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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Biomed Mater Res A. Author manuscript; available in PMC 2017 August 1.
Published in final edited form as:
PMCID: PMC5258117
NIHMSID: NIHMS842661

Intracellular calcium and cyclic nucleotide levels modulate neurite guidance by microtopographical substrate features

Abstract

Objective

Micro- and nano-scale surface features have emerged as potential tools to direct neurite growth into close proximity with next generation neural prosthesis electrodes. However, the signaling events underlying the ability of growth cones to respond to topographical features remain largely unknown. Accordingly, this study probes the influence of [Ca2+]i and cyclic nucleotide levels on the ability of neurites from spiral ganglion neurons (SGNs) to precisely track topographical micropatterns.

Approach

Photopolymerization and photomasking were used to generate micropatterned methacrylate polymer substrates. Dissociated SGN cultures were plated on the micropatterned surfaces. Calcium influx and release from internal stores were manipulated by elevating extracellular K+, maintenance in calcium-free media, or bath application of various calcium channel blockers. Cyclic nucleotide activity was increased by application of cpt-cAMP or 8-Br-cGMP.

Main results

Elevation of [Ca2+]i by treatment of cultures with elevated potassium reduced neurite alignment to physical microfeatures. Maintenance of cultures in Ca2+-free medium or treatment with the non-selective voltage-gated calcium channel blocker cadmium or L-type Ca2+ channel blocker nifedipine did not signficantly alter SGN neurite alignment. By contrast, ryanodine or xestospongin C, which block release of internal calcium stores via ryanodine-sensitive channels or inositol-1,4,5-trisphosphate receptors respectively, each significantly decreased neurite alignment. Cpt-cAMP significantly reduced neurite alignment while 8-Br-cGMP significantly enhanced neurite alignment.

Conclusion

Manipulation of [Ca2+]i or cAMP levels significantly disrupts neurite guidance while elevation of cGMP levels increases neurite alignment. The results suggest intracellular signaling pathways similar to those recruited by chemotactic cues are involved in neurite guidance by topographical features.

Keywords: calcium, cyclic nucleotide, nerve guide, spiral ganglion neuron, surface topography, micropatterning, photopolymerization

1. Introduction

Precise spatial targeting of neurite growth is critical to the formation of functional neuronal circuits during neural development or regeneration. Analogously, precisely directing de novo neurite growth into close proximity, or even contact, with stimulating or recording electrodes will enable significant performance improvements in next generation neural prosthetics. Neurite pathfinding is accomplished as the growth cone senses and responds to a range of environmental information including biochemical and biophysical (e.g. topographical) cues. Accordingly, neural engineers have sought to create specific patterns of neural guidance cues to precisely guide neurite growth toward desired targets. For example, gradients or patterns of bioactive proteins that influence growth cone turning are used to specify the trajectory of neurite regeneration 13.

More recently, micro- and nano-scale surface topographical features have emerged as potential tools to control neurite growth, neural polarity and circuitry 413. For example, microtopographical features generated by photopolymerization of methacrylate polymer systems direct neurite growth of spiral ganglion neurons (SGNs), trigeminal ganglion neurons, dorsal root ganglion neurons, cerebellar granular neurons, and PC-12 cells 12,1416. However, unlike the mechanisms underlying biochemical guidance of neurite growth that have been thoroughly investigated 17,18, our understanding of how growth cones sense topographical features and transduce those cues into directed neurite growth is limited 19,20. Recently, microtopographical features were shown to activate TRPV1 channels and, subsequently, RhoA signaling pathways to direct neurite growth 21.

Accumulating evidence reveals the essential role of [Ca2+]i in chemotropic growth cone guidance 2225. Whether attractive or repulsive, chemical guidance cues elicit an asymmetric [Ca2+]i response in growth cones with the higher [Ca2+]i concentration at the side facing the source of guiance cues. Furthermore, the baseline calcium levels alter the attractive and repulsive reponses of growth cones to guidance cues 26. In addition to [Ca2+]i, cyclic nucleotides including cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) also play an important role in growth cone steering 27,28,2931.

