Simultaneous electrophysiological and morphological assessment of functional damage to neural networks in vitro after 30\u2013300\u2009g impacts

Abstract

An enigma of mild traumatic brain injury are observations of substantial behavior and performance deficits in the absence of bleeding or other observable structural damage. Altered behavior and performance reflect changes in action potential (AP) patterns within neuronal networks, which could result from subtle subcellular responses that affect synaptic efficacy and AP production. The aim of this study was to investigate and quantify network activity changes after simulated concussions in vitro and therewith develop a platform for simultaneous and direct observations of morphological and electrophysiological changes in neural networks. We used spontaneously active networks grown on microelectrode arrays (MEAs) to allow long-term multisite monitoring with simultaneous optical observations before and after impacts delivered by a ballistic pendulum (30 to 300 g accelerations). The monitoring of AP waveshape templates for long periods before and after impact provided an internal control for cell death or loss of cell-electrode coupling in the observed set of neurons. Network activity patterns were linked in real-time to high power phase contrast microscopy. There was no overt loss of glial or neuronal adhesion, even at high-g impacts. All recording experiments showed repeatable spike production responses: a loss of activity with recovery to near reference in 1 hr, followed by a slow activity decay to a stable, level plateau approximately 30–40% below reference. The initial recovery occurred in two steps: a rapid return of activity to an average 24% below reference, forming a level plateau lasting from 5 to 20 min, followed by a climb to within 10% of reference where a second plateau was established for 1 to 2 hrs. Cross correlation profiles revealed changes in firing hierarchy as well as in Phase 1 in spontaneous network oscillations that were reduced by as much as 20% 6–8 min post impact with only a partial recovery at 30 min. We also observed that normally stable nuclei developed irregular rotational motion after impact in 27 out of 30 networks. The evolution of network activity deficits and recovery can be linked with microscopically observable changes in the very cells that are generating the activity. The repeatable electrophysiological impact response profiles and oscillation changes can provide a quantitative basis for systematic evaluations of pharmacological intervention strategies. Future expansion to include fluorescent microscopy should allow detailed investigations of damage mechanisms on the subcellular level.