Bailey, Joanne Louise
Using Hi-Spots to investigate in vitro network
dysfunction in Cysteine String Protein ?
University of Southampton, School of Biological Sciences,
Hi-Spot are highly re-aggregated neural cultures grown on PTFE membrane at the liquidair interface. The Hi-Spot protocol was developed by scientists at Capsant Neurotechnologies using embryonic neural tissue. We characterised Hi-Spots made using existing protocols to confirm they represented a maturing neural network with molecular, cellular and functional signatures. We have additionally modified existing protocols to allow use of postnatal tissue as the source for dissociation and re-aggregation. Hi-Spots made from postnatal day zero (P0) rats self organised into a complex 3D tissue-like structure containing anatomically synaptically-interconnected neurons, astroglia and microglia. This CNS analogue of brain tissue provides for the emergence of a co-ordinated excitatory and inhibitory network, demonstrating a maturing pattern of activity involving single spikes developing into bursting behaviour driven by intrinsic synaptic activity. This activity can be represented as frequency or averaged amplitude (RMS), increases with time in culture and is blocked by glutamate receptor antagonists and stimulated by the inhibitory receptor antagonist bicuculline. The validity of its more in vivo-like organization, not observed in more conventional dissociated cultures, is evidenced by a glutamate toxicity resistance in the Hi-Spot cultures. Further, the thesis describes the optimisations to the Hi-Spot protocol to allow viable high density re-aggregated cultures to be made from individual rather than pooled brain dissociations. Modifications to the protocol included dissociation of the mouse tissue using a protease papain kit and cell plating at an increased cell density. Using this as a platform we have gone on to investigate the neuronal dysfunction occurring in Cysteine String Protein (CSP)?-/- mice. CSP? is a presynaptic protein thought to have co-chaperone like functions, mice lacking CSP? are born alive but show progressive weakness and neuronal degeneration soon after birth (Fernandez-Chacon, Wolfel et al. 2004). Hi-Spots formed from CSP? -/- tissue did not show overt neurodegenerative characteristics compared to +/+ controls and functional analysis demonstrated that at~DIV14 CSP? +/+ and -/- mice displayed equal levels of basal spontaneous network activity. Addition of bicuculline (50?M) to +/+ cultures lead to a significantly increasedfrequency and RMS value. However, in -/- cultures there was no increase induced by bicuculline. This may be due to an inability of CSP? -/- cultures to sustain high frequency synaptic transmission that is associated with bursting activity, or a selective degeneration of a sub-population of inhibitory neurons and a homeostatic network plasticity. The data suggest CSP? may act to protect the ability of neurons for high frequency synaptic transmission and/ or protect inhibitory neurons from degeneration.
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