Neuronal activity-dependent development of layer 4 pyramidal cells in the primary visual cortex

Intricate neuronal circuits are at the base of several vital functions and complex behaviours. The proper functioning of these networks relies foremost on the correct development of these circuits, which emerge from an interplay of innate genetic programs and activity-dependent plasticity mechanisms. The extent to which neuronal activity plays a role, in particular the contributions of activity that is spontaneously generated by immature neuronal circuitry, is not precisely known. Furthermore, while the dendritic morphology is an important morphological feature that determines how a neuron is integrated in the circuitry, much remains to be understood on how the dendritic tree is formed and reshaped during development. The current thesis provides insight into these questions, by providing a detailed description of the development of both thalamocortical (TC) axon ingrowth in layer 4 (L4) of the primary visual cortex (V1) and the development of basal dendritic morphology of L4 pyramidal V1 neurons. Furthermore, we explored the influence of spontaneous activity on the basal dendritic development of L4 pyramidal neurons of V1. Central for studying these developmental processes was the development of a pioneering surgical protocol to virally introduce the expression of fluorescent proteins and molecular silencers in the dorsal Lateral Geniculate Nucleus (dLGN) at postnatal day 0 (P0). The proposed surgical protocol for viral injections is therefore the first stereotactic protocol that accomplishes transgene expression in a spatially restricted target region in the neonate brain without the use of the Cre/loxP expression system.
Using this novel stereotactic injection protocol, we introduced the expression of a fluorescent protein in neurons from the dLGN and studied TC axon ingrowth in L4 of V1. TC axon ingrowth was not yet complete at birth; instead, the number of axons/axonal arborizations increased until P10 after which axonal coverage remained stable. Furthermore, the number of axonal varicosities that colocalized with the presynaptic marker vlgut2 appeared to increase in parallel with axonal ingrowth, suggesting that the number of presynaptic structures increases during early postnatal development.
Several trends emerged when evaluating the dendritic morphological development of sparsely labelled L4 neurons. First, the dendritic length increased throughout the first two weeks of postnatal development. Second, the dendritic complexity appeared to increase between P4 and P10, as the number of secondary and tertiary dendrites, along with the number of branch points increased. Interestingly, these dendritic parameters showed a tendency to decrease between P10 and P15, suggesting that part of the dendritic complexity previously gained is subsequently lost. This raises the hypothesis that the loss in dendritic complexity is caused by the pruning of aberrant secondary and tertiary branches. We then markedly reduced TC activity through the expression of the molecular silencer Kir2.1 in the dLGN and studied the effect on basal dendritic morphology at P13. Silencing TC activity seemed to be associated with an increased dendritic length. Furthermore, the basal dendritic structure appeared to be more complex, indicated by an increase in the number of tertiary dendrites, branch points and Sholl radius. The number of primary dendrites, which also remained constant during development, was not affected by silencing TC activity. This raises the hypothesis that the number of primary dendrites is established early in development independent from neuronal activity from the dLGN. Though the results presented are preliminary, the trend emerges that silencing thalamocortical activity leads to an increased level of dendritic complexity. Considering the role attributed to activity-dependent genetic programs in dendritic pruning, we hypothesize that the apparent increase in dendritic complexity is caused by the failure of pruning aberrant connections, in absence of spontaneous thalamocortical input. Future studies will have to be carried out to test this hypothesis.