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• W3009999788 abstract "•IP3 receptor type 3 (IP3R3) controls axonal sensitivity to IP3-based guidance cues•IP3R3−/− growth cones are not attracted to NGF due to global Ca2+ responses•Lower NGF concentrations can polarize IP3R3−/− growth cones for attractive turning•NGF knockdown in vivo can revert abnormal trajectory of IP3R3−/− axons During neurodevelopment, the growth cone deciphers directional information from extracellular guidance cues presented as shallow concentration gradients via signal amplification. However, it remains unclear how the growth cone controls this amplification process during its navigation through an environment in which basal cue concentrations vary widely. Here, we identified inositol 1,4,5-trisphosphate (IP3) receptor type 3 as a regulator of axonal sensitivity to guidance cues in vitro and in vivo. Growth cones lacking the type 3 subunit are hypersensitive to nerve growth factor (NGF), an IP3-dependent attractive cue, and incapable of turning toward normal concentration ranges of NGF to which wild-type growth cones respond. This is due to globally, but not asymmetrically, activated Ca2+ signaling in the hypersensitive growth cones. Remarkably, lower NGF concentrations can polarize growth cones for turning if IP3 receptor type 3 is deficient. These data suggest a subtype-specific IP3 receptor function in sensitivity adjustment during axon navigation. During neurodevelopment, the growth cone deciphers directional information from extracellular guidance cues presented as shallow concentration gradients via signal amplification. However, it remains unclear how the growth cone controls this amplification process during its navigation through an environment in which basal cue concentrations vary widely. Here, we identified inositol 1,4,5-trisphosphate (IP3) receptor type 3 as a regulator of axonal sensitivity to guidance cues in vitro and in vivo. Growth cones lacking the type 3 subunit are hypersensitive to nerve growth factor (NGF), an IP3-dependent attractive cue, and incapable of turning toward normal concentration ranges of NGF to which wild-type growth cones respond. This is due to globally, but not asymmetrically, activated Ca2+ signaling in the hypersensitive growth cones. Remarkably, lower NGF concentrations can polarize growth cones for turning if IP3 receptor type 3 is deficient. These data suggest a subtype-specific IP3 receptor function in sensitivity adjustment during axon navigation. During neurodevelopment, an axon in search of its appropriate target relies on the navigational activity of its distal end called the growth cone, which senses molecular guidance cues in the immediate environment and makes path-finding decisions at choice points. These guidance cues can be presented as a shallow gradient among a noisy background, with spatial and temporal variations in cue concentrations. Consequently, the growth cone needs to not only amplify guidance signals but also adjust its sensitivity as it migrates through different segments along the chemical gradient. One important challenge for understanding axon guidance is to decipher whether and how intracellular second messengers, critical components mediating signal amplification and growth cone polarization for turning, can also control its sensitivity to guidance signals. Both attractive and repulsive cues instruct growth cone turning through, in most cases, asymmetric Ca2+ elevations with higher Ca2+ concentrations on the side of the growth cone facing the source of the cues (reviewed in Gomez and Zheng, 2006Gomez T.M. Zheng J.Q. The molecular basis for calcium-dependent axon pathfinding.Nat. Rev. Neurosci. 