Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 Mar 15;23(6):2274-83.
doi: 10.1523/JNEUROSCI.23-06-02274.2003.

Direct cAMP signaling through G-protein-coupled receptors mediates growth cone attraction induced by pituitary adenylate cyclase-activating polypeptide

Carmine Guirland  1 , Kenneth B Buck, Jean A Gibney, Emanuel DiCicco-Bloom, James Q Zheng
Affiliations

Direct cAMP signaling through G-protein-coupled receptors mediates growth cone attraction induced by pituitary adenylate cyclase-activating polypeptide

Carmine Guirland et al. J Neurosci. .

Abstract

Developing axons are guided to their appropriate _targets by environmental cues through the activation of specific receptors and intracellular signaling pathways. Here we report that gradients of pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide widely expressed in the developing nervous system, induce marked attraction of Xenopus growth cones in vitro. PACAP exerted its chemoattractive effects through PAC1, a PACAP-selective G-protein-coupled receptor (GPRC) expressed at the growth cone. Furthermore, the attraction depended on localized cAMP signaling because it was completely blocked either by global elevation of intracellular cAMP levels using forskolin or by inhibition of protein kinase A using specific inhibitors. Moreover, local direct elevation of intracellular cAMP by focal photolysis of caged cAMP compounds was sufficient to induce growth cone attraction. On the other hand, blockade of Ca2+, phospholipase C, or phosphatidyl inositol-3 kinase signaling pathways did not affect PACAP-induced growth cone attraction. Finally, PACAP-induced attraction also involved the Rho family of small GTPases and required local protein synthesis. Taken together, our results establish cAMP signaling as an independent pathway capable of mediating growth cone attraction induced by a physiologically relevant peptide acting through GPCRs. Such a direct cAMP pathway could potentially operate in other guidance systems for the accurate wiring of the nervous system.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Expression of PACAP-selective receptors inXenopus neurons and neurite outgrowth. a,b, Fluorescent images of Xenopus neurons stained using a specific antibody against PAC1 receptors. Two images were acquired using a 20× objective (a) and a 40× objective (b). The asteriskin a marks a muscle cell without PAC1 expression, andarrows indicate intense PAC1 staining at the growth cone. c, Fluorescent staining of the plasma membrane of a neuron using DiIC18. Scale bars, 20 μm.d, Box and whisker plots of neurite outgrowth in the presence of control medium and media containing PACAP and VIP, respectively.
Fig. 2.
Fig. 2.
Attractive turning of growth cones induced by PACAP gradients. a, b, DIC images of representative growth cones that responded to the PACAP (a) and VIP (b) gradients. The concentration of PACAP or VIP in the pipette was 1 μm. Asterisks indicate the application pipette. Dashed lines indicate the original direction of growth cone extension, and dotted lines represent the corresponding position of the growth cone at the onset of the gradient application. Scale bar, 50 μm. c, Superimposed traces of the trajectory of neurite extension during the 30 min turning assay for a sample population of 15 neurons for each condition. The origin is the center of the growth cone at the onset of the gradient, and the original direction of growth cone extension was vertical.Arrows indicate the direction of the gradient.d, Scatter plots depict all data collected for each condition. Each point depicts final angular position of a growth cone (abscissa) and its total net neurite extension (ordinate) during the 30 min assay period.e, Cumulative histogram shows the distribution of the turning angles for each condition. Each point represents the percentage of the growth cones with final turning angles of equal or smaller values. Attractive turning response is represented by the distribution being shifted toward positive turning angles.f, Average turning angles of different groups of growth cones exposed to control, VIP, PACAP, and maxadilan, the PAC1-specific agonist. The values on the abscissarepresent the concentrations (in micromolar) used in the pipette.
Fig. 3.
Fig. 3.
