Event Abstract Back to Event The effect of exogenous human α-synuclein on neuronal network functionality Gerco Hassink1*, Christian C. Raiss2, Mireille Claessens2 and Joost Le Feber1 1 University of Twente, Biomedical Signal and Systems, MIRA Institute for Biomedical Technology and Technical Medicine, The Netherlands 2 University of Twente, Nanobiophysics Group, MESA+ Institute for Nanotechnology, The Netherlands Motivation The formation of protein aggregates called Lewy Bodies (LB) and Lewy Neurites (LN) is a pathological hallmark of Parkinson’s disease (PD) or Lewy Body Dementia (LBD). The main component of these protein aggregates is alpha-synuclein (AS), and elevated levels of AS found in the cerebrospinal fluid are associated with the disease onset. Little is known about the mechanisms by which increased levels of AS and the appearance of AS inclusions contribute to development of disease symptoms. Here, we hypothesize that AS is not directly toxic to neurons, but they may affect synaptic functioning. To validate this hypothesis, we exposed cultured rat cortical networks to human recombinant AS. Material and Methods We recorded spontaneous and stimulus induced activity in cultured cortical networks older than 3 weeks, during 8 days after administration of 100uM human recombinant AS. We determined the temporal evolution of synaptic and neuronal functioning in relation to the formation of LB like deposits and viability. We assessed synaptic functioning by the network response to electrical stimulation at 15-300 ms latencies (the late phase response), which has been shown to depend on synaptic transmission (1). Neuronal functioning was monitored by the number of neurons that remained active throughout the experiment (although spiking frequencies generally dropped) and the shape of recorded action potentials. Combined neuronal and synaptic functioning were observed through the array wide firing rate. Results Exposure to AS did not significantly change viability or metabolic activity. Monitoring the neuronal activity over a time period of 7 days showed an initial increase in firing rate, directly after administration of AS, which lasted from a few hours up to a full day. The magnitude of the increase varied per experiment. After this initial increase, the array wide firing rate decreased and dropped below baseline values in approximately 24 hours. The firing rate further decreased until day 5, when hardly any neuronal activity could be recorded. In parallel cultures, we quantified the amount of AS aggregates. The number AS deposits >500 nm increased in time, but after 5 days only a third of the cells harbored AS deposits. Remarkably, most action potential wave shapes, if changed at all, did not change gradually over time, but changed rather suddenly during the last hours before a particular neuron stopped firing. In contrast to the observed firing rate, the late phase of stimulus responses started to decrease within 12 hours after AS administration. This decrease continued for 4 days until we were no longer able to measure any late responses. Very similar results were obtained with 50 uM AS. Discussion The above results suggest that only extracellular increase of AS is enough to reduce the overall firing rate of the network. The fast decline of late phase stimulus responses suggests that the observed reduction in firing rate is a result of disruption of synaptic transmission. Despite this reduction, the cultures are otherwise healthy in terms of percentage of cell death, metabolism and quality of the produced action potentials. This suggest that elevated AS concentrations are not toxic on the short term. Cells may however ultimately die due to lack of synaptic input (2,3). While cells develop AS deposits relatively slowly, the first electrophysiological effects of increased AS already become visible within the first 24 hours. This suggests that the AS deposits are unlikely to cause the loss in synaptic transmission. Conclusion Combined, these data support the idea that the observed decreased neuronal activity due to elevated AS concentration results from synaptic failure rather than neuronal dysfunction. We speculate that prolonged synaptic inactivity ultimately results in neural death.