Previous studies focus on the growth from the DRG to the cord, or the projection of these fibers into the grey matter. Accordingly, there is much less analysis on the “waiting period” between those two events. However, this waiting period is not a stagnant period. The present study investigated whether cAMP activity regulates the longitudinal extension during the waiting period between axonal entry into the CNS and extension into the grey matter. These data implicate a role for cAMP in regulating the extension of sensory afferents in the developing spinal cord, namely that forskolin causes a decrease in longitudinal extension while H-89 in turn increases longitudinal extension. Our data further demonstrate that cAMP mediates this inhibition of sensory spinal axonal extension through downstream activation of PKA.
The concentration of intracellular cyclic nucleotides, namely cAMP and cGMP, has been shown to modulate the effects of chemoattractants and chemorepellants on axonal development (Nishiyama et al., 2003
; Song et al., 1997
; Song and Poo, 1999
). By pharmacologically altering the concentration of intracellular cAMP, we observed differences in longitudinal extension of sensory spinal axons with a decrease in rostrocaudal extension due to forskolin. Alternatively, inhibiting PKA and creating the effect of diminished endogenous intracellular cAMP led to greater longitudinal growth.
These findings complement studies that indicate membrane-bound G-protein coupled receptors play a role in sensory spinal outgrowth. Watanabe et al. (2006)
, attribute aspects of the waiting period of primary afferent development to inhibitory cues from transient dorsally derived netrin-1. Correspondingly, Chalasani et al. (2003)
show that the chemokine SDF-1 and its activation of the G-protein coupled receptor CXCR4 modulates murine DRG axonal responsiveness to chemorepellants such as netrin-1, sema3A and slit-2. Slit/Robo signaling has been shown to specifically affect the guidance of one of the two branches of sensory axons as they bifurcate upon entry into the spinal cord (Ma and Tessier-Lavigne, 2007
). Kreibich et al. (2004)
describe activation of the glutamate receptor, mGluR1, and its reversal of DRG axon repulsive turning response to sema3A. Activation of this metabotropic glutamate receptor leads to an increase in intracellular cAMP. In our study, forskolin was used in ovo to bypass this type of membrane-bound receptor to induce intracellular production of cAMP. Forskolin has been reported to stimulate adenylyl cyclase (AC) subtypes AC1–AC8, weakly stimulate AC9, and have no effect on the soluble subtype SAC. AC1–AC9 subtypes are present in the nervous system (Sunahara and Taussig, 2002
). For that reason, the addition of forskolin is less specific than the activation of any one type of G-protein coupled receptor; therefore, forskolin can have a greater impact on cellular signaling by mimicking the effects of stimulation of many different types of G-protein coupled receptors simultaneously. Consequently, H-89, which also operates in a ubiquitous fashion, verified that the abnormal growth effect of elevated cAMP was functioning through the downstream target PKA. These findings are consistent with Kreibich et al. (2004)
, who showed that mGluR1 activation in the mouse spinal cord activates PKA. The blockade of the effect of endogenous cAMP through the inhibition of PKA produced the opposite outcome to that of increased cAMP.
There are multiple types of chemoattractants and chemorepellants present in and around the spinal cord, in addition to netrins and semaphorins, which could potentially be affected by an alteration of cAMP levels. Such a widespread activation of the cAMP pathway could augment any or all of these attractants and repellants, leading to abnormal growth in the sensory spinal pathway. Liu and Halloran (2005)
show that knock-down of the glycosyl-phosphatidylinositol-linked (GPI-linked) membrane protein TAG-1 retards the growth of centrally extending axon branches of Zebrafish Rohon-Beard neurons. While control axons show a steady advance at the rate of approximately 24μm/h, axons in TAG-1 knock-down embryos slow to the rate of approximately 10μm/h. Interestingly, these axons do not advance at a steady rate, although they are still highly active with many advances and retreats. The decrease in growth rate is due to the aberrant extensions and backtrackings of the axon, rather than merely slower extension. A similar model could explain the decreased length of axons in our forskolin-treated embryos.
Previous studies investigating sensory axon outgrowth have reported a range of growth rates ranging from 20 μm/hour for embryonic peripheral trigeminal axons in vivo (Davies 1987
) to ~40 μm/hour for DRG cells that were cultured and then transplanted into the adult CNS (Davies et al., 1997
). Embryonic chicken DRG cells in culture vary their rate of growth depending on the substratum, with laminin supporting a rate of approximately 54 μm/hour, and polylysine supporting a rate of approximately 36 μm/hour (Ketschek et al., 2007
). Disruption of myosin II function with blebbistatin in these cultures decreased the rate of extension on laminin, but increased the rate of extension on polylysine (Ketschek et al., 2007
). Control of rate of axon extension in the embryo is likely to be complex and depend upon varying substrates and dynamic molecular interactions as the axons extend. The estimated rates of extension calculated from our data of sensory axons extending in vivo are similar to the range of reported data from other studies. The mechanism underlying the diminished growth following forskolin treatment and the enhanced growth following H-89 treatment are unknown. These drugs may act directly on the neurons or may alter the substrate upon which they extend.
The results of our study contrast the findings of injury and regeneration models. Qiu et al. (2002)
attribute the regeneration of lesioned dorsal column axons in the spinal cord to an elevation of cAMP. The elevated cAMP facilitates regeneration through the reversal of regeneration inhibition by myelin and myelin associated glycoprotein (MAG). Unlike the adult animal regeneration model, the embryonic chicken at E6 and E7 has not yet undergone myelination of the spinal cord; this process does not occur until around E13 (Keirstead et al, 1992
). In our study, myelin/MAG repulsion does not exist, and therefore sensory spinal growth is not affected by the same factors and may be regulated by using alternative mechanisms. In addition, our study examined the initial growth of sensory afferents, rather than their regeneration.
We have presented evidence that cAMP plays a critical role in longitudinal extension of spinal sensory afferents in the chicken embryo through the activation of PKA. These findings are consistent with previous studies that have shown that increased cAMP switches the repulsive turning response into an attractive response. Based on our data we propose a role for cAMP/PKA controlled extension of sensory afferents in the spinal cord of the chicken embryo. This regulation provides insight into normal embryonic sensory development and the possibility of manipulation to correct for poorly developing reflex pathways and perception of sensory information. The study also suggests that mechanisms that guide initial growth are different from those after injury in an adult.