2006; 7: 115-125Crossref PubMed Scopus (242) Google Scholar). Whether the growth cone turns toward the higher Ca2+ side (attraction) or the lower Ca2+ side (repulsion) depends, in principle, on the source of Ca2+ signals: Ca2+ release from the ER triggers attraction, whereas Ca2+ influx through plasma membrane channels induces repulsion (reviewed in Tojima et al., 2011Tojima T. Hines J.H. Henley J.R. Kamiguchi H. Second messengers and membrane trafficking direct and organize growth cone steering.Nat. Rev. Neurosci. 2011; 12: 191-203Crossref PubMed Scopus (139) Google Scholar). Inositol 1,4,5-trisphosphate (IP3) is one important second messenger produced from membrane phospholipids and, upon binding to tetrameric IP3 receptors (IP3Rs) on the ER, can elicit Ca2+ release from the ER into the cytosol (reviewed in Mikoshiba, 2007Mikoshiba K. IP3 receptor/Ca2+ channel: from discovery to new signaling concepts.J. Neurochem. 2007; 102: 1426-1446Crossref PubMed Scopus (290) Google Scholar). This process is commonly termed IP3-induced Ca2+ release (IICR). In the skin, nerve growth factor (NGF) produced by epidermal keratinocytes (Botchkarev et al., 2006Botchkarev V.A. Yaar M. Peters E.M. Raychaudhuri S.P. Botchkareva N.V. Marconi A. Raychaudhuri S.K. Paus R. Pincelli C. Neurotrophins in skin biology and pathology.J. Invest. Dermatol. 2006; 126: 1719-1727Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) participates in target field innervation by sensory neurons (Albers et al., 1994Albers K.M. Wright D.E. Davis B.M. Overexpression of nerve growth factor in epidermis of transgenic mice causes hypertrophy of the peripheral nervous system.J. Neurosci. 1994; 14: 1422-1432Crossref PubMed Google Scholar, Patel et al., 2000Patel T.D. Jackman A. Rice F.L. Kucera J. Snider W.D. Development of sensory neurons in the absence of NGF/TrkA signaling in vivo.Neuron. 2000; 25: 345-357Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Also, in vitro studies demonstrated that NGF acts as an attractive axon guidance cue through binding to tropomyosin receptor kinase A (TrkA) receptor and activating phospholipase C, an enzyme that catalyzes the hydrolytic conversion of membrane phospholipids into diacylglycerol and IP3 (Gallo et al., 1997Gallo G. Lefcort F.B. Letourneau P.C. The trkA receptor mediates growth cone turning toward a localized source of nerve growth factor.J. Neurosci. 1997; 17: 5445-5454Crossref PubMed Google Scholar, Ming et al., 1999Ming G. Song H. Berninger B. Inagaki N. Tessier-Lavigne M. Poo M. Phospholipase C-gamma and phosphoinositide 3-kinase mediate cytoplasmic signaling in nerve growth cone guidance.Neuron. 1999; 23: 139-148Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Our subsequent work showed that, in a growth cone migrating in an extracellular NGF gradient, phospholipase-C-dependent production of IP3 and ensuing IICR occurs on the side of the growth cone facing higher NGF concentrations (Akiyama et al., 2009Akiyama H. Matsu-ura T. Mikoshiba K. Kamiguchi H. Control of neuronal growth cone navigation by asymmetric inositol 1,4,5-trisphosphate signals.Sci. Signal. 2009; 2: ra34Crossref PubMed Scopus (37) Google Scholar) and that this asymmetric IICR causes polarized membrane dynamics leading to attractive axon turning toward NGF (Akiyama and Kamiguchi, 2010Akiyama H. Kamiguchi H. Phosphatidylinositol 3-kinase facilitates microtubule-dependent membrane transport for neuronal growth cone guidance.J. Biol. Chem. 2010; 285: 41740-41748Crossref PubMed Scopus (30) Google Scholar). In this way, IP3-induced asymmetric Ca2+ signals across the growth cone mediate attractive guidance responses to physiological cues such as NGF. Although a growth cone can adjust its sensitivity to a wide range of guidance cue concentrations via multiple mechanisms such as receptor internalization and turnover (reviewed in Gallo and Letourneau, 2002Gallo G. Letourneau P. Axon guidance: proteins turnover in turning growth cones.Curr. Biol. 2002; 12: R560-R562Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, Ming et al., 2002Ming G.L. Wong S.T. Henley J. Yuan X.B. Song H.J. Spitzer N.C. Poo M.M. Adaptation in the chemotactic guidance of nerve growth cones.Nature. 