Resilience of network activity in preconditioned neurons exposed to 'stroke-in-a-dish' insults

Exposing cultured cortical neurons to stimulatory agents - the K⁺ channel blocker 4-aminopyridine (4-ap), and the GABA_A receptor antagonist bicuculline (bic) - for 48 h induces down-regulated synaptic scaling, and preconditions neurons to withstand subsequent otherwise lethal 'stroke-in-a-dish' insults; however, the degree to which usual neuronal function remains is unknown. As a result, multi-electrode array and patch-clamp electrophysiological techniques were employed to characterize hallmarks of spontaneous synaptic activity over a 12-day preconditioning/insult experiment. Spiking frequency increased 8-fold immediately upon 4-ap/bic treatment but declined within the 48 h treatment window to sub-baseline levels that persisted long after washout. Preconditioning resulted in key markers of network activity - spiking frequency, bursting and avalanches - being impervious to an insult. Surprisingly, preconditioning resulted in higher peak NMDA mEPSC amplitudes, resulting in a decrease in the ratio of AMPA:NMDA mEPSC currents, suggesting a relative increase in synaptic NMDA receptors. An investigation of a broad mRNA panel of excitatory and inhibitory signaling mediators indicated preconditioning rapidly up-regulated GABA synthesis (GAD67) and BDNF, followed by up-regulation of neuronal activity-regulated pentraxin and down-regulation of presynaptic glutamate release (VGLUT1). Preconditioning also enhanced surface expression of GLT-1, which persisted following an insult. Overall, preconditioning resulted in a reduced spiking frequency which was impervious to subsequent exposure to 'stroke-in-a-dish' insults, a phenotype initiated predominantly by up-regulation of inhibitory neurotransmission, a lower neuronal postsynaptic AMPA: NMDA receptor ratio, and trafficking of GLT-1 to astrocyte plasma membranes.