Given the recent evidence that implicates intracellular signaling events similar to those recruited by chemorepulsive biochemical cues as a mechanism whereby topographical surface features direct neurite growth 21, we sought to determine the influence of [Ca2+]i and cyclic nucleotide levels on the ability of neurites to sense and respond to simple topographical micropatterns. The spatial and temporal control of photopolymerization is used to fabricate micropatterned methacrylate polymer substrates to probe this crucial relationship. Dissociated cultures of spiral ganglion neurons (SGNs), the target neurons of the cochlear implant which is the most widely used and successful neural prosthesis, were plated on the micropatterned substrates. We find that elevation of [Ca2+]i or cAMP levels disrupts neurite alignment to physical cues while elevation of cGMP levels enhances neurite alignment.

2. Materials and methods

Photopolymerization to generate micropatterned substrates

To investigate neurite growth cone sensing of biophysical cues, photopolymerization was used to generate micropatterned surfaces with various widths and depths as described elsewhere 12,14,16. Briefly, the monomer mixtures of 39 wt% hexyl methacrylate and 60 wt% 1,6-hexanediol dimethacrylate (Sigma-Aldrich, St. Loius, MO) with 1 wt% of 2,2-dimethoxy-2-phenylacetophenone (Ciba, Tarrytown, NY) as the photoinitiator were spread on glass microscope slides and subsequently covered by photomasks with alternating transparent or reflective bands with a periodicity of 50 μm. Monomer samples were cured under a high pressure mecury vapor arc lamp (Omnicure S-1500) equipped with a homogenizing light pipe and collimating adapter. Radiation dosage was varied by altering UV exposure time to attain specific microfeature depths as previously described 12,14. Micropatterns substrates consisted of parallel ridges and grooves with 1–3 μm amplitudes and 50 μm periodicities for neurite contact guidance studies. Channel amplitudes and periodicities were characterized by white light interferometry using a Wyko NT 1100 optical profiling system (Veeco, Plainview, NY).

Spiral ganglion dissociated cultures

The micropatterned substrates were sterilized by 70% ethanol and UV light, then coated with poly-L-ornithine solution (Sigma-Aldrich) for 1 hour at room temperature and laminin (20 μg/ml, Sigma-Aldrich), an extracellular matrix protein, for 2 hours at 37°C. Spiral ganglion dissociated cultures were prepared as previously described 32,33. NIH guidelines for the care and use of laboratory animals (NIH Publication #85-23 Rev. 1985) have been observed. Briefly, spiral ganglia from P3-5 rat pups were enzymatically dissociated in Ca2+/Mg2+-free HBSS with 0.1% collagenase (Sigma-Aldrich) and 0.125% trypsin-EDTA (Thermo Fisher Scientific, Waltham, MA), followed by mechanical dissociation using gentle blowing with pipettes. The cell suspension was plated in glass cylinders mounted on polymer substrates by sterile silicone glues. Cultures were maintained in Dulbecco’s modified Eagle Medium (DMEM, Life Technologies) supplemented with N2 supplement (Life Technologies), 5% fetal bovine serum (Thermo Fisher Scientific), 10 μg/ml insulin (Sigma-Aldrich), 50 ng/ml Neurotrophin-3 (NT-3, R&D Systems, Minneapolis, MN) and 50 ng/ml Brain Derived Neurotrophic Factor (BDNF, R&D Systems) at 37°C in a 6.5% CO2 incubator. The cultures were maintained for total of 72 hours and then fixed with 4% paraformaldehyde for 20 minutes at room temperature.