2002; 417: 411-418Crossref PubMed Scopus (342) Google Scholar, Piper et al., 2005Piper M. Salih S. Weinl C. Holt C.E. Harris W.A. Endocytosis-dependent desensitization and protein synthesis-dependent resensitization in retinal growth cone adaptation.Nat. Neurosci. 2005; 8: 179-186Crossref PubMed Scopus (132) Google Scholar), a potential role for second messenger signaling in this adjustment process remains to be determined. In the current study, we investigate NGF-dependent axon guidance using mice lacking each of the three IP3R subunits identified in mammals—type 1 (Furuichi et al., 1989Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400.Nature. 1989; 342: 32-38Crossref PubMed Scopus (803) Google Scholar), type 2 (Südhof et al., 1991Südhof T.C. Newton C.L. Archer 3rd, B.T. Ushkaryov Y.A. Mignery G.A. Structure of a novel InsP3 receptor.EMBO J. 1991; 10: 3199-3206Crossref PubMed Scopus (318) Google Scholar), and type 3 (Blondel et al., 1993Blondel O. Takeda J. Janssen H. Seino S. Bell G.I. Sequence and functional characterization of a third inositol trisphosphate receptor subtype, IP3R-3, expressed in pancreatic islets, kidney, gastrointestinal tract, and other tissues.J. Biol. Chem. 1993; 268: 11356-11363Abstract Full Text PDF PubMed Google Scholar), abbreviated respectively as IP3R1, IP3R2, and IP3R3. These subunits can form a homo- or heterotetrameric IP3R that acts as a functional Ca2+ channel upon IP3 binding to each subunit (Maes et al., 2001Maes K. Missiaen L. Parys J.B. De Smet P. Sienaert I. Waelkens E. Callewaert G. De Smedt H. Mapping of the ATP-binding sites on inositol 1,4,5-trisphosphate receptor type 1 and type 3 homotetramers by controlled proteolysis and photoaffinity labeling.J. Biol. Chem. 2001; 276: 3492-3497Crossref PubMed Scopus (43) Google Scholar, Monkawa et al., 1995Monkawa T. Miyawaki A. Sugiyama T. Yoneshima H. Yamamoto-Hino M. Furuichi T. Saruta T. Hasegawa M. Mikoshiba K. Heterotetrameric complex formation of inositol 1,4,5-trisphosphate receptor subunits.J. Biol. Chem. 1995; 270: 14700-14704Crossref PubMed Scopus (191) Google Scholar, Nucifora et al., 1996Nucifora Jr., F.C. Sharp A.H. Milgram S.L. Ross C.A. Inositol 1,4,5-trisphosphate receptors in endocrine cells: localization and association in hetero- and homotetramers.Mol. Biol. Cell. 1996; 7: 949-960Crossref PubMed Scopus (53) Google Scholar, Wojcikiewicz and He, 1995Wojcikiewicz R.J. He Y. Type I, II and III inositol 1,4,5-trisphosphate receptor co-immunoprecipitation as evidence for the existence of heterotetrameric receptor complexes.Biochem. Biophys. Res.Commun. 1995; 213: 334-341Crossref PubMed Scopus (86) Google Scholar). Navigational behavior of dorsal root ganglion (DRG) neurons derived from IP3R1 knockout (R1KO) and IP3R2 knockout (R2KO) mice are indistinguishable from that of wild-type (WT) neurons. However, DRG neuronal growth cones of IP3R3 knockout (R3KO) mice cannot respond properly to normal concentration ranges of NGF in vitro and in the skin in vivo, most likely because they are hypersensitive to IP3 and therefore exhibit global Ca2+ elevations in response to NGF gradients. These data raise the possibility that growth cones could adjust their sensitivity at the IP3R level such that they continue to produce polarized signals