Auditory brainstem neurons as components of a biohybrid system
Neuronal cell culture systems have many advantages for studies on cellular and smallscale network level. But a conceptual problem in analysing functional neuronal networks in dissociated cultures is, that no biological function can be attributed to the system. Thus, no prediction of the output of the circuit after a given input can be made. This dilemma can be solved with the approach to reconstruct a neuronal network in vitro after a model circuit of known biological function. The sound localisation circuit from the auditory brainstem of birds is an ideal candidate for this. It consist of two neuronal populations (Nucl. magnocellularis, NM and Nucl. laminaris, NL), which are highly specialised in their physiology. The embryonic development of these nuclei and their connections have already been studied in vivo. This study was undertaken to prove that neurons from the auditory brainstem of the chicken can be taken into a primary culture and to characterise their properties in vitro. This knowledge provides the basis for later attempts to reconstruct the NM/NL circuit as a biohybrid system. Neurons from NM and NL were excised at an early stage of development (E6.5) and cultivated for up to 15 days in vitro. The cells in the culture formed networks of neurites, expressed neuronal marker proteins and differentiated into dendritic and axonal compartments. The outgrowth of the neurites could be successfully guided with micro-contact printed protein structures. Moreover, the neurons were compatible with metal/semiconductor surfaces as culture substrate, if coated with protein. I could show that about one third of the neurons expressed calretinin, a more specific marker of auditory brainstem neurons. This was true for neurons from the chicken as well as barn owl brain of corresponding developmental stages. Also, a smaller amount of neurons were found to be positive for specialised voltage gated potassium channels. A significant maturation of basic membrane properties (resting membrane potential, membrane resistance, membrane capacity) could be determined in whole-cell patch experiments. Levels comparable to that in late embryonic/hatch neurons of NM/NL were reached. About 25% of the neurons showed action potential (AP) firing patterns similar to those seen in NM/NL in slice recordings. However, damper as well as true phasic firing types were found, which can be attributed to early and late developmental states of NM/NL neurons. It was proven by prelabeling of NM/NL neurons with fluorescent tracers, that neurons of those firing types in the culture are of auditory origin. AP amplitudes and voltage activated inward currents recorded in voltage clamp experiments developed towards a mature phenotype in vitro. These data suggest, that the general maturation of neuronal function is an cell-autonomous process in the auditory brainstem neurons of the chicken. However, a detailed analysis of the temporal dynamics of APs revealed impaired development of specialised features. This was corroborated in voltage clamp recordings. Though characteristic voltage-activated outward currents were found and did increase during in vitro development, the currents were an order of magnitude too low. I suppose that initiation and strengthening of expression of these characteristic channel proteins are different developmental processes in the auditory brainstem neurons. Functional synaptogenesis is a prerequisite for the reconstruction of in vitro circuits. With a variety of methods I could prove that synaptogenesis took place in the culture. Synaptic marker proteins were colocalised in punctiform domains on dendrites and somata of the neurons. A functional imaging of vesicle uptake with styryl dyes revealed punctiform domains of similar size, indicating synaptic zones. Recordings of excitatory postsynaptic potentials and APs after extracellular stimulation and recordings of miniature excitatory postsynaptic currents provided further evidence for synaptic transmission between neurons in the culture. This study demonstrates that the neurons from the avian auditory brainstem circuit are well suited as the biological component of a biohybrid system to analyse principles of neuronal connectivity. My results also show the differentiation of NM and NL neurons to be determined by cell-autonomous and activity-regulated processes. More insight could be provided with long-term examination of the development of the biohybrid in vitro version of the circuit.
info:eu-repo/classification/ddc/570, Zelldifferenzierung, Neurobiologie, Hirnstamm, Auditorisches System, Zellkultur, Elektrophysiologie, Biokompatibilität, Biowissenschaften, Biologie, biohybrid technology, potassium channels, synaptogenesis
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