cAMP signaling in PACAP-induced growth cone attraction. a, Superimposed traces of the trajectory of neurite extension during the 30 min turning assay in a PACAP gradient (1 μm in pipette) for a sample population of 15 neurons with bath application of 50 μm Rp-cAMP, 200 nm KT 5720, and 10 μm forskolin. The origin is the center of the growth cone at the onset of the gradient, and the original direction of growth cone extension was vertical.Arrows indicate the direction of the gradient.b, Scatter plots depict all data collected for each condition. Each point depicts final angular position of a growth cone (abscissa) and its total net neurite extension (ordinate) during the 30 min assay period.c, Cumulative histogram shows the distribution of the turning angles for each bath application experiment. Each point represents the percentage of the growth cones with final turning angles of equal or smaller values.
Fig. 4.
Fig. 4.
Growth cone turning induced by focal photoactivated release of caged cAMP. a, Representative DIC images showing a control growth cone (not loaded with caged cAMP) exposed to repetitive UV illumination (50 msec duration, every 10 sec). The dotted circles indicate the position and size of the UV illumination. b, Representative DIC images showing a growth cone exposed to focal photoactivated release of caged cAMP. Scale bar, 10 μm. c, d, Superimposed traces of the trajectory of neurite extension of neurons during the 30 min repetitive focal UV illumination without (c) and with (d) caged cAMP loaded. Tick marks represent 10 μm. The three-dimensional plot depicts the fluorescence intensity generated by the focal photolysis of caged fluorescein-dextran and illustrates the spatial gradient of focal uncaging. e, Average turning angles of groups of growth cones exposed to control UV illumination and focal cAMP release.
Fig. 5.
Fig. 5.
Examination of Ca2+, PLC, and PI-3 signaling pathways in PACAP-induced growth cone attraction.a, Ca2+ responses ofXenopus neurons to bath-applied PACAP at 1 or 100 nm final concentrations. The fura-2 ratio (340/380 nm excitation) was normalized against the average value from the control recording period. Error bars represent SD. b,c, Cumulative histograms represent the distribution of growth cone turning angles to glutamate gradients (b) or PACAP gradients (c).Symbols indicate whether growth cones were preloaded with BAPTA (+BAPTA) to buffer [Ca2+]i changes or treated with 15 μm LY-294002 (+LY) to inhibit PI-3 kinase or with 10 μmU73122 (+U73122) to inhibit PLC.
Fig. 6.
Fig. 6.
Involvement of Rho GTPases and local protein synthesis in PACAP-induced growth cone attraction.a–c, Scatter plots depict all data collected for growth cones exposed to bath application of toxin B (100 pg/ml) (a), anisomycin (Aniso; 40 μm) (b), and cycloheximide (Cyclo; 25 μm) (c). For all three conditions, a PACAP gradient (1 μm PACAP in pipette) was used to induce turning. Each point depicts the final angular position of a growth cone (abscissa) and its total net neurite extension (ordinate) during the 30 min assay period.d, The cumulative histogram shows the distribution of the turning angles for each bath application experiment. Eachpoint represents the percentage of the growth cones with final turning angles of equal or smaller values.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Arimura A. Pituitary adenylate cyclase activating polypeptide (PACAP): discovery and current status of research. Regul Pept. 1992;37:287–303. - PubMed
    1. Bolsover SR, Gilbert SH, Spector I. Intracellular cyclic AMP produces effects opposite to those of cyclic GMP and calcium on shape and motility of neuroblastoma cells. Cell Motil Cytoskeleton. 1992;22:99–116. - PubMed
    1. Buck KB, Zheng JQ. Growth cone turning induced by direct local modification of microtubule dynamics. J Neurosci. 2002;22:9358–9367. - PMC - PubMed
    1. Campbell DS, Holt CE. Chemotropic responses of retinal growth cones mediated by rapid local protein synthesis and degradation. Neuron. 2001;32:1013–1026. - PubMed
    1. Chartrel N, Tonon MC, Vaudry H, Conlon JM. Primary structure of frog pituitary adenylate cyclase-activating polypeptide (PACAP) and effects of ovine PACAP on frog pituitary. Endocrinology. 1991;129:3367–3371. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources

  NODES
twitter 2