Dendritic mRNAs encode diversified functionalities in hippocampal pyramidal neurons

Abstract

Background: Targeted transport of messenger RNA and local protein synthesis near the synapse are important for synaptic plasticity. In order to gain an overview of the composition of the dendritic mRNA pool, we dissected out stratum radiatum (dendritic lamina) from rat hippocampal CA1 region and compared its mRNA content with that of stratum pyramidale (cell body layer) using a set of cDNA microarrays. RNAs that have over-representation in the dendritic fraction were annotated and sorted into function groups.

Results: We have identified 154 dendritic mRNA candidates, which can be arranged into the categories of receptors and channels, signaling molecules, cytoskeleton and adhesion molecules, and factors that are involved in membrane trafficking, in protein synthesis, in posttranslational protein modification, and in protein degradation. Previously known dendritic mRNAs such as MAP2, calmodulin, and G protein gamma subunit were identified from our screening, as were mRNAs that encode proteins known to be important for synaptic plasticity and memory, such as spinophilin, Pumilio, eEF1A, and MHC class I molecules. Furthermore, mRNAs coding for ribosomal proteins were also found in dendrites.

Conclusion: Our results suggest that neurons transport a variety of mRNAs to dendrites, not only those directly involved in modulating synaptic plasticity, but also others that play more common roles in cellular metabolism.

Figure 1

A schematic diagram of the experiment. Stratum pyramidale and stratum radiatum were dissected from hippocampal slices of adult rats. Total RNA extracted from each fraction was reverse primed with a T7 promoter-conjugated oligo-d(T) primer and labeled with either Cyanine 3 or Cyanine 5 through in vitro transcription. Equal amount of labeled probes were mixed and hybridized to a set of five replicates of the Agilent 22 K rat oligonucleotide microarray. Enlarged views of the microarrays are presented showing reproducible hybridization.

Figure 2

The spinophilin mRNA is transported to dendrites. RNA in situ hybridization on rat brain sections using digoxigen-labeled antisense (A) and sense (B) ribo-probes shows that the spinophilin mRNA is located to the dendritic lamina of hippocampal CA1 region (indicated by an arrow). Phase-contrast (C-E) and plain views (F-H) of in situ hybridization using antisense (C,D,F,G) or sense (F,H) spinophilin probes also revealed its dendritic localization in cultured hippocampal neurons (indicated by arrows). In addition, an increase in spinophilin mRNA level was observed after KCl depolarization (D,G).

Figure 3

In situ hybridization showing the dendritic localization of the Arc mRNA (A), rat Pumilio 2 mRNA (B), a putative potassium channel I2RF5 mRNA (C), and secretogranin III mRNA (D). Arrows point to punctate staining in the dendritic lamina of rat hippocampal CA1 region.

Figure 4

Ribosomal protein L9 mRNA is localized in dendrites and is induced by kainate seizure. In situ hybridization using a sense (A) and an antisense (B,C) probe specific for L9 mRNA, showing L9 mRNA is localized in dendrites of pyramidal neurons (indicated by arrows), in addition to its localization in interneuron and glial cell bodies (indicated by arrowheads). An increased level of L9 mRNA has been observed in brain slices of kainic acid treated animals (C).