We counted the total number of excitatory synapses, DG synapses, and CA synapses formed onto different neuron types over time.

At all time points, dendrites of CA1 and CA3 neurons developed very similar numbers of excitatory synapses. This indicates that neither check details cell type has any more synaptogenic potential than the other (Figure 4D). However, CA3 neurons developed significantly more DG synapses (up to 2.4 times greater) than CA1 neurons at all time points (Figure 4E). During our analyses we noticed that, like mossy fiber terminals in vivo, SPO-positive synapses were often much larger than typical excitatory presynaptic sites. Therefore, we determined whether these extra-large excitatory presynaptic

terminals were also preferentially located on CA3 neurons. Indeed, when we limited our analysis to synapses greater than 1.0 μm2, we discovered that CA3 neurons have up to 4.4 times more extra-large DG synapses than CA1 neurons (Figure 4F), and the average size of a DG synapse is greater on CA3 neurons (Figure 4G). We also observed that CA1 neurons developed BMS-354825 mouse significantly more CA synapses than CA3 neurons, which indicates that specificity may not be limited to DG synapses but that other types of synapses also undergo selective formation in culture (Figure 4H). Together, these experiments support the conclusion that mechanisms driving specific synapse formation in culture function without spatial cues present in the brain. Because we observe a strong synaptic bias as early as 8 DIV, it suggests that this specificity is largely driven

by selective synapse formation onto correct targets, and not by elimination of synapses from incorrect targets. below To identify molecules that might regulate the formation of DG-CA3 synapses, we analyzed expression patterns of genes that encode transmembrane proteins with extracellular domains that could mediate cell-cell interactions. The initial analysis was based on gene expression data published in the Allen Brain Atlas (http://www.brain-map.org/) and led to identification of the cadherin gene family as potential mediators of connectivity. There are about 20 classic cadherin genes thought to mediate cell-cell interactions, although the specific function of most cadherins is unknown. Several cadherins are expressed in the hippocampus, but only one, cadherin-9, is strongly and specifically expressed in DG and CA3 regions (Figure 5A) (Bekirov et al., 2002). Therefore, we hypothesized that cadherin-9 interactions between DG axons and CA3 dendrites may be important for regulating mossy fiber synapse development but not other types of synapses in the hippocampus. Cadherin-9 is a relatively uncharacterized gene predicted to encode a classic type II cadherin, and therefore, cadherin-9 may signal via homophilic binding.