even in the presence of local variations in guidance cue concentrations. Although it is known that asymmetric IICR across the growth cone plays a crucial role in axon guidance mediated by extracellular cues such as NGF, which one(s) of the three mammalian IP3R subtypes participates in this process remains unclear. We employed focal laser-induced photolysis (FLIP) of caged IP3 to generate spatially localized IP3 signals in growth cones derived from IP3R-subtype-specific knockout mice. For loading, DRG neurons were incubated with 0.5 μM solution of caged IP3. In our previous study using chicken neurons (Akiyama et al., 2009Akiyama H. Matsu-ura T. Mikoshiba K. Kamiguchi H. Control of neuronal growth cone navigation by asymmetric inositol 1,4,5-trisphosphate signals.Sci. Signal. 2009; 2: ra34Crossref PubMed Scopus (37) Google Scholar), FLIP of caged IP3 resulted in a sustained increase in IP3 and its consequent Ca2+ elevation on the side of the growth cone receiving laser irradiation (“near side”). These asymmetric signals caused growth cone attractive turning toward higher IP3 and Ca2+. In the current experiments, DRG growth cones from WT mice also turned attractively toward higher IP3, whereas control growth cones that had not been loaded with caged IP3 showed no detectable turning after laser irradiation (Figures 1A and 1B ). As the occurrence of IICR depends on cytosolic levels of cyclic adenosine monophosphate (cAMP), chicken neurons cultured on a laminin substrate in our previous study, which caused a reduction in cAMP levels in growth cones, failed to respond to IP3 signals (Akiyama et al., 2009Akiyama H. Matsu-ura T. Mikoshiba K. Kamiguchi H. Control of neuronal growth cone navigation by asymmetric inositol 1,4,5-trisphosphate signals.Sci. Signal. 2009; 2: ra34Crossref PubMed Scopus (37) Google Scholar). Consistently, IP3-induced turning of mouse neuronal growth cones was also dependent on cAMP because the cAMP antagonist Rp-cAMPS (20 μM) blocked the effect of IP3 uncaging (Figure 1B). Analyses of neurons derived from IP3R-subtype-specific knockout mice showed that R1KO or R2KO growth cones turned toward higher IP3 (Figure 1B). By contrast, R3KO growth cones showed no significant turning (Figures 1A and 1B), indicating that IP3R3 is necessary for turning responses to IP3 under this experimental condition. We next tested whether an extracellular NGF gradient was attractive to DRG growth cones from WT and each of the three knockout mice. In these assays, NGF concentrations near the growth cones were approximately 0.1% of in-pipette concentration of 20 μg/mL (Lohof et al., 1992Lohof A.M. Quillan M. Dan Y. Poo M.M. Asymmetric modulation of cytosolic cAMP activity induces growth cone turning.J. Neurosci. 1992; 12: 1253-1261Crossref PubMed Google Scholar). The NGF gradient attracted WT, R1KO and R2KO growth cones, but not R3KO growth cones (Figures 1C and 1D). The migration speed of growth cones in NGF gradients was not affected by the loss of each IP3R subtype (Figure S1). Because attractive turning responses of WT and R1KO growth cones may potentially be different quantitatively, we calculated Cohen's d between WT and R1KO to be 0.23 for IP3 uncaging (Figure 1B) and 0.69 for NGF gradients (Figure 1D), suggesting that the effect of R1KO is small to moderate. Nonetheless, we concluded that, at least qualitatively, R1KO growth cones are indistinguishable from WT growth cones in their responses to IP3 and NGF signals. As a control to show that R3KO growth cones were still able to turn, we examined the effect of myelin-associated glycoprotein (MAG) known to attract nascent axons via Ca2+ release from the ER through another class of Ca2+ channels, ryanodine receptors (Henley et al., 2004Henley J.R. Huang K.H. Wang D. Poo M.M. Calcium mediates bidirectional growth cone turning induced by myelin-associated glycoprotein.Neuron. 