Manipulation of calcium influx and release from internal stores

Four to six hours after plating, experimental manipulations were carried out by changing culture medium. Calcium influx was increased by depolarizing with elevated extracellular K+ - 25 mM (25K) or 50 mM (50K) - as previously described 33,34. To decrease calcium influx, calcium-free culture media was used, in which DMEM was substituted with calcium-free DMEM (Life Technologies) with 1mM EDTA. Cadmium (Sigma-Aldrich), a non-selective voltage-gated calcium channel blocker, was added into the medium to a final concentration of 10 μM. Nifedipine (Sigma-Aldrich), an L-type Ca2+ channel blocker, was added to a final concentration of 1 μM. To disturb calcium release from internal stores, application of 100 μM Ryanodine (Sigma-Aldrich) or 2 μM Xestospongin C (Sigma-Aldrich) was used to block ryanodine-sensitive channels or inhibit inositol-1,4,5-trisphosphate (IP3) receptors, respectively.

Cyclic nuecleotides

To investigate the role of cyclic nucleotides in neurite guidance by microptterned features, we used cpt-cAMP (1 mM, Sigma-Aldrich), a membrane-permeable cAMP analog, to increase cAMP activity or 8-Br-cGMP, a cell-permeable cGMP analog (20, 200 μM, Sigma-Aldrich), to increase cGMP activity.

Immunostaining

Immunolabeling of SGNs and spiral ganglion Schwann cells (SGSCs) was performed as previously described 12,35. Anti-neurofilament 200 antibody (NF200, 1:400, Sigma-Aldrich), which labels heavy neurofilaments expressed in SGN somata and peripheral processes, and anti-S100 antibody (1:400, Sigma-Aldrich), which labels a calcium-binding protein in SGSCs, were used to identify SGNs and SGSCs, respectively. Primary antibody labeling was detected by Alexa Fluor-488 or Alexa Fluor-546 (1:800, Thermo Fisher Scientific) conjugated secondary antibodies. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI) in the mounting media.

Determination of SGN survival

SGN survival was determined as previously described by scoring the total of neurons in each condition and expressed as percent of control32,36. Criteria for neuronal viability were NF200 immunoreactivity and a nucleus that was visible and not pyknotic.

Assessment of neurite and cell alignment

SGN neurite and SGSC alignment was measured as described previously 12,16,21. The entire area of each culture was imaged with the micropatterns positioned parallel to horizontal and digitally stitched as one image by the “scan slide” function of Metamorph software (Molecular Devices, Silicon Valley, CA). Total neurite length (TL) and aligned length (AL), or the end-to-end distance of the neurite only in the direction of the micropattern features, were measured with ImageJ as previously described. The alignment ratio of TL/AL was used to assess the extent of neurite alignment to micropatterns. Neurites that align to and track micropattern features along their entire length result in alignment ratios close to one, while neurites that do not strongly align to the pattern yield significantly higher ratios. SGSC alignment was measured as the angle (θ) between the micropattern direction and the major axis of an ellipse drawn around the cell body using ImageJ software (National Institutes of Health, Bethesda, MD). Angles approaching zero degrees indicate significant SGSC alignment to the micropattern.

3. Results

Intracellular calcium levels modulate neurite alignment to micropatterned features

To fabricate micropatterned substrates for neurite contact guidance studies, HMA/HDDMA formulations containing photoinitiator were exposed to UV radiation through a photomask. Photomasks used in this work have alternating parallel transparent (glass) and reflective (chrome) bands each 25 μm wide (Fig. 1A). Spatially controlling the polymerization in this manner enables micropattern formation with simple tuning of the reaction parameters facilitating modulation of micropattern features during their development (Fig. 1) 12,14. Side view (Fig. 1B) and top-down view (Fig. 1C) scanning electron microscopic images confirm the consistency, periodicity, and amplitude of the micropatterns thus generated and used in these studies.

Figure 1
Generation of micropatterned HMA-co-HDDMA polymer surfaces. a. Schematic of photopatterning process. A photomask allows selectively exposure of photopolymerizable monomer to UV light resulting in a pattern of raised microfeatures across the surface. b, ...

