The proposed research will investigate the role of neurotrophins and neuronal activity in the development of the barrel cortex in mice. As characterized in previous work, connections in this region of the somatosensory cortex form during a critical period lasting from approximately postnatal day 6 to postnatal day 14. Because neurotrophins and their receptors have been shown to be present during this period, and their level of expression is regulated by neuronal activity, it is proposed that neurotrophins play a key role in the development of local connections in the barrel cortex.
Three specific aims are proposed to test this hypothesis. In the first aim, the patterns of expression of the neurotrophins, BDNF, NT3, and NT4/5, and their receptors, trkB and trkC, will be characterized between P6 and P21. Both in situ hybridization to detect mRNA and immunohistochemistry to detect protein are proposed. These experiments are the necessary first step for determining which molecules are in the right place at the right time during development, and serve as a prelude to other experiments aimed at determining whether they play a role in the development of local connections. Antibodies and nucleic acid probes for most of the molecules of interest have been identified. Although there may be some technical difficulties, these experiments are likely to be feasible. The experimental approach is strengthened by the parallel approaches of in situ hybridization and immunohistochemistry. In the second aim, formation of cortical connections will be disrupted by sectioning the infraorbital nerve at P6 and the effect on subsequent expression of neurotrophins in the barrel cortex will be determined. Although these experiments may provide correlative data linking neurotrophin expression data levels to the formation of synaptic connections, such evidence will be insufficient to establish a direct role for neurotrophins in the process. Antisense experiments are proposed to help fill this gap. However, such experiments can be extremely tricky to perform and may be difficult to interpret if neurotophins play multiple signalling roles. In the third aim, the role of activity in the development of synaptic connections will be explored in the presence of bicuculline, an antagonist for GABAa receptors. The drug will be applied locally over the left barrel cortex. Knocking out the action of a major inhibitory transmitter should increase activity in the region. Changes in the expression of neurotrophins and the formation of connections will be monitored. Again, correlative information may be obtained, but it will be very difficult to provide direct evidence for the existence of a pathway in which activity controls neurotrophin levels which in turn control synapse formation.
