Characterization of three-dimensional rat central nervous system culture maturation, with applications to monitor cholinergic integrity

Studying age-related neuropathologies in vitro requires a three-dimensional (3D) culture system presenting mature phenotypes. In this study, we aimed to determine whether aged reaggregate cultures physiologically represent mature brain tissue. Results support that embryo-derived rat central nervous system (CNS) reaggregate cultures develop into mature-like tissues, comparable to in vivo maturation, including the following characteristics: (a) progressive reduction in cell proliferation (reduced anti-Ki-67 immunoreactivity), (b) progressive restriction of long neurite growth potential (as explant cultures), and (c) increased and sustained synaptic enzyme (acetylcholine esterase, AChE) activity. The acquisition of mature-like reaggregate cultures has allowed us to pursue the hypothesis that the physiological integrity of 3D CNS cultures may be monitored by synaptic enzyme activity. To assess this hypothesis, mature-like reaggregates were exposed to H2O2, glutamate, or amyloid beta(1-42); each resulted in diminished AChE activity. H2O2 exposure resulted in nuclear fragmentation. Glutamate and amyloid beta(1-42) exposure resulted in acetylcholine content reduction. Simultaneous reduction of AChE activity and acetylcholine content verified diminished cholinergic integrity. This scheme exploiting synapse enzyme activity of mature-like 3D CNS tissue is therefore applicable to age-related neuropathology research including in vitro screening of conditions potentially affecting synapse integrity, including the promotion of dementia.