On unpatterned surfaces, which were exposed to UV through a blank glass slide, SGN neurites grow in random trajectories (Fig. 2A) as evidenced by a high alignment ratio, as previously described 12,14. SGNs plated on micropatterned methacrylate substrates develop neurites that turn and align to the parallel ridge-groove line space grating (Fig. 2B) yielding an alignment ratio close to one.

Figure 2
Membrane depolarization reduces SG neurite alignment to micropatterns. a, b. Representive images of SG neurite alignment in cultures plated on unpatterned surface (a) and micropatterned surfaces (b). c–d. Representive images of SG neurite alignment ...

To examine the influence of intracellular Ca2+ ([Ca2+]i) on SGN neurite alignment to micropatterned features, dissociated SG cultures were plated on 3 μm amplitude, 50 μm periodicity micropatterns while maintaining the culture in media with 5.4 mM K+ or elevated K+ (25 mM, 25 K or 50 mM, 50K). Elevation of extracellular potassium (K+) depolarizes SGNs leading to chronic elevation of [Ca2+]i via influx from voltage sensitive Ca2+ channels 33,34. Treatment of SGN cultures with 25K or 50K significantly reduced neurite alignment compared to control cultures maintained in normal non-depolarizing media (Fig. 2C, D). Further, 50K disrupted alignment to a greater extent than 25K demonstrating a dose-response effect of elevated extracellular K+. These results demonstrate that membrane depolarization, which results in elevation of [Ca2+]i due to entry through voltage sensitive Ca2+ channels, reduces SGN alignment to microtopographical features.

Interestingly, SGN neurite alignment in cultures maintained in Ca2+-free medium was similar to the alignment of neurites in medium with normal Ca2+ levels (Fig. 3E). Further bath application of the non-selective voltage-gated calcium channel blocker cadmium (10 μM) or L-type Ca2+ channel blocker nifedipine (1 μm) did not signficantly alter SGN neurite alignment (Fig. 3). Taken together, these results indicate that while entry of extracellular Ca2+ disrupts SGN neurite pathfinding in response to biophysical cues, it is not necessarily required within the growth cones extracellular environment to effect guidance to biophysical features.

Figure 3
Inhibition of calcium influx fails to disturb SG neurite alignment to micropatterns. a. Images of SG neurite alignment in cultures plated on micropatterned surfaces (a). b–d. Representive images of SG neurite alignment in cultures plated on micropatterned ...

By contrast, bath application of 100 μM Ryanodine or 2 μM Xestospongin C, which block release of internal Ca2+ stores via ryanodine-sensitive channels or inositol-1,4,5-trisphosphate (IP3) receptors respectively, both significantly decrease neurite alignment (Fig. 4). Thus release of Ca2+ from internal stores appears necessary for SGN neurite guidance by microtopographical cues.

Figure 4
Inhibition of calcium release from internal calcium store reduces SG neurite alignment to micropatterns. a–c. Images of SG neurite alignment in cultures plated on micropatterned surfaces in control medium (a), in the presence of ryanodine (b) ...

On unpatterned tissue culture plastic coated with laminin, elevation of [Ca2+]i due to membrane depolarization exerts a biphasic response on SGN survival and decreases SGN neurite growth in a dose dependent manner 33,34. Thus, we determined SGN survival and neurite length for the various conditions used in these experiments. Table I reports these results. Consistent with prior results, elevation of extracellular K+ to 50 mm significantly reduced SGN survival and overall neurite length 33,34. However, neurite alignment did not appear to systematically vary with SGN survival or overall neurite length in the various treatment conditions.

Table I
SGN survival and neurite length.