2004; 44: 909-916Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, Tojima et al., 2014Tojima T. Itofusa R. Kamiguchi H. Steering neuronal growth cones by shifting the imbalance between exocytosis and endocytosis.J. Neurosci. 2014; 34: 7165-7178Crossref PubMed Scopus (34) Google Scholar). Consistent with such differences in the requirement of ion channels mediating Ca2+ release, MAG caused attractive turning of R3KO growth cones (Figure 1E). Therefore, the loss of IP3R3 rendered growth cones unresponsive to an IP3R-based guidance cue such as NGF. Although IP3R1 and IP3R3 are expressed by neurons, IP3R2 is detectable only in glial cells in the nervous system (Sharp et al., 1999Sharp A.H. Nucifora Jr., F.C. Blondel O. Sheppard C.A. Zhang C. Snyder S.H. Russell J.T. Ryugo D.K. Ross C.A. Differential cellular expression of isoforms of inositol 1,4,5-triphosphate receptors in neurons and glia in brain.J. Comp. Neurol. 1999; 406: 207-220Crossref PubMed Scopus (159) Google Scholar, Taylor et al., 1999Taylor C.W. Genazzani A.A. Morris S.A. Expression of inositol trisphosphate receptors.Cell Calcium. 1999; 26: 237-251Crossref PubMed Scopus (231) Google Scholar). Consistent with this glial-specific expression pattern of IP3R2, our results showed that IP3R2 was dispensable for IP3-dependent growth cone turning. Therefore, we decided to leave out R2KO mice in subsequent experiments. To investigate a correlation between growth cone turning and intracellular signaling asymmetry, we monitored spatiotemporal dynamics of Ca2+ in response to localized IP3 signals. In WT and R1KO growth cones, IP3 uncaging resulted in Ca2+ elevations on the near side but no detectable Ca2+ increases on the opposite “far” side (Figures 2A–2C ). Such asymmetric Ca2+ signals are consistent with the ability of these growth cones to turn toward the side with IP3 production. By contrast, R3KO growth cones showed widespread increases in Ca2+ spanning both the near and far sides after localized IP3 uncaging on the near side (Figures 2A–2C). To capture a potential timepoint when Ca2+ gradients could be formed in R3KO growth cones, we compared Ca2+ levels between the near and far sides during earlier periods after IP3uncaging. As shown in Figure 2D, localized IP3 production caused a transient asymmetry in Ca2+ concentrations across the R3KO growth cone at 30–60 s but could not sustain the Ca2+ gradient after 1 min of the onset of IP3 uncaging (Figures 2C and 2D), suggesting that R3KO growth cones have symmetric IICR on the timescale for axon turning processes. We also tested whether extracellular NGF gradients cause similar spatiotemporal dynamics of Ca2+ in growth cones, using ratiometric Oregon Green 488 BAPTA-1 (OGB-1)–Fura-red (FR) imaging as a measure of Ca2+ levels. NGF gradients induced Ca2+ elevations only on the near side of WT and R1KO growth cones but caused widespread Ca2+ increases on both sides of R3KO growth cones (Figure 3). These results suggest that the failure of R3KO growth cones to turn toward NGF-induced IP3 signals may be due to symmetric Ca2+ elevations, a distribution pattern that is unlikely to act as a polarizing signal.Figure 3Spatiotemporal Dynamics of Ca2+ in Growth Cones Responding to NGF GradientsShow full caption(A) Pseudo-color Ca2+ images in WT, R1KO, and R3KO growth cones 1 min before and 3.5 min after the onset of repetitive NGF ejection from the direction indicated by the white arrows. The growth cones were preloaded with a ratiometric pair of calcium indicators, OGB-1 and FR, and the OGB-1/FR emission ratio (FOGB-1/FFR, defined as R) was determined. Shown are relative changes in R (R/Rbase, defined as R′) used as a measure of cytosolic Ca2+ levels. The near (red) and far (blue) ROIs were defined as described in Figure 2. Scale bar, 5 μm.