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.To integrate synaptic input, neurons develop a specific dendritic branching pattern that determines their function (1). Neuronal activity modifies the formation and stabilization of dendritic processes (2, 3). Dendrites are motile structures that contain high concentrations of filamentous actin. By controlling the stability and assembly of the actin cytoskeleton, members of the Rho family of small GTPases regulate neuronal morphogenesis (4). Rac and Cdc42 facilitate the outgrowth of dendrites, dendritic branches, filopodia, and spines, whereas RhoA and Rho kinase (ROCK) 3 attenuate it (5-9). In Xenopus optic tectal neurons, the neurotransmitter glutamate changes the activity of Rho GTPases by acting on ionotropic NMDA (NMDAR) and L-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors. It facilitates dendrite formation by inhibiting RhoA and activating Rac (10). Because calcium signaling plays an important role in dendrite formation (11,12), mainly the effects of NMDAR stimulation seem to be important. In hippocampal neurons, the guanine exchange factor Tiam1 couples NMDARs to the activity-dependent development by activating Rac1 and inhibiting RhoA (13,14).In vitro, class I phosphatidylinositol 3-kinases (PI3Ks) support neurite formation by producing membrane-bound phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (15-17). PI3Ks stimulate dendrite and branch outgrowth by inhibiting the RhoA/...

Gabapentin is currently used as a therapeutic agent against epilepsy as well as neuropathic pain. In contrast to gabapentin, its derivative gabapentin-lactam has a pronounced neuroprotective activity. We have studied in cultured hippocampal neurons whether gabapentin-lactam has also neurotrophic effects. Gabapentin-lactam enhanced the formation of dendritic filopodia, which are necessary for synapse formation. It also induced a network of F-actin-containing neurites. In studies with time lapse microscopy, gabapentin-lactam increased the addition but also the elimination of new branches. Affinity precipitation assays showed that gabapentin-lactam increased the GTP binding of the small GTPases Rac and Cdc42, which facilitate branch addition. Gabapentin-lactam also activated RhoA and phosphatidylinositol 3-kinases. In neurons transfected with dominant-negative RhoA or treated with the RhoAinactivating C3 toxin, gabapentin-lactam increased the number of dendrites and branches. In the presence of Y-27632, which inhibits Rho kinase, newly added branches induced by gabapentin-lactam were no longer eliminated so that gabapentinlactam increased the number of branches. Y-27632 [(ϩ)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide] also prevented the gabapentin-lactam induced activation of phosphatidylinositol 3-kinases. The phosphatidylinositol 3-kinase inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride] reduced the elimination of newly added branches caused by gabapentin-lactam and thus facilitated branch formation. In contrast to gabapentin-lactam, gabapentin had no effect on dendritic filopodia or motility. The effects exerted by gabapentin-lactam on dendritic arborization may be of potential therapeutic interest.

GluN receptors are heteromers of obligatory GluN1 subunits and GluN2(A-D) subunits. In the present study, we addressed the question whether GluN2A and GluN2B subunits play distinct roles in the formation of filopodia and dendrites during the early development of hippocampal neurons. In hippocampal neurons brought into culture at embryonic day 17, we studied 2-3 days later the effects of N-methyl-D-aspartic acid (NMDA) on the numbers of filopodia, growth cones, and primary as well as secondary dendrites. Antagonists specific for GluN2A and GluN2B subunits were applied together with NMDA. NMDA only induced the formation of filopodia and secondary dendrites. Whereas filopodia were generated within 15 min by NMDA alone, secondary dendrites were only induced by the joint application of NMDA and the Rho kinase inhibitor Y-27632 for 24 h. The GluN2B antagonists ifenprodil and Ro 25-6981 prevented the NMDA-induced formation of filopodia, whereas the GluN2A antagonists NVP-AAM007 and EAA-090 prevented the formation of secondary dendrites. We conclude that both GluN2 subunits have differential roles in dendritic arborization.

