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Developing sensory axons grow into the spinal cord in a three step process: the axons extend toward and into the cord, then branch rostrally and caudally to establish a longitudinal pathway, and finally grow into the grey matter. This study investigated regulation by cAMP of the longitudinal extension of this pathway within the spinal cord. The cAMP pathway was pharmacologically altered in chicken embryos to determine its effects on establishing the longitudinal extension of the dorsal funiculus. A forskolin-induced increase in cAMP in ovo inhibited longitudinal growth by sensory afferents. Furthermore, blocking cAMP activation of Protein Kinase A (PKA) in ovo with H-89 substantially increased longitudinal extension. These results demonstrate a specific role for the cAMP/PKA pathway in the initial longitudinal spinal afferent growth in the chicken embryo.
Somatosensory information from the body reaches the central nervous system (CNS) via primary spinal afferents that extend from the dorsal root ganglion (DRG) and enter the spinal cord in the dorsal root entry zone (DREZ) at each segmental level. These afferents contact neurons in the grey matter at the segment of entry and at several segments rostral and caudal to entry. The rostrocaudal extension of sensory afferents is important for the intersegmental communication that enables appropriate perception and reflexes (Sprague, 1958). During development, the longitudinal extension of afferents within the spinal cord occurs prior to the simultaneous extension of collateral branches into the grey matter at multiple segments (Davis et al., 1989; Eide and Glover, 1995; Ozaki and Snider, 1997). The precise mechanisms that regulate longitudinal extension of primary afferents in the CNS are unknown.
Extracellular cues such as neurotrophins and axon guidance molecules bind to receptors on the growth cones of developing axons and act as attractive and repulsive cues that guide the axon to its final destination (Giger and Kolodkin, 2001; Huang and Reichardt, 2001; Tessier-Lavigne and Goodman, 1996). Cyclic nucleotides can have both attractive and repulsive effects, depending on the concentration, although they are neither sufficient attractants nor repellents on their own. High concentrations of cyclic nucleotides support an attractive turning response, while low concentrations support a repulsive turning response (Ming et al., 1997; Song et al., 1997; Song and Poo, 1999). In Xenopus, for example, DRG axons with higher levels of intracellular cyclic guanine monophosphate (cGMP) are less responsive to repellent effects of semaphorin 3A (sema3A) (Chalasani et al., 2003). In animal injury models, alterations in cyclic adenosine monophosphate (cAMP) signaling can also enable re-growth of sensory afferents in the rat spinal cord by overcoming myelin-induced inhibition of regeneration (Qiu et al., 2002).
Previous studies investigating sensory axon outgrowth have reported a range of growth rates depending on the system and whether the studies were conducted in vivo or in vitro. Davies (1987) calculated a growth rate of 20 μm per hour for the peripheral processes of mammalian trigeminal axons in vivo. Embryonic chicken DRG cells in culture have been shown to extend axons at varying rates depending on the substratum, with laminin supporting a rate of approximately 54 μm/hour, while growth on polylysine progresses more slowly at approximately 36 μm/hour (Ketschek et al., 2007). The in vivo rate of growth of DRG cell axons during the initial establishment of the longitudinal pathway in the CNS has not been quantified.
The present study investigated the longitudinal growth of spinal sensory afferent fibers in chicken embryos in the presence of altered cAMP activity induced by forskolin and H-89. Forskolin causes a direct increase in intracellular cAMP by bypassing the membrane-bound receptor to activate adenylyl cyclase directly (Seamon and Daly, 1981). Alternatively, the downstream effect of cAMP can be blocked by inhibiting activation of Protein Kinase A (PKA) with H-89 (Hidaka and Kobayashi, 1992). The use of chicken embryos in ovo provided precise control of the temporal component of pharmacological alterations. This system ensures that the treatment was applied after the afferents reached the cord, during the time that the intersegmental reflex pathways are initially forming, and before the final projection into the grey matter. Thus the results speak specifically to the longitudinal extension of spinal sensory afferents as they initially extend to establish the dorsal funiculus of the spinal cord.