(B) Time course changes in R′ in the near (red line) and far (blue line) ROIs positioned on WT (dotted line), R1KO (finely dotted line), and R3KO (solid line) growth cones. Data from the first minute after the start of NGF ejection were excluded because of a lack of stable NGF gradients. Data are represented as mean ± SEM.(C) The mean amplitude of R′ over the last 1 min of repetitive NGF ejection shown in (B). Each gray line connecting two dots represents data from the near and far ROIs of a single growth cone, and each colored bar represents the mean. ∗p < 0.05; ∗∗∗p < 0.001; ns, not significant; Wilcoxon matched pairs signed rank test.See also Figure S2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Pseudo-color Ca2+ images in WT, R1KO, and R3KO growth cones 1 min before and 3.5 min after the onset of repetitive NGF ejection from the direction indicated by the white arrows. The growth cones were preloaded with a ratiometric pair of calcium indicators, OGB-1 and FR, and the OGB-1/FR emission ratio (FOGB-1/FFR, defined as R) was determined. Shown are relative changes in R (R/Rbase, defined as R′) used as a measure of cytosolic Ca2+ levels. The near (red) and far (blue) ROIs were defined as described in Figure 2. Scale bar, 5 μm. (B) Time course changes in R′ in the near (red line) and far (blue line) ROIs positioned on WT (dotted line), R1KO (finely dotted line), and R3KO (solid line) growth cones. Data from the first minute after the start of NGF ejection were excluded because of a lack of stable NGF gradients. Data are represented as mean ± SEM. (C) The mean amplitude of R′ over the last 1 min of repetitive NGF ejection shown in (B). Each gray line connecting two dots represents data from the near and far ROIs of a single growth cone, and each colored bar represents the mean. ∗p < 0.05; ∗∗∗p < 0.001; ns, not significant; Wilcoxon matched pairs signed rank test. See also Figure S2. One possible explanation for the lack of Ca2+ signal asymmetry in R3KO growth cones is widespread distribution of IP3 due to faster diffusion or slower degradation of IP3. To test for this possibility, we monitored spatiotemporal dynamics of IP3 that had been photo-released from caged IP3, using the IP3 sensor IRIS-2.3 (Matsu-ura et al., 2019Matsu-ura T. Shirakawa H. Suzuki K.G.N. Miyamoto A. Sugiura K. Michikawa T. Kusumi A. Mikoshiba K. Dual-FRET imaging of IP3 and Ca2+ revealed Ca2+-induced IP3 production maintains long lasting Ca2+ oscillations in fertilized mouse eggs.Sci. Rep. 2019; 9: 4829Crossref PubMed Scopus (8) Google Scholar). This sensor contains enhanced green fluorescent protein (EGFP) and HaloTag- tetramethylrhodamine (TMR), and the efficiency of fluorescence resonance energy transfer (FRET) from EGFP to TMR decreases upon IP3 binding to IRIS-2.3. Therefore, we calculated the inverse FRET ratio in IRIS-2.3, i.e., the ratio of EGFP emission compared with TMR emission (FEGFP/FTMR) as a measure of IP3 levels. In both WT and R3KO neurons, IP3uncaging on one side of the growth cone resulted in asymmetric increases in IP3, with IP3 levels on the near side being significantly higher than those on the far side (Figures 4A–4C ). Similarly, NGF gradients induced IP3 elevations only on the near sides of both WT and R3KO growth cones (Figures 4D–4F). These data indicate that spatiotemporal dynamics of IP3 is indistinguishable between WT and R3KO growth cones and that the lack of Ca2+ signal asymmetry in R3KO growth cones is not attributable to altered IP3 dynamics. Another possible