” In this assay, hippocampal neurons from neonatal rats were dissociated and plated to glial microislands on glass coverslips so that each island had approximately 10–30 neurons (Segal and Furshpan, 1990) (Figure 2A). Cultures were transfected with synaptophysin-GFP to visualize synaptic terminals of transfected cells. For analysis only islands containing one transfected

DG neuron were selected so that every synaptophysin-GFP punctum could be uniquely associated with the transfected neuron (Figure 2A). To determine whether DG neurons recognize correct targets in microcultures, we used antibodies to identify hippocampal cell types in culture. Anti-Prox1 specifically labels DG neurons (Bagri et al., 2002), anti-PY BIBW2992 supplier labels CA3 pyramidal neurons and PD0325901 concentration some interneurons (Woodhams et al., 1989), and anti-CTIP2 labels CA1 pyramidal neurons and most DG neurons (Arlotta et al., 2005). These antibodies, when used in combination, uniquely identify each principal cell type in the hippocampus (Figures 2B and 2C; see Figure S1 available online), and every island analyzed included both correct and incorrect targets so that DG neurons always had a target choice. An island with one transfected DG neuron and several surrounding untransfected

neurons is shown in Figure 2D. Synaptophysin-GFP puncta are represented in yellow and were expanded for visibility at this magnification. An example of each cell type is Cediranib (AZD2171) shown at higher magnification with the anti-GFP signal (Figure 2E). Although positioned nearby on the island, many more synaptophysin-GFP puncta are found on the dendrites of the CA3 neuron compared to the DG or CA1 neuron (Figure 2E). To quantify this, the total number of synaptophysin-GFP puncta on every neuron from 21 islands was counted, normalized to dendrite length, and sorted by cell type. Analysis of the data indicates that DG neurons form significantly more synapses with CA3 neurons than with DG or CA1 neurons (Figure 2F). Furthermore, we calculated the expected number of synapses if all cells were innervated equally and represented this average by the dotted line

in Figure 2F. Because CA3 neurons are innervated significantly above this value, it suggests that there is a signal that promotes DG synapse formation onto these cells. Conversely, CA1 neurons were innervated significantly below this average value, suggesting the potential presence of a negative cue that inhibits synapse formation by DG neurons. DG neurons formed synapses with other DG neurons near the frequency expected by chance (Figure 2F). To determine if the synaptic bias of DG neurons for CA3 dendrites measured by synaptophysin-GFP reflects a bias in functional connectivity, we analyzed synaptic responses of neurons in microcultures by whole-cell recordings. For these experiments, pairs of neurons on an island were recorded simultaneously and tested for synaptic connectivity.