Fertilized eggs (White Leghorn chicken, Oliver Merrill & Sons) were stored at 4°C until incubation at 37.5°C. To determine the stage at which thoracic primary afferents reach the spinal cord, eggs were removed at various intervals of incubation and embryonic stage was assessed by somite number. For all treated embryos, eggs were removed after three days of incubation (E3) and windowed by cutting a small hole above the air cell, exposing the embryo and piercing the inner membrane to create a drug entrance area. The hole was covered with tape except during drug application. The eggs were divided into groups that were assigned to specific drug concentrations for the remainder of the trial.
Drug concentrations are described in molarity prior to application, instead of molarity within the egg, because the distribution of the treatment within the contents of the egg may not have been homogenous. Initial forskolin (Sigma) concentrations were based on those found in the literature (Nishikawa et al., 1989; DeFouw and DeFouw, 1999), and adjusted for in ovo application. Survival rates were determined to decide experimental dosages. The solubility limit of forskolin in 5% DMSO solution is listed by Sigma© as 0.2mM. We used a concentration close to the solubility limit that produced an effect on survival without causing complete lethality: 0.18 mM in a vehicle of 5% Dimethyl Sulfoxide (DMSO, Sigma)/95% saline (0.9% NaCl). H-89 (Calbiochem) was diluted to 25μM in a vehicle of 5%DMSO/95% saline. Both groups were compared to vehicle-treated embryos. DMSO was used for solubility and greater cellular permeability.
Each egg received five 100μL applications of solution of its assigned drug concentration. The five drug applications were administered to each egg at twelve hour intervals starting on the morning of E4 and ending on the morning of E6. For each round of applications, the eggs were moved from their incubator to a temporary work-space incubator. After all eggs received drug or vehicle applications, they were returned to the regular incubator.
Embryos (E2–E4) were killed and fixed in 4% paraformaldehyde (Fisher) overnight at 4ºC. Embryos were then embedded in 1.5% agarose and sectioned transversely at 200uM on a vibratome. A capillary pipette containing 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes) was transiently placed in the DRG on each side of the thoracic spinal cord. The sections were then placed into a depression well and coverslipped in a mounting solution of 50 % glycerol/4% paraformaldehyde (pH 10). The sections were incubated overnight at 37ºC to allow the dye to travel and subsequently imaged on a BioRad Multiphoton Microscope. The z series images were then flattened using ImageJ and measurements were taken with LSM Browser.
E6 embryos were killed two hours following the final drug application; embryos designated as E7 dissections were sacrificed 24 hours after the final drug application. Embryo development was assessed to determine the developmental stage at sacrifice according to Hamburg and Hamilton (1951) staging criteria. On the assigned day, the embryos were decapitated in cold, oxygenated chicken Ringer’s physiological saline solution and eviscerated so that only the wings, legs, and trunk remained. Sensory afferent fibers were labeled with either fluorescein dextran amine (FDA; Molecular Probes) or DiI.
FDA labeling occurred in in vitro preparations of the thoracic region of the embryos on the day of dissection, as previously described (Forehand et al., 1994). Briefly, peripheral spinal nerves in a thoracic segment (third to fifth) were severed at the lateral edge of the dorsal root ganglia and FDA was applied to the transected nerve. The corresponding left and right sides of the embryo were labeled and counted separately. The tissue was placed in a bath of circulating oxygenated chicken Ringer’s solution at room temperature for 16–19 hours to allow the fluorescent tag to travel. The tissue was then fixed with 4% paraformaldehyde for four to six hours and cryoprotected in 30% sucrose until being embedded and frozen in OCT. The frozen tissue was cryosectioned transversely at 30 microns for analysis. Axonal length was measured by counting transverse sections that contained labeled axons. The longitudinal extent of labeled DRG was also assessed to determine whether treatment altered development of the DRG. No difference in DRG extent was observed in any of the treatment groups.
