorescence changes (∆F/F) of the membrane impermeable calcium indicator Oregon Green were measured. The dye was pressure injected into the soma of neurons under investigation. ∆F/F caused by a single action potential (AP) in mechanosensory neurons had approximately the same amplitude and time course in the soma and in distal processes. By contrast, in other neurons such as the Anterior Pagoda neuron, the Annulus Erector motoneuron, the L motoneuron and other motoneurons, APs evoked by passing depolarizing current in the soma produced much larger fluorescence changes in distal processes than in the soma. When APs were evoked by stimulating one distal axon through the root, ∆F/F was large in all distal processes, but very small in the soma. These results confirm and extend previous electrophysiological data which demonstrate that the soma of a motoneuron in the leech, as in many other invertebrates, does not generate action potentials (Stuart, 1970; Muller and Nicholls, 2 1974; Goodman and Heitler, 1979). Impulses recorded in the soma are normally only a few millivolts in amplitude. The AP of a motoneuron propagates to muscles of the body wall along segmental nerves that emerge from ganglia. The site of impulse initiation has been found to be at a distance from the soma but within the ganglion (Melinek and Muller, 1996; Gu et al. 1991). Our experiments with fluorescent transients are in accord with the concept that they result from calcium entry through voltage sensitive channels. Thus at sites where APs are found to be large, the calcium signals are large (as in peripheral axons), while at sites where spikes are small, (as in motoneuronal cell bodies) signals were weak, or non existent.