Ryanodine and Xestospongin C disrupt SGSC alignment to micropatterns

SGSCs support SGN neurite growth and SC topographic features have been used to enhance directed regeneration of dorsal root ganglia axons35,37,38. Further, SGSCs strongly align to micropatterns, an effect that can be disrupted by inhibition of RhoA/ROCK signaling similar to the ability of RhoA and ROCK inhibitors to disrupt neurite alignment. These observations raise the possibility that SGSCs may rely on similar intracellular second messenger systems as SGNs to respond to topographical cues. To further explore this possibility, we treated SGSC cultures plated on micropatterned substrates with 3 μm amplitude and 50 μm periodicity features with ryanodine (100 μM) or Xestospongin C (2 μM). SGSC aligment was measured as the angle (θ) between the micropattern direction and the major axis of an ellipse drawn around the cell body. Angles close to zero degrees represent close alignment to the micropattern while random alignment would be yield angles near 45°. As with SGN neurites, treatment with ryanodine or Xestospongin C each significantly reduced SGSC alignment to the micropatterns (Fig. 5), suggesting that release of Ca2+ from internal stores is necessary for SGSC orientation to microtopographical cues.

Figure 5
Inhibition of calcium release from internal calcium store reduces SGSC alignment to micropatterns. a–c. Images of SGSC alignment in cultures plated on micropatterned surfaces in control medium (a), in the presence of ryanodine (b) or Xestospongin ...

cAMP and cGMP levels modulate SGN neurite and SGSC guidance to topographical cues

Cyclic nucleotides, including cAMP and cGMP, modulate growth cone responses to chemotactic stimuli 27,28,2931. To determine if they similarly contribute to SGN neurite pathfinding in response to topographical features, SGN cultures plated on micropatterned substrates were treated with the cell permeant cAMP analog, cpt-cAMP or the cGMP analog, 8-Br-cGMP. Treatment of SGN cultures with cpt-cAMP (1 mM) significantly reduces SGN neurite alignment on patterned substrates with the specified amplitude and periodicity (Fig. 6).

Figure 6
Elevation of cAMP levels disrupts SGN neurite alignment to micropatterns. a. Treatment of SGNs with cpt-cAMP (1 mM, n = 200), a cell permeant cAMP analog, significantly reduces SGN neurite alignment to micropatterns compared to SGN neurites maintained ...

As cAMP and cGMP often exert opposing effects on neurite guidance in response to chemotactic stimuli, we asked if elevation of cGMP could enhance SGN neurite alignment to topographical features. To uncover an enhancing effect of cGMP on neurite alignment, it is necessary to plate the cultures on micropatterns that do not exert a strong influence alignment that would obscure an enhancing effect. SGN alignment was previously shown to correlate with pattern depth and spacing such that shallow or widely spaced features are less effective at inducing neurite alignment compared to deep or frequent features12,14. Accordingly, SGN cultures were maintained on substrates with shallow amplitude (e.g. 1 μm) features and wide pattern spacing so that the pattern did not exert as strong of influence on neurite growth compared to the micropatterns with 3 μm amplitudes. The alignment ratio in control cultures on these relatively shallow features with 1 μm amplitude is 1.43±0.05 (mean±SEM) in contrast to neurites that strongly align to similar patterns with a 3 μm amplitude yielding an alignment ratio of 1.11±0.01 (mean±SEM) (Figs. 24). Interestingly, treatment of SGN cultures with 8-Br-cGMP (20 μM) significantly increases neurite alignment on substrates with the described shallow feature pattern (Fig. 7), implying that elevation of cGMP enhances growth cone responses to topographical features. Thus, cAMP and cGMP levels appear to exert opposing influences on growth cone response to topographical cues.

Figure 7
Elevation of cGMP levels enhances SGN neurite alignment to micropatterns. a. Treatment of SGNs with 8-Br-cGMP (cGMP, 20 μM, n = 119; 200 μM, n = 136) significantly increases SGN neurite alignment to micropatterns compared to SGN neurites ...

Similar to the effect on SGN neurites, cpt-cAMP significantly decreases SGSC alignment to micropatterns with 3 μm amplitude features (Fig. 8), increasing the mean alignment angle from 27.4±2.0 (mean±SEM) to 41.4±2.9 on micropatterned surfaces. Meanwhile, treatment of SGSC cultures plated on with 8-Br-cGMP significantly decreases the mean alignment angle (i.e. improves alignment) on micropatterns with 1 μm amplitude (Fig. 8). Thus, elevation of cAMP levels reduces the responsiveness of both SGN neurites and SGSCs to micropatterned surfaces while elevation of cGMP levels enhances the responsiveness of SGN neurites and SGSCs to these biophysical cues.

