Primary neuronal culture of Locusta migratoria for construction of networks on microelectronic recording devices

In this thesis a basis for the construction of simple neuronal networks of Locusta migratoria was developed. For the study of synaptogenesis and signal transmission over a long time period and at many recording sites simultaneously, it is desired to couple those networks with extracellular recording devices like field-effect transistors or metal electrode arrays. The present work describes a protocol for a primary culture of locust neurons. The neurons regenerated neurites, which also connected to adjacent neurons. Cell culture conditions were optimised by supplementing the medium with insulin and the neurohormone 20-hydroxyecdysone. Concanavalin A was proved as optimum coating molecule concerning soma attachment and neurite outgrowth. This cell culture protocol enabled long-term culture for up to four weeks. The electrophysiological characterisation by patch-clamp recordings from the soma revealed three different response classes to positive current injection: nonspiker, which showed graduated responses, spiker, which were able to generate one action potential, and burster, which generated series of action potentials. These different electrophysiological behaviours reflected intrinsic properties of the neurons and were possibly due to different conductances for potassium ions. Recordings from identified motoneurons revealed changes of soma excitability due to the isolation procedure or the culture conditions. The former described three responses classes in vitro can not represent in vivo electrophysiological classes since those cultured motoneurons showed single spikes and also series of action potentials. Nonspiking local interneurons probably kept their nonspiking behaviour in vitro and neurons, which had the appropriate channel composition for generating action potentials showed one or more action potentials at the soma. Pharmacological experiments revealed that voltage activated Ca2+-channels were required to induce bursting behaviour. For the first time synaptic transmission in insect neuronal cell culture was observed directly in simultaneous patch-clamp recordings. Chemical as well as electrical synapses were demonstrated. GABA was identified as neurotransmitter in the cell culture. The electrical synapses showed voltage-rectifying behaviour. Signals of insect neurons were coupled successfully to microelectronic recording systems. Action potentials and ionic currents were recorded by field-effect transistors. Also ionic currents of neurons were recorded by metal microelectrodes. The extracellular signal was dominated by ionic currents in the cleft between the cellular membrane and the gate. Action potentials were recorded as biphasic signals corresponding to the C-type responses described elsewhere. It was shown that the amplitude of field-effect transistor signals strongly depends on distance and position of the neuron to the gate. For geometrical control of network formation micro-contact printing of concanavalin A on star-shaped PEG was shown to be the method of choice. Soma position and neurite outgrowth were controlled successfully