Abstract
Dendrite morphogenesis is highly dynamic and characterized by the addition and elongation of processes and also by their selective maintenance, retraction, and elimination. Glutamate can influence these events via N-methyl-d-aspartic acid (NMDA) receptors. The neuropeptides vasoactive intestinal peptide and pituitary adenylyl cyclase-activating polypeptide-38 (PACAP38) affect neurogenesis and differentiation in the developing nervous system. We report here that the peptides and NMDA acted synergistically on dendrite and branch formation. In stage III hippocampal neurons, NMDA increased not only the addition but also the elimination of new dendrites and branches by activating Rac and Cdc42 and phosphatidylinositol 3-kinases, respectively. When applied alone, the neuropeptides did not influence dendrite or branch formation. However, they reduced the elimination of newly formed dendrites and branches caused by NMDA by preventing the NMDA-induced activation of phosphatidylinositol 3-kinases. This led to the formation of persistent dendrites and branches. Additional timelapse studies on the dynamics of dendrite elongation showed alternating periods of elongation and retraction. Phosphatidylinositol 3-kinases increased the velocities of dendrite elongation and retraction, whereas the neuropeptides prolonged the periods of elongation. By modifying NMDA-induced activation of Rho GTPases and phosphatidylinositol 3-kinases, vasoactive intestinal peptide and PACAP38 could play an important role in the control of dendrite growth and branching during development and in response to neuronal activity.
Highlights
Dendrites, dendritic branches, filopodia, and spines, whereas RhoA and Rho kinase (ROCK)3 attenuate it (5–9)
N-methyl-D-aspartic acid (NMDA) and Vasoactive intestinal polypeptide (VIP)/pituitary adenylyl cyclase-activating peptide-38 (PACAP38) Enhance the Activities of Rac and Cdc42—Because Rac and Cdc42 are essential in glutamate-induced dendrite and branch formation (10), we studied how NMDA changed the GTP binding of Rac and Cdc42 in the hippocampal neurons
VIP and Y-27632 Inhibit the NMDA-induced phosphatidylinositol 3-kinases (PI3Ks) Activation—Because VIP and Y-27632 reduced the elimination of new branches generated by NMDA, we examined whether they affected PI3K activity
Summary
Materials—NMDA and LY294002 were from Tocris (Koln, Germany). Y-27632, H89, KN93, KN92, nimodipine, and dizocilpine (MK801) were from Calbiochem, and wortmannin was from Sigma. For F-actin staining, cells were incubated with Alexa 594-conjugated phalloidin (Molecular Probes, Heidelberg, Germany) and washed with PBS. Time-lapse Light Microscopy—To determine the dynamics of branch formation as well as dendrite elongation, hippocampal neurons were plated on poly-L-lysine-coated glass-bottom microwell dishes (MatTek, Ashland, MA) and used for timelapse light microscopy. Morphometry of Fixed Cells—The dendrites were distinguished from the axon of a stage III hippocampal neuron by use of morphological criteria (36). To quantify the number of dendrites, their branches, and the dendritic length, we took images of the phalloidin staining for F-actin and of the -tubulin III staining. The hippocampal culture contained less than 1% astrocytes, as determined with glial fibrillary acidic protein immunohistochemistry for astrocytes and -tubulin III immunohistochemistry for neurons. If samples showed normal distribution, analysis of variance and Scheffe’s test or Student’s t test were used
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
