, 2006), and endothelial cells ( Daneman et al., 2010) also did not exhibit any in situ signal in neuronal dendrites ( Figures S7E–S7I). Analysis of the different mRNA distribution patterns

indicates that the dendrite to soma ratio for distinct mRNAs is not a constant value and is not solely dependent on the apparent somatic concentration of an mRNA (see Figure 6B). In addition, Dabrafenib ic50 we quantified the ratio of the Dlg4 mRNA between the dendrites and the cell body in single neurons and found a dendrite: soma ratio of ∼30:70 ( Figure S5). The above experiments validate the presence of mRNAs we identified via deep sequencing in the dendrites of cultured hippocampal neurons. To examine the localization of a subset of mRNAs in a more realistic context, we adapted the high resolution in situ hybridization technique for use in rat hippocampal slices and combined it Selleckchem Panobinostat with immunohistochemical labeling of dendrites using an antibody to MAP2 (Figure 7A). We focused our analysis on area CA1, the region from which we microdissected

tissue for deep sequencing and the site of several forms of plasticity that require local translation. We examined the localization of 19 (Dlg4, Map1a, Cacng2, Shank3, Psd, Shank1, Cacna1i, Hpcal4, Nlgn3, Kcnd2, Camk4, Gria2, Cyfip2, Grin2a, Grik2, Kif5a, Kcna2, Actb, and Pclo; 11 are shown in Figure 7) different transcripts and found positive evidence for their presence within the synaptic neuropil ( Figures

7A and 7B). In some fortuitous cases, the MAP2-labeled dendrites were sufficiently well-resolved to allow us to visualize labeled mRNAs associated with dendrites ( Figure 7A). Taken together, these data indicate that the mRNAs identified by deep sequencing can be observed by high-resolution imaging to reside in the synaptic neuropil of hippocampal area CA1. The proteome of an individual synapse is the physical entity that determines the response of either a given synapse to an input, and it is clear that, like other proteomes (e.g., Ingolia et al., 2009), the synaptic proteome is subject to ongoing and dynamic modification by regulated protein synthesis and degradation. The prior identification of mRNAs resident in dendrites and axons have yielded a largely heterogeneous mix of a small number (100 or so) transcripts that did not suggest an ongoing role for local protein synthesis in synaptic function, but rather suggested that local synthesis might be used in special cases during some forms of synaptic plasticity. Here, using next generation sequencing of hippocampal neuropil RNA samples we reveal a surprisingly large number of previously undetected neuropil mRNAs, suggesting that mRNA localization may be more of a rule, than the exception. In addition, many of the proteins that populate the synapse may originate from a local source. Based on current reference databases (NCBI, Rattus norvegicus transcriptome version rn4.