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AMPA receptors regulate experience-dependent dendritic arbor growth in vivo.

ABSTRACT: The size and shape of neuronal dendritic arbors affect the number and type of synaptic inputs, as well as the complexity and function of brain circuits. In the intact brain, dendritic arbor growth and the development of excitatory glutamatergic synapse are concurrent. Consequently, it has been difficult to resolve whether synaptic inputs drive dendritic arbor development. Here, we test the role of AMPA receptor (AMPAR)-mediated glutamatergic transmission in dendrite growth by expressing peptides corresponding to the intracellular C-terminal domains of AMPAR subunits GluR1 (GluR1Ct) and GluR2 (GluR2Ct) in optic tectal neurons of the Xenopus retinotectal system. These peptides significantly reduce AMPAR synaptic transmission in transfected neurons while leaving visual system circuitry intact. Daily in vivo imaging over 5 days revealed that GluR1Ct or GluR2Ct expression dramatically impaired dendrite growth, resulting in less complex arbors than controls. Time-lapse images collected at 2-h intervals over 6 h show that both GluR1Ct and GluR2Ct decrease branch lifetimes. Ultrastructural analysis indicates that synapses formed onto neurons expressing the GluRCt are less mature than synapses onto control neurons. These data suggest that the failure to form complex arbors is due to reduced stabilization of new synapses and dendritic branches. Although visual stimulation increases dendritic arbor growth rates in control tectal neurons, a weak postsynaptic response to visual experience in GluRCt-expressing cells leads to retraction of branches. These results indicate that AMPAR-mediated transmission underlies experience-dependent dendritic arbor growth by stabilizing branches, and support a competition-based model for dendrite growth.

SUBMITTER: Haas K

PROVIDER: S-EPMC1525049 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Different families of auxiliary subunits regulate the function and trafficking of native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the central nervous system. While a facilitatory role of auxiliary subunits in ER export and forward trafficking of newly synthesized AMPA receptors is firmly established, it is unclear whether auxiliary subunits also control endosomal receptor turnover in dendrites. Here, we manipulated the composition of AMPA receptor complexes in cultured hippocampal neurons by overexpression of two auxiliary subunits, transmembrane AMPAR regulatory protein (TARP) γ-8 or cysteine knot AMPAR-modulating protein (CKAMP) 44a, and monitored dendritic receptor cycling in live-cell imaging experiments. Receptor surface delivery was assayed using a modified AMPA receptor subunit carrying the pH-dependent fluorophore superecliptic pHluorin (SEP-GluA1), which regains its fluorescence during receptor exocytosis, when transiting from the acidic lumen of transport organelles to the neutral extracellular medium. Strikingly, we observed a dramatic reduction in the spontaneous fusion rate of AMPA receptor-containing organelles in neurons overexpressing either type of auxiliary subunit. An analysis of intracellular receptor distribution also revealed a decreased receptor pool in dendritic recycling endosomes, suggesting that incorporation of TARPγ-8 or CKAMP44a in receptor complexes generally diminishes cycling through the endosomal compartment. To directly analyze dendritic receptor turnover, we also generated a new reporter by N-terminal fusion of a self-labeling HaloTag to an AMPA receptor subunit (HaloTag-GluA1), which allows for selective, irreversible staining of surface receptors. Pulse chase-experiments with HaloTag-GluA1 indeed demonstrated that overexpression of TARPγ-8 or CKAMP44a reduces the constitutive internalization rate of surface receptors at extrasynaptic but not synaptic sites. Thus, our data point to a yet unrecognized regulatory function of TARPγ-8 and CKAMP44a, by which these structurally unrelated auxiliary subunits delay local recycling and increase surface lifetime of extrasynaptic AMPA receptors.

Project description:The patterning of dendrites is regulated by many factors, such as brain-derived neurotrophic factor (BDNF), which our laboratory has previously shown alters the dendritic arbor uniquely depending on the mode of extracellular application. In the current work, we examine how BDNF affects dendritogenesis in hippocampal neurons when it is overexpressed intracellularly by transcripts previously reported to be transported to distinct cellular compartments. The BDNF gene is processed at two different polyadenylation sites, leading to mRNA transcription with two different length 3' untranslated regions (UTRs), and therefore, different mRNA localization preferences. We found that overexpression of BDNF mRNA with or without 3' UTRs significantly alters dendritic branching compared to branching in control neurons as analyzed by Sholl distribution curves. Unexpectedly, we found that the overexpression of the shorter BDNF mRNA (reported to be preferentially targeted to the cell body) results in similar changes to Sholl curves compared to overexpression of the longer BDNF mRNA (reported to be preferentially targeted to both the cell body and dendrites). We also investigated whether the BDNF receptor TrkB mediates these changes and found that inhibiting TrkB blocks increases in Sholl curves, although at different distances depending on the transcript's UTR. Finally, although it is not found in nature, we also examined the effects of overexpressing BDNF mRNA with the unique portion of the longer 3' UTR since it was previously shown to be necessary for dendritic targeting of mRNA. We found that its overexpression increases Sholl curves at distances close to the cell body and that these changes also depend on TrkB activity. This work illustrates how the mRNA spatial code affects how BDNF alters local dendritogenesis and how TrkB may mediate these effects. Finally, our findings emphasize the importance of intracellular transport of BDNF mRNAs in the regulation of dendrite morphology.

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AMPA receptors regulate experience-dependent dendritic arbor growth in vivo.

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