Pituitary adenylyl cyclase-activating polypeptide 38 (PACAP38) plays an important role in the proliferation and differentiation of neural cells. In the present study, we have investigated how PACAP38 inhibits the proliferation of cultured neocortical astroglial cells. When applied to synchronized cells during the G 1 phase of the cell cycle, PACAP38 diminished the subsequent nuclear uptake of bromodeoxyuridine. When applied for 2 days, it reduced the cell number. PACAP38 did not exert its antiproliferative effect by activating protein kinase A. It also did not reduce the activity of mitogen-activated protein kinases essential for G 1 phase progression. Instead, PACAP38 acted on a member of the Rho family of small GTPases. It reduced the activity of RhoA as was shown with a Rhotekin pull-down assay. The decrease in endogenous RhoA activity induced by treatment of the cells with C3 exotoxin or by expression of dominant negative RhoA also reduced the nuclear uptake of bromodeoxyuridine. In contrast, expression of constitutively active RhoA prevented the effect of PACAP38. Our data show a novel signal transduction pathway by which the neuropeptide influences cell proliferation.Pituitary adenylyl cyclase-activating polypeptide 38 (PACAP38) 1 and vasoactive intestinal polypeptide (VIP) are neuropeptides of the secretin-glucagon family (1). Whereas both peptides stimulate the G protein-coupled receptors VPAC1 and VPAC2 at subnanomolar concentrations, only PACAP38 stimulates PAC1 receptors with a potency in the same range (2, 3). During the embryonic and postnatal period, the neuropeptides and the receptors are expressed in cells of the rodent neocortex and its growth zones. There is strong evidence for the involvement of these peptides in neural cell proliferation, phenotypic determination, differentiation, and survival (4 -12).Activation of PAC1 receptors by PACAP38 can change the proliferation of neural cells. However, inhibitory as well as stimulatory effects mediated by protein kinase A (PKA) have been observed in cultured neocortical or cerebellar neurons and spinal cord glial cells (3,4,(12)(13)(14)(15)(16). These contradictory effects may be related to the diversity in receptor isoforms and their signal transduction pathways. The 7 isoforms reported differ in their third intracellular loop and can couple to adenylyl cyclase as well as phospholipases C and D (12, 17).In the cellular proliferation cycle, cyclins and cyclin-dependent kinases (CDKs) determine the progression through the G 1 phase into the S phase. Thus, the association of cyclin D with CDK4/6 and of cyclin E with CDK2 is important for G 1 progression and G 1 /S transition, respectively. The complexes induce the phosphorylation of the retinoblastoma gene product that allows the transcription of E2F-regulated genes (18). Upon activation, extracellular signal-related kinases 1 and 2 (ERK1 and ERK2) increase the activity of cyclin D1. However, only the sustained activation of ERKs and cyclin D1 leads to G 1 progression (19,20). In primary cells, such a ...

In cells cultured from neocortex of newborn rats, phosphoinositide-3-kinases of class I regulate the DNA synthesis in a subgroup of astroglial cells. We have studied the location of these cells as well as the kinase isoforms which facilitate the S phase entry. Using dominant negative (dn) isoforms as well as selective pharmacological inhibitors we quantified S phase entry by nuclear labeling with bromodeoxyuridine (BrdU). Only in astroglial cells harvested from the marginal zone (MZ) of the neocortex inhibition of phosphoinositide-3-kinases reduced the nuclear labeling with BrdU, indicating that neocortical astroglial cells differ in the regulation of proliferation. The two kinase isoforms p110α and p110β were essential for S phase entry. p110α diminished the level of the p27Kip1 which inactivates the complex of cyclin E and CDK2 necessary for entry into the S phase. p110β phosphorylated and inhibited glycogen synthase kinase-3β which can prevent S-phase entry. Taken together, both isoforms mediated S phase in a subgroup of neocortical astroglial cells and acted via distinct pathways.