explanation for the lack of Ca2+ signal asymmetry in R3KO growth cones is increased expression of NGF downstream signaling components such as TrkA and IP3R1, in which the far side of R3KO growth cones could respond to lower concentrations of NGF. However, these possibilities are unlikely because we could not detect any substantial increase in the amount of TrkA and IP3R1 expressed in DRGs of R3KO mice (Figure S2). In R3KO growth cones, symmetric Ca2+ elevations in spite of asymmetric IP3 signals suggest the presence of hypersensitive IP3Rs that can be maximally activated by physiological IP3 signals, such as those in the growth cone near side, and also generate substantial Ca2+ release in response to low levels of IP3, such as those in the growth cone far side. To test for this hypothesis, we monitored Ca2+ responses to low-amplitude IP3 signals generated by FLIP of a smaller amount of caged IP3, i.e., 0.1 μM in contrast to 0.5 μM in previous experiments (Figures 1A, 1B, and 2). These low-amplitude IP3 signals attracted R3KO growth cones but had no detectable effect on directional preference of WT growth cones (Figure 5A). Consistent with these turning responses, R3KO growth cones exhibited asymmetric Ca2+ elevations with higher Ca2+ on the side with IP3 uncaging, whereas WT growth cones showed no detectable Ca2+ increases presumably because the amplitude of IP3 signals were below the threshold for IICR (Figures 5B and 5C). We also examined the effect of lower-concentration NGF gradients, i.e., 0.4 μg/mL NGF in pipette in contrast to 20 μg/mL in previous experiments (Figures 1C, 1D, and 3). These NGF gradients induced neither Ca2+ elevations nor turning responses in WT growth cones but caused asymmetric Ca2+ signals and attractive turning responses in R3KO growth cones (Figures 5D–5F). These data support our notion that R3KO growth cones are hypersensitive to IP3-based guidance cues such as NGF. To determine growth cone sensitivity more comprehensively, we analyzed the effect of other NGF concentrations on axon turning responses using WT- and IP3R-subtype-specific knockout neurons. Dose-response curves were generated by plotting axon turning angles against log-scale NGF concentrations in micropipette (Figure 6). WT and R1KO growth cones were responsive to similar concentration ranges of NGF, but the curve for R3KO growth cones shifted to lower concentration ranges of NGF. Collectively, our results suggest that growth cones can respond to different concentration ranges of IP3-based guidance cues depending on whether IP3R3 participates in the generation of IICR. If R3KO axons are hypersensitive, partial inhibition of a remaining IP3R subtype in these axons, IP3R1, should restore their sensitivity to NGF into normal ranges. It is known that cAMP-dependent phosphorylation of IP3R1 is necessary for IICR (Nakade et al., 1994Nakade S. Rhee S.K. Hamanaka H. Mikoshiba K. Cyclic AMP-dependent phosphorylation of an immunoaffinity-purified homotetrameric inositol 1,4,5-trisphosphate receptor (type I) increases Ca2+ flux in reconstituted lipid vesicles.J. Biol. Chem. 1994; 269: 6735-6742Abstract Full Text PDF PubMed Google Scholar) and that the cAMP antagonist Rp-cAMPS blocks axonal responses to IP3-mediated guidance signals such as NGF (Akiyama et al., 2009Akiyama H. Matsu-ura T. Mikoshiba K. Kamiguchi H. Control of neuronal growth cone navigation by asymmetric inositol 1,4,5-trisphosphate signals.Sci. Signal. 