Age-Dependent Failure of Axon Regeneration in Organotypic Culture of Gerbil Auditory Midbrain

ABSTRACT: Inferior colliculus (IC) slice cultures from postnatal (P) day 6–8 gerbils exhibit axonal regen-eration across a lesion site, and these regrowing pro-cesses can form synapses. To determine whether regen-erative capacity is lost in older tissue, as occurs in vivo, slices from P12–21-day animals were grown under sim-ilar conditions. While these cultures displayed a near complete loss of neurons over 6 days in vitro, glutamate receptor antagonists (AP5 and/or CNQX) significantly enhanced survival, particularly at P12–15. In contrast, several growth factors or high potassium did not im-prove neuron survival. Therefore, axonal regeneration was assessed following complete transection of the com-missure in AP5/CNQX-treated IC cultures from P12 animals. Neurofilament staining revealed that transected commissural axons survived for 6 days in vitro, but only a few processes crossed the lesion site and these axons did not extend into the contralateral lobe. In contrast, there was robust axonal sprouting and growth within one lobe of the IC, remote from the lesion site. When P6 and P12 tissue was explanted onto a coated substrate, the P6 axons grew onto the substrate, but the P12 axons were seemingly prevented from reaching the substrate by a veil of nonneuronal cells. Coculture of the IC and one of its afferent populations, the lateral supe-rior olive, provided a similar finding, indicating that failure to regenerate was a general property at the age examined. These data show that neuron survival is not sufficient to permit axon regeneration at P12, and sug-gest that P12 lesion sites manufacture a prohibitive sub-strate since process outgrowth is blocked specifically a