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Triggering of high-speed neurite outgrowth using an optical microheater.

Oyama K, Zeeb V, Kawamura Y, Arai T, Gotoh M, Itoh H, Itabashi T, Suzuki M, Ishiwata S - Sci Rep (2015)

Bottom Line: This high-speed, persistent elongation of neurites was suppressed by inhibitors of both microtubule and actin polymerization, indicating that the thermosensitive dynamics of these cytoskeletons play crucial roles in this heat-induced neurite outgrowth.Furthermore, we showed that microheating induced the regrowth of injured neurites and the interconnection of neurites.These results demonstrate the efficacy of optical microheating methods for the construction of arbitrary neural networks.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.

ABSTRACT
Optical microheating is a powerful non-invasive method for manipulating biological functions such as gene expression, muscle contraction, and cell excitation. Here, we demonstrate its potential usage for regulating neurite outgrowth. We found that optical microheating with a water-absorbable 1,455-nm laser beam triggers directional and explosive neurite outgrowth and branching in rat hippocampal neurons. The focused laser beam under a microscope rapidly increases the local temperature from 36 °C to 41 °C (stabilized within 2 s), resulting in the elongation of neurites by more than 10 μm within 1 min. This high-speed, persistent elongation of neurites was suppressed by inhibitors of both microtubule and actin polymerization, indicating that the thermosensitive dynamics of these cytoskeletons play crucial roles in this heat-induced neurite outgrowth. Furthermore, we showed that microheating induced the regrowth of injured neurites and the interconnection of neurites. These results demonstrate the efficacy of optical microheating methods for the construction of arbitrary neural networks.

No MeSH data available.


Related in: MedlinePlus

Involvement of cytoskeletal components in neurite outgrowth during microheating.Summary of the elongation lengths of neurites after 60 s heat treatment in the absence of chemical agents (Control), and in the presence of 10 nM vinblastine (Vinb), 30 μg·mL−1 colchicine (Col), 10 μM nocodazole (Noco), 20 μM taxol, 10 μM cytochalasin D (CytoD), 10 μM latrunculin B (LatB), 30 μg·mL−1 colchicine and 10 μM cytochalasin D (Col + CytoD), 30 μg·mL−1 colchicine and 10 μM latrunculin B (Col + LatB), 25 μM ciliobrevin D (CilioD), 25 μM blebbistatin (Blebbi), 20 μM ML-7, or 10 μM Y-27632. Bars indicate average values. The laser power was 11 mW, the ΔT was 4.9 ± 0.4 °C (means ± s.d.), and the T0 was 36 °C. Elongation lengths were compared by one-way ANOVA with Tukey-Kramer tests (**p < 0.01, ***p < 0.001; NS, not significant).
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f4: Involvement of cytoskeletal components in neurite outgrowth during microheating.Summary of the elongation lengths of neurites after 60 s heat treatment in the absence of chemical agents (Control), and in the presence of 10 nM vinblastine (Vinb), 30 μg·mL−1 colchicine (Col), 10 μM nocodazole (Noco), 20 μM taxol, 10 μM cytochalasin D (CytoD), 10 μM latrunculin B (LatB), 30 μg·mL−1 colchicine and 10 μM cytochalasin D (Col + CytoD), 30 μg·mL−1 colchicine and 10 μM latrunculin B (Col + LatB), 25 μM ciliobrevin D (CilioD), 25 μM blebbistatin (Blebbi), 20 μM ML-7, or 10 μM Y-27632. Bars indicate average values. The laser power was 11 mW, the ΔT was 4.9 ± 0.4 °C (means ± s.d.), and the T0 was 36 °C. Elongation lengths were compared by one-way ANOVA with Tukey-Kramer tests (**p < 0.01, ***p < 0.001; NS, not significant).

