The influence of neuronal activity on cortical axon branching was studied by imaging axons of layer 2/3 neurons in organotypic slice cultures of rat visual cortex. Upper layer neurons labeled by electroporation of plasmid encoding yellow fluorescent protein were observed by confocal microscopy. Time-lapse observation of single-labeled axons showed that axons started to branch after 8 -10 d in vitro. Over the succeeding 7-10 d, branch complexity gradually increased by both growth and retraction of branches, resulting in axon arbors that morphologically resembled those observed in 2-to 3-week-old animals. Electrophysiological recordings of neuronal activity in the upper layers, made using multielectrode dishes, showed that the frequency of spontaneous firing increased dramatically ϳ10 d in vitro and remained elevated at later stages. To examine the involvement of spontaneous firing and synaptic activity in branch formation, various blockers were applied to the culture medium. Cultures were silenced by TTX or by a combination of APV and DNQX but exhibited a homeostatic recovery of spontaneous activity over several days in the presence of blockers of either NMDA-type or non-NMDA-type glutamate receptors alone. Axonal branching was suppressed by TTX and AMPA receptor blockade but not by NMDA receptor blockade. We conclude that cortical axon branching is highly dynamic and that neural activity regulates the early developmental branching of upper layer cortical neurons through the activation of AMPA-type glutamate receptors.

During development, sensory thalamocortical (TC) axons grow into the neocortex and terminate primarily in layer 4. To study the molecular mechanism that underlies lamina-specific TC axon termination, we investigated the responsiveness of TC axons to ephrin-A5, semaphorin-7A (Sema7A) and kit ligand (KL), which are expressed in the upper layers of the developing cortex. Dissociated cells of the dorsal thalamus from embryonic rat brain were cultured on dishes that were coated with preclustered Fc-tagged extracellular domains of these molecules. Each protein was found to promote TC axon growth in a dose-dependent fashion of a bell-shaped curve. Any combination of the three proteins showed a cooperative effect in lower concentrations but not in higher concentrations, suggesting that their growth-promoting activities act in a common pathway. The effect of spatial distributions of these proteins was further tested on a filter membrane, in which these proteins were printed at a size that recapitulates the scale of laminar thickness in vivo, using a novel protein-printing technique, Simple-To-mAke Micropore Protein-Printing (STAMP2) method. The results demonstrated that TC axons grew massively on the laminin-coated region but were prevented from invading the adjacent ephrin-A5-printed region, suggesting that TC axons detect relative differences in the growth effect between these regions. Moreover, the inhibitory action of ephrin-A5 was enhanced by copresence with KL and Sema7A. Together, these results suggest that the lamina-specific TC axon targeting mechanism involves growth-inhibitory activity by multiple molecules in the upper layers and detection in the molecular environments between the upper and deep layers.

Neocortical neuronal circuits are refined by experience during the critical period of early postnatal life. The shift of ocular dominance in the visual cortex following monocular deprivation has been intensively studied to unravel the mechanisms underlying the experience-dependent modification. Synaptic plasticity is considered to be involved in this process. We previously showed in layer 2/3 pyramidal neurons of rat visual cortex that low-frequency stimulation-induced long-term potentiation (LTP) at excitatory synapses, which requires the activation of Ni(2+)-sensitive (R-type or T-type) voltage-gated Ca(2+) channels (VGCCs) for induction, shared a similar age and experience dependence with ocular dominance plasticity. In this study, we examined whether this LTP is involved in ocular dominance plasticity. In visual cortical slices, LTP was blocked by mibefradil, kurtoxin and R-(-)-efonidipine, T-type VGCC blockers, but not by SNX-482, an R-type VGCC blocker, indicating that LTP induction requires T-type VGCC activation. Mibefradil did not affect synaptic transmission even at a dose about 30 times higher than that required for LTP blockade. Therefore, this drug was used to test the effect of T-type VGCC blockade on ocular dominance shift produced by 6 days of monocular deprivation during the critical period using visual evoked potentials (VEPs). Although this monocular deprivation commonly produced both depression of deprived eye responses and potentiation of nondeprived eye responses, only the former change occurred when mibefradil was infused into the visual cortex during monocular deprivation. Mibefradil infusion produced no acute effects on VEPs. These results suggest that T-type VGCC-dependent LTP contributes to the experience-dependent enhancement of visual responses.

Immature excitatory synapses often have NMDA receptors but not AMPA receptors in central neurons, including visual cortical pyramidal neurons. These synapses, called silent synapses, are converted to functional synapses with AMPA receptors by NMDA receptor activation during early development. It is likely that this process underlies the activity-dependent refinement of neuronal circuits and brain functions. In the present study, we investigated postnatal development of excitatory synapses, focusing on the role of visual inputs in the conversion of silent to functional synapses in mouse visual cortex. We analyzed presumably unitary excitatory postsynaptic currents (EPSCs) between a pair of layer 2/3 pyramidal neurons, using minimal stimulation with a patch pipette attached to the soma of one of the pair. The proportion of silent synapses was estimated by the difference in the failure rate between AMPA- and NMDA-EPSCs. In normal development, silent synapses were present abundantly before eye opening, decreased considerably by the critical period of ocular dominance plasticity, and almost absent in adulthood. This decline in silent synapses was prevented by dark rearing. The amplitude of presumably unitary AMPA-EPSCs increased with age, but this increase was suppressed by dark rearing. The quantal amplitude of AMPA-EPSCs and paired-pulse ratio of NMDA-EPSCs both remained unchanged during development, independent of visual experience. These results indicate that visual inputs are required for the conversion of silent to functional synapses and this conversion largely contributes to developmental increases in the amplitude of presumably unitary AMPA-EPSCs.

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