2009; 2: ra34Crossref PubMed Scopus (37) Google Scholar). Therefore, instead of a commonly used Rp-cAMPS concentration of 20 μM, we treated R3KO neurons with 0.4 μM Rp-cAMPS that corresponded to 5% of a reported Ki of 8 μM (Van Haastert et al., 1984Van Haastert P.J. Van Driel R. Jastorff B. Baraniak J. Stec W.J. De Wit R.J. Competitive cAMP antagonists for cAMP-receptor proteins.J. Biol. Chem. 1984; 259: 10020-10024PubMed Google Scholar). Such mild treatment caused R3KO axons to recover attractive turning responses to a normal concentration range of NGF (20 μg/mL in pipette): turning angle (mean ± SEM) = 1.7° ± 3.0° (19 untreated axons) versus 10.5° ± 2.7° (19 Rp-cAMPS-treated axons), p < 0.05; student's t test. These data further support our notion that IP3R sensitivity, which can be regulated by cAMP-dependent phosphorylation, is an important determinant of optimal concentration ranges of NGF for axon guidance. To validate in vivo physiological significance of our findings, we compared projection patterns of NGF-responsive TrkA-expressing sensory axons in the hindpaw skin of WT versus R3KO mice. Sensory axons in the hindpaw skin is one of the most well-studied models, and axon projection patterns are relatively easy to quantify (Patel et al., 2000Patel T.D. Jackman A. Rice F.L. Kucera J. Snider W.D. Development of sensory neurons in the absence of NGF/TrkA signaling in vivo.Neuron. 2000; 25: 345-357Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, Wang et al., 2013Wang T. Jing X. DeBerry J.J. Schwartz E.S. Molliver D.C. Albers K.M. Davis B.M. Neurturin overexpression in skin enhances expression of TRPM8 in cutaneous sensory neurons and leads to behavioral sensitivity to cool and menthol.J. Neurosci. 2013; 33: 2060-2070Crossref PubMed Scopus (18) Google Scholar). We analyzed four pairs of WT/R3KO littermates from IP3R3 heterozygous-heterozygous mating. The border between epidermis and dermis was evident because the epidermis except for its basal layer, but not the dermis, is immunopositive for NGF (Botchkarev et al., 2006Botchkarev V.A. Yaar M. Peters E.M. Raychaudhuri S.P. Botchkareva N.V. Marconi A. Raychaudhuri S.K. Paus R. Pincelli C. Neurotrophins in skin biology and pathology.J. Invest. Dermatol. 2006; 126: 1719-1727Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). We observed that TrkA-positive axons in the epidermis of WT and R3KO mice had different morphologies: WT axons were straighter and well organized (relatively evenly spaced), whereas R3KO axons were undulating and appeared to follow meandering paths (Figure 7A). Such abnormal trajectories of R3KO axons in the skin were also observed in cleared whole-mount tissues where TrkA-positive axons were three-dimensionally reconstructed (Videos S1 and S2). To quantify axon morphological characteristics on skin sections, we employed a “curviness index” of each TrkA-positive axon in the NGF-positive epidermis (see Transparent Methods). The curviness index of 1 indicates the completely straight axon trajectory, whereas larger values represent more curly axons. As shown in Figures 7B and 7C, R3KO axons had significantly larger curviness index than WT axons, confirming our observation that R3KO axons have abnormal trajectories in the skin. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJjODA2MzJhMmU5ZTdlYjcwYTE1NGQ3ODYzZTY2ZjgyNiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjM2NzI3Nzc1fQ.ggIWCfcvc4hFnoD_Kqw8OpPu870gxtVwbkDnVcIG0OiZdePVa2PvEWsjTo59qKnTlVXg2p65E3f4R5Sg4Gkyr8jQmt9B4YxGY-uEAYl1T8uoxYbSnSlHi2CRo9e6KdPsZrBCygXZH9OD_-ZJB_pMdp_aWjCWDGVIEjHcGeHFWQOaI_OoUKAObJr58cV9HUYk8-vXOu_uX6I2kzoMVfAPGq7MHcaWq7tAr4iiWwh_o4y_ZHPqKAbsAxenXhHpQGkW" @default.
• W3009999788 created "2020-03-13" @default.
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• W3009999788 title "Inositol 1,4,5-Trisphosphate Receptor Type 3 Regulates Neuronal Growth Cone Sensitivity to Guidance Signals" @default.
• W3009999788 cites W1496221368 @default.
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