Primary brain cells cultured on flat surfaces, i.e., in a two-dimensional fashion, have a long history of use as an experimental model system in neuroscience research. However, it is questionable to which extent these cultured brain cells resemble their in vivo counterparts. Mainly, it has been claimed that the non-oxidative glucose metabolism reflected by lactate production is unphysiologically high. Furthermore, it is known that culturing in 2D alters the phenotype of cells. Here we present diphenylalanine peptide nanowires (PNWs) as a culturing substrate for primary neocortical neurons from mice. The topology of the PNWs leads to neuronal cultures developing in 2.5D environment and hence improved culturing conditions. We investigate the effect of different concentrations of PNWs and different cell densities of neurons on the culturing conditions. The neocortical neurons were examined through scanning electron microscopy in order to study the effect of PNW concentrations and neuron densities on the structural appearance of the cells. Then employing the optimal combination of neuron density and PNW concentration, the neurons were evaluated functionally and metabolically by comparison with neocortical neurons standard culturing methods in 2D. Specifically, we tested neuronal viability, capacity for vesicular release of neurotransmitter GABA, as well as oxidative and non-oxidative glucose metabolism. It was evident that neurons cultured on PNWs exhibited increased viability combined with an increased capacity for neurotransmitter release and a lower fraction of non-oxidative metabolism than neurons cultured in 2D. Hence, neocortical neurons cultured in 2.5D on PNWs appear to be healthier and less glycolytic than neurons cultured in 2D.