Blackiston DJ et al. (2015), A novel method for inducing nerve growth via mo...

XB-ART-49959

Neurotherapeutics 2015 Jan 01;121:170-84. doi: 10.1007/s13311-014-0317-7.

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A novel method for inducing nerve growth via modulation of host resting potential: gap junction-mediated and serotonergic signaling mechanisms.

Blackiston DJ , Anderson GM , Rahman N , Bieck C , Levin M .

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A major goal of regenerative medicine is to restore the function of damaged or missing organs through the implantation of bioengineered or donor-derived components. It is necessary to understand the signals and cues necessary for implanted structures to innervate the host, as organs devoid of neural connections provide little benefit to the patient. While developmental studies have identified neuronal pathfinding molecules required for proper patterning during embryogenesis, strategies to initiate innervation in structures transplanted at later times or alternate locations remain limited. Recent work has identified membrane resting potential of nerves as a key regulator of growth cone extension or arrest. Here, we identify a novel role of bioelectricity in the generation of axon guidance cues, showing that neurons read the electric topography of surrounding cells, and demonstrate these cues can be leveraged to initiate sensory organ transplant innervation. Grafts of fluorescently labeled embryological eye primordia were used to produce ectopic eyes in Xenopus laevis tadpoles. Depolarization of host tissues through anion channel activation or other means led to a striking hyperinnervation of the body by these ectopic eyes. A screen of possible transduction mechanisms identified serotonergic signaling to be essential for hyperinnervation to occur, and our molecular data suggest a possible model of bioelectrical control of the distribution of neurotransmitters that guides nerve growth. Together, these results identify the molecular components of bioelectrical signaling among cells that regulates axon guidance, and suggest novel biomedical and bioengineering strategies for triggering neuronal outgrowth using ion channel drugs already approved for human use.

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Species referenced: Xenopus laevis
Genes referenced: htr7 kcnj10 slc6a4l tecta.2