Figure 8
Elevation of cAMP levels disrupts SGSCs alignment to micropatterns while elevation of cGMP levels enhances alignment. a. Treatment of SGSC cultures with cpt-cAMP (1 mM, n = 197) significantly reduces SGSC alignment to micropatterns compared to SGSCs maintained ...

4. Discussion

Neural prostheses suffer from poor spatial resolution compared to native neural circuits due, at least in part, to the physical distances separating the stimulating electrodes from the target neurons that they stimulate. For example, in normal cochleae, afferent SGN fibers are in intimate synaptic contact (~10 nm) with the inner hair cells. However, cochlear implant electrodes are located in the scala tympani 250–>1000 μm away from the SGNs that they activate 39. This gap results in current spread and poor spatial signal resolution between adjacent electrodes. Thus, cochlear implant recipients typically experience poor hearing in noise and music appreciation due, in part, to non-specific neural stimulation 40,41. Other neural prostheses (e.g. retinal implant) likewise suffer from non-specific neural stimulation and poor spatial resolution 42.

To improve the interface between neural prostheses and their target neurons, neural engineers have sought to develop biomaterials with micropatterned biochemical or, more recently, topographical surface features capable of directing neurite growth into close proximity or even contact with the electrodes 19. For example, efforts have been made to guide SGN neurite growth towards the cochlear implant electrode arrays by topographical cues and chemotactic molecules 1416,4345. Understanding the cellular processes that mediate topographical neurite guidance will enhance the ability of neural engineers to precisely guide neurite growth. Our data demonstrate that intracellular calcium and cyclic nucleotide second messenger systems participate in growth cone pathfinding in response to biophysical cues found in the developing neural microenvironment.

Many in vitro and in vivo studies demonstrate that cells in general, and neurons and their processes in particular, respond to biophysical features in their microenvironment 19, yet little is known about how cells sense topography and then respond to it. Understanding the cellular signaling events that underlie such responses will facilitate the design of surface features aimed at enhancing the integration of biomaterials with native tissue and influencing the response of the target cells to these surface features. For example, it may be possible to use small molecule inhibitors or activators of second messenger systems, such as cyclic nucleotides, to enhance the effects of topographical features to precisely guide axon regeneration.

Here we used the spatial and temporal control of photopolymerization to fabricate methacrylate platforms with precise micropatterns to probe biochemical effects on physical signaling and, ultimately, alignment of SGN neurite growth and SGSC orientation. As previously demonstrated, this photopolymerization process produces micropatterns with gradual sloping transitions between ridges and grooves. These gradual transitions facilitate studies aimed at identifying signaling events involved in topographical neurite guidance as they do not physically constrain neurites. By contrast, patterns prepared by photolithography have sharp transitions that can constrain the growth cone 46. Without such physical constraints, the neurite response can change following manipulation of intracellular signaling events. For example, micropatterns produced by photopolymerization similar to those used in these studies facilitated the identification of transient receptor potential (TRP) cation channel subfamily V member 1 (TRPV1) and RhoA/Rho associated kinase signaling as key mediators of neurite alignment to micropatterns 21.

Calcium as a mediator of topographical guidance

Calcium is a ubiquitous second messenger that has been shown to be a critical mediator of growth cone responses to chemotactic guidance molecules. Ca2+ concentrations in growth cones are regulated by influx of extracellular Ca2+ through specific channels and by Ca2+ release from Ca2+ stores in the endoplasmic reticulum 25. Ca2+ channels on the plasma membrane, such as TRP channels, voltage-dependent Ca2+ channels (VDCCs), and cyclic nucleotide-gated ion channels (CNGCs), mediate “Ca2+ influx” from the extracellular space, whereas Ca2+ channels on the endoplasmic reticulum (ER) membrane, such as RyRs and IP3 receptors (IP3Rs), mediate “Ca2+ release” from the ER Ca2+ stores 47. Ca2+ signals from these different resources mediate growth cone steering induced by various chemotactic factors. For example, TRP channels, L-type VDCCs, and RyRs are involved in netrin-1-induced attraction 48,49; IP3Rs are involved in NGF- and BDNF-induced attraction 50,51; and CNGCs are involved in Sema3A-induced repulsion 52.