Two methods of labeling with DiI were used to assess axonal trajectories in the longitudinal plane. The first was performed based on methods of Ozaki and Snider (1997). Briefly, embryos were fixed in 4% paraformaldehyde, and injected with a supersaturated solution of DiI between the third and fifth thoracic segments using a picospritzer and a capillary pipette. The labeled embryonic tissue was placed in a 37ºC incubator for one week. Spinal cords were then removed, embedded in 1.5% agarose, and sectioned longitudinally on a vibratome at 700μm; sections were placed in depression well slides and coverslipped with glycerol/paraformaldehyde medium (pH 10). A second method involved removing the notochord after embryo fixation, transiently inserting a capillary pipette containing DiI into DRG between the third and fifth thoracic segment. Unsectioned tissue was placed in a depression well slide and coverslipped in glycerol/paraformaldehyde medium (pH 10) and incubated for one week at 37ºC. Tissue from both labeling methods was imaged with a BioRad Multiphoton Microscope. The z series images were flattened using ImageJ and reassembled in Adobe Photoshop.
Groups were compared using One-Way Analysis of Variance (ANOVA). When the ANOVA indicated a significant difference, Tukey’s Post Test (two-tailed) was used to determine which groups were significantly different from each other. The calculations were made using Prism Scientific Graphing, Curve-fitting and Statistics software from GraphPad (version 4).
The current study sought to assess longitudinal extension of the developing dorsal funiculus. In order to accurately address this issue, treatments had to be applied after the axons had reached the cord, branched, and begun to establish a longitudinal pathway. DiI labeling indicated that thoracic DRG axons that penetrate the cord to establish the dorsal root entry zone (DREZ) reach the cord at Hamburger and Hamilton (1951) stage 19, on the 3rd day of incubation (Fig 1). This time point is consistent with reports of E3 in the cervical cord (Fraher et al., 2007) and E4 in lumbar cord (Eide and Glover, 1995). Given the variation in embryonic age within the same period of incubation, the optimal time to start treatment was determined based on the time of incubation when the majority of embryos were older than stage 19. The percentage of embryos stage 20 or older at 78 hours of incubation was 0 (n=5), 50% at 89 hours of incubation (n=8), and 60% at 97 hours of incubation (n=5). At 97 hours of incubation, only one out of 5 embryos was younger than stage 19. Treatments were subsequently started between 98 and 100 hours of incubation.
Treated embryos that survived appeared morphologically normal. Although cardiovascular abnormalities have been reported in chicken embryos following treatments with forskolin (Nishikawa et al., 1989), the dosages used in this study were lower than those in previous reports and did not appear to cause any major cardiovascular malformations by E6. However, cardiovascular abnormalities may have contributed to embryo toxicity for embryos that did not survive treatment. It was noted that blood vessels in the surrounding yolk appeared healthier and brighter in color in the H-89 treated embryos than with either the vehicle-treated or forskolin-treated embryos, though there were no apparent embryonic malformations in the heart or embryonic vasculature. The survival of forskolin-treated embryos was decreased in a dose-dependent manner (Fig. 2A). Only one dose of H-89 was used; survival of embryos treated with H-89 was not different from that of vehicle-treated controls (p>0.05, data not shown). No developmental lag was noted in surviving embryos in any of the treatment groups (Fig. 2B).
The longitudinal length of primary afferents was measured by counting the number of transverse sections (30μm) containing labeled axons in the dorsal funiculus (Fig 3). Significant changes in length of longitudinal extension of sensory spinal axons assayed by FDA labeling were seen with altered cAMP levels in the E6 chicken embryo (Fig. 4A). In the presence of increased cAMP levels induced by five 100μL applications of 0.18 mM forskolin, longitudinal extension was decreased approximately two-fold compared to vehicle control-treated embryos. The total longitudinal projection within the spinal cord was 55% shorter than that in vehicle controls (p = 0.036); growth in the rostral direction was 59% shorter (p = 0.010) and growth in the caudal direction was 55% shorter, but not statistically significant (p = 0.12).