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	Fig. 1. Ectopic eyes hyperinnervate hosts in response to chloride channel activation. (a) Diagram of eye primordium graft location (red eye) and location imaged for innervation analysis (yellow shaded area =40Ã—; gray shaded area =100Ã—). (b) Eye primordium removed from a donor tdTomato-expressing Xenopus and transplanted to caudal locations of a wild-type (WT) recipient produces ectopic eyes at the site of the graft (scale bar =500Â Î¼m). (c) Fluorescent labeling of donor tissue reveals that ectopic eyes occasionally innervate the host, with small numbers of axons observed in the fin and trunk of the tadpole (scale bar =100Â Î¼m). (d), In the presence of the glutamate-gated chloride channel activator ivermectin (IVM), ectopic eyes hyperinnervate the host, with labeled axons present throughout large portions of the fin and trunk of the recipient. (e) High magnification reveals extensive branching of donor axons in response to IVM. (f) Sholl analysis comparing WT (n =15) and IVM-exposed (n =14) embryos reveals significantly more axons (P <0.01) in treated animals at both proximal and distal locations to the transplanted eye. (g) Time lapse imaging of ectopic eye innervation shows extensive axon remodeling in response to IVM, including extension, retraction, and degradation over 4Â days. Loopback structures were observed in which axons appear to self-cross, after which the axon shows blebbing and degradation (white arrows; scale bar =100Â Î¼m). Error bars indicate Â±1 SEM. Asterisks represent values which significantly differ from controls (2-way analysis of variance, followed by Bonferroni post hoc analysis)
	Fig. 2. Ectopic eye hyperinnervation is location-specific and requires depolarization of the host. (a) Donor eye primordium treated with ivermectin (IVM) prior to implantation does not result in hyperinnervation of the host (eye is off frame to the left of the image). (ai) High magnification of donor eyes treated with IVM reveals a small number of eyes extend few projections into the host, but not in significant levels on hyperinnervation. (b) When native tadpole eyes are extirpated and replaced with labeled donor eye tissue, optic nerves (white arrow) emerge from replacement structures and target the optic tecta of the host. (c) When native eyes are replaced with labeled donor eyes in the presence of IVM, hyperinnervation is not observed. Transplanted eyes send single optic nerves to the optic tecta of the host (white arrow). (d) Xenopus eyes can be surgically removed at tadpole stages, resulting in eyeless animals. (e) Ectopic eyes induced in eyeless animals do not hyperinnervate the host. WT = wild-type
	Fig. 3. Ectopic eye hyperinnervation is a result of membrane voltage depolarization, not restricted to chloride flux. (a) While ectopic eyes hyperinnervate hosts in response to ivermectin (IVM), supplementing the media with high chloride levels (hyperpolarizing cells affected by the drug) inhibits the extent of innervation and branching pattern. (b) Injecting mRNA coding for the hyperpolarizing potassium channel Kir4.1 inhibits hyperinnervation of ectopic eyes in response to IVM exposure. (c) Treating animals with the sodium transport inhibitor tricaine mesylate, which hyperpolarizes affected cells, inhibits hyperinnervation of ectopic eyes in response to IVM exposure
	Fig. 4. Membrane voltage control of ectopic eye innervation functions through serotonin (5-HT) signaling. (a) Supplementing the Xenopus media with 5-HT can induce hyperinnervation, even in the absence of membrane voltage alteration. (b) Disrupting 5-HT production in Xenopus embryos through para-chlorophenylalanine (TPH) exposure inhibits ectopic eye innervation of host animals. (c) Inhibition of the 5-HT transporter (SERT) with fluoxetine blocks hyperinnervation in response to membrane depolarization. (d) Selectively blocking 5-HT receptor 3 (5-HT3) with exposure to tropisetron has no effect on ectopic eye innervation of the host. (e) Inhibition of the 5-HT receptors 1, 2, and 7 (5-HT1, 2, 7)_with the broad-spectrum antagonist metergoline abolishes ectopic eye innervation of the host. (f) Disruption of 5-HT receptor 1A and 1B (5-HT1) with the antagonist cyanopindolol does not inhibit host innervation by ectopic eyes. (g) Inhibiting 5-HT receptor 2A (5-HT2) with the compound altanserin has no effect on ectopic eye innervation of the host. (h) Selectively blocking 5-HT receptor 7 (5-HT7) with exposure to SB258719 does not inhibit ectopic eye innervation of the host. (i) Using a combination of altanserin and cyanopindolol, innervation of hosts by ectopic eyes could be suppressed, indicating a downstream role of 5-HT receptors 1 and 2 (5-HT1/2). IVM = ivermectin
	Fig. 5. Gap junction (GJ) communication is essential for ectopic eye innervation of the host. (a) In the presence of the ivermectin (IVM), hyperinnervation by ectopic eyes could be suppressed by exposure to the GJ antagonist lindane. (b) Similar to chemical exposure, hyperinnervation by ectopic eyes in response to IVM could also be suppressed by early mRNA injections of the dominant negative (DN) GJ H7. (c) Animals treated with both the gap junction inhibitor lindane and supplemented serotonin (5-HT) resulted in hyperinnervation
	Fig. 6. Model of ectopic eye innervation in response to membrane voltage changes. (a) In untreated animals, serotonin (5-HT) is produced across time space during development. The positively charged 5-HT moves between cells via gap junctions and accumulates in negatively hyperpolarized cells, which act as sinks for the molecule. In the absence of extracellular 5-HT, the 5-HT receptors of ectopic eye neurons are not activated and the growth cones show minimal extension. (b) In treated animals 5-HT also accumulates in negatively hyperpolarized cells. However, exposure to ivermectin (IVM) activates glycine-gated chloride channels (GlyCl), allowing chloride to exit the cell along its concentration gradient, depolarizing the cell. In response to depolarization, the accumulated 5-HT diffuses out of the cell via the 5-HT transporter (SERT). Extracellular 5-HT then binds 5-HT1/2 receptors on the surface of ectopic eye retinal ganglion cells, leading to extension of the growth cone

References [+] :

Adams, H+ pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induce Xenopus tail regeneration. 2007, Pubmed, Xenbase