Further, the Ca2+ level in the growth cone can alter attractive and repulsive reponses of growth cones to guidance cues 26. In some cases, Ca2+ release through RyRs or IP3Rs generates a steep Ca2+ elevation and initiates growth cone attraction 48,50,53 while Ca2+ influx through plasma membrane channels without Ca2+ release from internal Ca2+ stores generates a shallow Ca2+ elevation and initiates growth cone repulsion 24,54. In other cases, a modest local Ca2+ elevation induces growth cone attraction, whereas a small local Ca2+ elevation (or shallow gradient across the growth cone) and large local Ca2+ transients induce growth cone repulsion 24,5557. Thus, there is compelling evidence that chemotactic guidance cues modulate calcium dynamics in the growth cone and that, in turn, Ca2+ levels modulate growth cone responses to chemotactic molecules.

In a previous report, rat hippocampal neurites were shown to grow perpendicular to shallow, narrow grooves (e.g. 130 nm deep, 1 μm wide) and parallel to similar features that are deeper and wider (e.g. 1,100 nm deep, 4 μm wide) 11. Perpendicular contact guidance was reduced by flunarizine, a N-type voltage-dependent calcium channels (VDCC), and was abolished by nifedipine and diltiazem, specific L-type VDCC antagonists suggesting that calcium influx via VDCCs is involved in perpendicular contact guidance. Conversely, parallel orientation was not perturbed by these VDCC antagonists 20. Apparently, perpendicular and parallel contact guidance of hippocampal neurites employ different Ca2+ signaling mechanisms.

In this study, we increased [Ca2+]i by depolarizing dissociated SGNs with elevated extracellular K+ - 25 mM (25K) or 50 mM (50K) - as previously described 33,34. SGN neurite alignment in cultures depolarized with 25K and 50K is significantly poorer than alignment in cultures in nondepolarized medium. Thus elevation of [Ca2+]i, by chronic membrane depolarization appears to disrupt SGN alignment to these micropatterns (Fig. 9). Thus, there could be an optimum intracellular calcium level necessary for contact guidance. However, entry of extracellular calcium does not appear to be necessary for contact guidance. Bath application of the general blocker of VDCCs, cadmium, or the L-type Ca2+ channel blocker, nifedipine, does not disrupt SGN neurite alignment, implying that external Ca2+ entry through VDCCs is not required for SGN process alignment to topographical features. Similarly, SGN neurite alignment in Ca2+-free medium is comparable to alignment in medium with normal [Ca2+]. These data suggest that while elevation of [Ca2+]i, from external sources, disrupts SGN neurite alignment to micropatterns, entry of Ca2+ via VDCCs is not required for SGN neurite alignment to micropatterns consisting of parallel ridges and grooves. This independence of SGNs from VDCCs to accurately track parallel features is similar to hippocampal neurites that align to parallel surface patterns 11.

Figure 9
Schematic of the effects of Ca2+ and cyclic nucleotide second messengers systems on growth cone sensitivity to micropatterns. Ridges of micropatterns function to repel neurite growth (red shade) and induce neurite extension within grooves (green shade). ...

Interestingly, bath application of ryanodine (100–300 μM) or Xestospongin C (2 μM), which inhibit RyR and IP3R, respectively, both significantly decrease neurite alignment suggesting that release of Ca2+ from internal stores is necessary for contact guidance. Thus, based on the data from this initial study, Ca2+ signaling in this system appears complex and likely depends on temporal and spatial dynamics.