The effects of enhanced cAMP persisted when forskolin treatments were stopped at E6 and embryos were allowed to develop until E7 (Fig. 4B). Cessation of treatment did not correct total longitudinal axon extension, which remained 57% shorter in forskolin-treated embryos compared to vehicle controls (p = .010). However, the E7 embryos showed significant shortening in both the rostral (49% shorter, p = 0.047) and caudal (67% shorter, p = 0.004) directions. On average, control embryos extended 869.4μm from E6 to E7, whereas forskolin-treated embryos only increased 327.3μm from E6 to E7. The E7 embryos were killed exactly 22 hrs after the E6 embryos. Thus a calculated average rate of extension between E6 and E7 is 39.5 μm per hour for vehicle-treated embryos and 14.9 μm per hour in forskolin-treated embryos.
To determine whether endogenous cAMP regulates the longitudinal projections of sensory afferents, H-89 was used to block endogenous cAMP from E4 to E6 in the chicken embryo. Embryos that received the five 100μL treatments of 25 uM H-89 were evaluated at E6. Total, rostral, and caudal longitudinal extension of the sensory afferents in the dorsal funiculus were significantly increased with H-89 treatments compared to vehicle controls by 46% (p = 0.025), 31% (p = 0.047), and 64% (p = 0.037), respectively (Fig. 4A). These results support the hypothesis that cAMP inhibits longitudinal axon extension of developing sensory afferents through the activation of PKA.
Drug treatments were initiated after axons had entered the DREZ and bifurcated to begin longitudinal growth, but the length of axons in the longitudinal funiculus at the time of first drug treatment is not known. However, relative maximum rates of extension of sensory axons in the longitudinal funiculus can be estimated assuming minimal longitudinal growth prior to treatment. At E6, exactly 50 hours after initial drug treatment, vehicle-treated axons had traversed 1122 μm, forskolin-treated axons 522 μm and H-89-treated axons 1905 μm. Calculated maximum rates of axon growth from E4 to E6 are thus 22 μm/hour for vehicle-treated axons, 10 μm/hour following forskolin treatment and 38 μm/hour following H-89 treatment.
In vitro labeling of longitudinal axon extension with FDA is a robust labeling technique that is easily quantified by counting the number of transverse sections traversed by the labeled axons. In the transverse plane, there was no indication of abnormal positioning of the DREZ or the dorsal funiculus, nor was there evidence of abnormal axonal projection out of the DREZ or dorsal funiculus among any of the treatment groups (data not shown). However, sectioning in the transverse plane is less than optimum for examining axon trajectory along the longitudinal extension, which is better visualized in whole mount preparations. Imaging the FDA label in whole mounts is difficult due to contamination with migratory macrophages that ingest the FDA in the live-labeling paradigm. Therefore, DiI labeling in fixed tissue was used to assess the trajectory of sensory axons in the longitudinal plane imaged in whole mounts or in 700 μm vibratome sections with multiphoton microscopy. In this technique, macrophages are not labeled (or migratory), but the labeling is difficult to visualize to the tips of the axons in these thick specimen due to sensitivity and resolution of the imaging technique. Thus this technique was used to assess longitudinal trajectory, but not for measurements. Imaging the whole mount DiI-labeled specimen demonstrated that axons travel in a predominantly linear pathway within the dorsal funiculus regardless of treatment (Fig. 5).
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.
Grant Sponsor: NIH; Grant Numbers: NS42816 and P20 RR16435.
We thank Jennifer Jaskolka, Francina Deason, and Lauren Arms for technical assistance, Victor May for advice regarding working concentrations of H-89, and Lynne Bianchi and Ralph Budd, for critical reading of the manuscript. The current address for Jaeda Coutinho-Budd is: Curriculum in Neurobiology, Campus Box #7320, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7320.