Abstract

The spatial and temporal expression of the protein-tyrosine kinase B (TrkB) receptor and its ligands has been correlated with the development of the neocortex. Activation of the receptor has been associated with neocortical neuronal survival, differentiation, connectivity and neurotransmitter release. Although such findings suggest an important role for TrkB signaling in corticogenesis, conclusive evidence from targeted gene deletion ("knockout"; TrkB -/-) mice has been limited, due in part to the neonatal lethality of most of these mutant mice and the confounding variables associated with the poor health of those few surviving slightly longer postnatally. In the present study, the effects of TrkB signaling on the survival, differentiation and integration of neocortical neurons was directly investigated in vitro and in vivo. First, we conducted a neuron-specific immunocytochemical analysis of TrkB -/- mice to determine whether early cortical structure and patterns of histogenesis were normal or perturbed. We then employed in vitro and in vivo approaches to extend the life of TrkB -/- neocortical neurons beyond the period possible in TrkB -/- mutant mice themselves: (i) dissociated cell culture to directly compare the developmental potential of TrkB -/-, +/- and +/+ neurons; and (ii) neural transplantation into homochronic wild-type recipients to investigate the cell-autonomous effects of the receptor knockout on the differentiation, growth and integration of neocortical neurons. These latter experiments allowed, for the first time, study of the survival and differentiation potential of TrkB -/- neocortical neurons beyond the initial stages of corticogenesis. Direct comparison of brains of TrkB -/-, +/- and +/+ littermates immunocytochemically labeled with antibodies to microtubule-associated protein-2, neurofilament and beta-tubulin III revealed subtle anatomical anomalies in the mutant mice. These anomalies include abnormally diffuse microtubule-associated protein-2 positive neurons just dorsal to the corpus callosum, and heterotopic aggregations of postmitotic neurons in the subventricular zones of the ganglionic eminences, both suggesting delayed neuronal migration and differentiation. Cell culture experiments revealed substantially reduced survival by TrkB -/- neocortical neurons, and a significant reduction in neurite outgrowth by surviving TrkB -/- neurons. In experiments where prelabeled embryonic or neonatal TrkB -/- neocortical neurons were transplanted into the cerebral cortices of neonatal wild-type recipients, a similar quantitatively significant defect in the formation of dendrites, as well as reduced integration of TrkB -/- neocortical neurons, was also evident. These findings demonstrate cell-autonomous abnormalities in the development of neocortical neurons from TrkB -/- mice, and the subtle, but potentially critical, role of protein-tyrosine kinase B signaling in neocortical neuronal survival, differentiation and connectivity.