Elevation of cGMP enhances SGN neurite alignment to microtopographical features while elevation of cAMP disrupts aligment

Cyclic nucleotides play an important role in growth cone steering by biochemical guidance cues. For example, elevation of cAMP levels is associated with attractive turning and neurite extension, even converting repulsive turning into an attractive response 24,2729. Likewise, elevation of cAMP levels in SGNs, as in other neurons, overcomes the inhibition of neurite growth by central glia 35,5860. Meanwhile, elevation of cGMP levels is generally associated with repulsive turning and growth cone collapse 2931,61,62. Furthermore, previous studies suggest a complex interactive relationship between cyclic nucleotides and calcium signaling in growth cone steering 61,62.

Here we found that elevation of cAMP disrupted SGN neurite alignment to the described photopolymerized micropatterns. We also found that increasing cGMP levels enhanced SGN neurite alignment to shallow (1 μm) micropatterns, which otherwise were not of sufficient amplitude to induce significant neurite alignment. Thus, cAMP and cGMP appear to exert opposing influences on neurite aligment to microtopographical features similar to their opposing effects on chemotactive biochemical cues 63,64.

Interestingly, SGSC alignment to the micropatterns, which, like to SGN neurite alignment, depends on RhoA/ROCK signaling 21, was likewise disrupted by ryanodine and IP3 receptor inhbitors and by treatment with a cAMP analog. Further, treatment with a cGMP analog enhanced SGSC alignment to micropatterns. These data support the notion that SGN neurites and SGSCs may engage some similar signaling mechanisms to achieve alignment to topographic features. Nevertheless, these observations remain correlative and that further experiments are required to firmly establish the signaling mechanisms recruited by both SGNs and SGSCs.

Ridges of topographical features appear to activate similar signaling cascades as chemorepulsive stimuli to repel SGN growth

Neurites prefer to grow in the grooves compared to the ridges of these micropatterned substrates 12,15. This observation implies that either the ridge features act as a repulsive signal and/or that the groove acts as a permissive substrate. We recently demonstrated that when SGN growth cones encounter the raised surfaces of the ridges intracellular signaling events are activated similar to those activated in response to chemorepulsive stimuli 21. For example, RhoA signaling is activated while Cdc42/Rac activity is suppressed 21. Further inhibition of TRPV1 channels reduces neurite alignment to micropatterns while induced expression of TRPV1 in 3T3 cells renders the cells sensitive to micropatterns implying that TRPV1 channels are essential for cellular guidance by physical cues. Significantly, both TRPV1 and RhoA signaling are implicated in growth cone turning in response to chemorepulsive cues. Consistent with the notion that the ridges function to repel growth cones by activating similar intracellular signaling events as chemorepulsive cues (e.g. RhoA)21 treatment of cultures with a cAMP analog disrupts SGN neurite alignment similar to the ability of elevated cAMP levels to overcome chemorepulsive cues 35,5557.

5. Conclusions

Here we used photopolymerization to generate specific micropatterns in methacrylate substrates that function as effective contact guidance cues to precisely direct SGN neurite growth. Such surface features could be used to guide the trajectory of regenerating SGN fibers in an effort to achieve a more intimate and precise interface with a cochlear implant electrode array. Elevation of intracellular calcium or cAMP levels significantly disrupts the alignment of SGN neurites to microtopographical patterns, while elevation of cGMP enhances alignment. The results suggest that similar intracellular signaling pathways as those recruited by chemorepulsive cues are involved in SGN neurite guidance by these topographical features. Manipulation of these intracellular signaling pathways will be useful to further probe the strength of biophysical cues engineered for directed neural outgrowth as well as inform efforts to engineer enhanced interfaces between a neural prosthesis and its target neurons.

Acknowledgments

Supported by NIDCD (R01 DC012578, P30 DC010362), National Natural Science Foundation of China (NSFC 81171482), and the Alpha Omega Alpha Carolyn L. Kuckein Student Research Fellowship.

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