Mentions: Directional changes of neurite outgrowth result from altered activities and distributions of actin networks and microtubules30. We therefore investigated the involvement of cytoskeletal components in the elongation of neurites during microheating. Treatment with a substoichiometric concentration of vinblastine (10 nM), which inhibits microtubule polymerization but does not affect the existing microtubule network3132 (Supplementary Fig. S11), had no effect on the elongation length of neurites subjected to microheating (10.7 ± 4.5 μm, n = 20) (Fig. 4). Thus, microtubule sliding plays a substantial role in the enhanced neurite elongation. In contrast, treatment with 30 μg·mL−1 colchicine (depolymerizer of microtubules) and 20 μM paclitaxel (taxol; stabilizer of microtubules), respectively, resulted in only slight (but not statistically significant) decreases in neurite elongation lengths (9.2 ± 4.8 μm, n = 30 and 8.1 ± 4.4 μm, n = 14, respectively). Likewise, the length of neurite elongation was decreased in the presence of 10 μM nocodazole (depolymerizer of microtubules) (5.4 ± 2.5 μm, n = 16). Furthermore, treatment with the actin polymerization inhibitor latrunculin B (10 μM) resulted in decreases in elongation length (7.8 ± 3.6 μm, n = 25). Although treatment with 10 μM cytochalasin D (inhibitor of actin polymerization, see Supplementary Fig. S10) did not significantly affect elongation length (9.3 ± 4.6 μm, n = 19), double treatment with colchicine and either cytochalasin D or latrunculin B strongly inhibited neurite elongation (3.0 ± 6.2 μm, n = 11, or 1.4 ± 1.8 μm, n = 19, respectively). The distinct effects produced by cytochalasin D and latrunculin B are attributable to the different effects of the reagents on actin. Cytochalasin D caps the barbed ends of F-actin, and inhibits both the polymerization/depolymerization and the direct coupling between F-actins. Besides, cytochalasin D has a potential to sever F-actin33. Therefore, treatment of cytochalasin D fragmented F-actin but did not reduce the amount of F-actin (Supplementary Fig. S10). On the other hand, latrunculin B binds G-actin, inhibits the polymerization and promotes the depolymerization34. Actually, the amount of rhodamine phalloidin-labelled F-actin was significantly decreased in the presence of latrunculin B (Supplementary Fig. S10). Meanwhile, treatment with 25 μM ciliobrevin D (inhibitor of dynein), 25 μM blebbistatin (a direct inhibitor of myosin II activity), and 20 μM ML-7 [an indirect inhibitor of myosin II activity via inhibition of myosin light chain kinase (MLCK)] diminished neurite elongation (0.2 ± 0.5 μm, n = 16, 7.0 ± 3.2 μm, n = 20, and 6.7 ± 3.9 μm, n = 17, respectively). Conversely, 10 μM Y-27632 [an indirect inhibitor of myosin II activity via inhibition of rho-kinase (ROCK)] had no effect on the elongation length compared to the control cells (10.7 ± 3.6 μm, n = 15). These results support the following conclusions: 1) neurite outgrowth induced by microheating is predominantly the result of enhanced microtubule and actin dynamics, and 2) interactions between the cytoskeletal networks and molecular motors are essential for rapid neurite elongation.


Triggering of high-speed neurite outgrowth using an optical microheater.

Oyama K, Zeeb V, Kawamura Y, Arai T, Gotoh M, Itoh H, Itabashi T, Suzuki M, Ishiwata S - Sci Rep (2015)

Involvement of cytoskeletal components in neurite outgrowth during microheating.Summary of the elongation lengths of neurites after 60 s heat treatment in the absence of chemical agents (Control), and in the presence of 10 nM vinblastine (Vinb), 30 μg·mL−1 colchicine (Col), 10 μM nocodazole (Noco), 20 μM taxol, 10 μM cytochalasin D (CytoD), 10 μM latrunculin B (LatB), 30 μg·mL−1 colchicine and 10 μM cytochalasin D (Col + CytoD), 30 μg·mL−1 colchicine and 10 μM latrunculin B (Col + LatB), 25 μM ciliobrevin D (CilioD), 25 μM blebbistatin (Blebbi), 20 μM ML-7, or 10 μM Y-27632. Bars indicate average values. The laser power was 11 mW, the ΔT was 4.9 ± 0.4 °C (means ± s.d.), and the T0 was 36 °C. Elongation lengths were compared by one-way ANOVA with Tukey-Kramer tests (**p < 0.01, ***p < 0.001; NS, not significant).
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Related In: Results  -  Collection

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Show All Figures
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f4: Involvement of cytoskeletal components in neurite outgrowth during microheating.Summary of the elongation lengths of neurites after 60 s heat treatment in the absence of chemical agents (Control), and in the presence of 10 nM vinblastine (Vinb), 30 μg·mL−1 colchicine (Col), 10 μM nocodazole (Noco), 20 μM taxol, 10 μM cytochalasin D (CytoD), 10 μM latrunculin B (LatB), 30 μg·mL−1 colchicine and 10 μM cytochalasin D (Col + CytoD), 30 μg·mL−1 colchicine and 10 μM latrunculin B (Col + LatB), 25 μM ciliobrevin D (CilioD), 25 μM blebbistatin (Blebbi), 20 μM ML-7, or 10 μM Y-27632. Bars indicate average values. The laser power was 11 mW, the ΔT was 4.9 ± 0.4 °C (means ± s.d.), and the T0 was 36 °C. Elongation lengths were compared by one-way ANOVA with Tukey-Kramer tests (**p < 0.01, ***p < 0.001; NS, not significant).
Mentions: Directional changes of neurite outgrowth result from altered activities and distributions of actin networks and microtubules30. We therefore investigated the involvement of cytoskeletal components in the elongation of neurites during microheating. Treatment with a substoichiometric concentration of vinblastine (10 nM), which inhibits microtubule polymerization but does not affect the existing microtubule network3132 (Supplementary Fig. S11), had no effect on the elongation length of neurites subjected to microheating (10.7 ± 4.5 μm, n = 20) (Fig. 4). Thus, microtubule sliding plays a substantial role in the enhanced neurite elongation. In contrast, treatment with 30 μg·mL−1 colchicine (depolymerizer of microtubules) and 20 μM paclitaxel (taxol; stabilizer of microtubules), respectively, resulted in only slight (but not statistically significant) decreases in neurite elongation lengths (9.2 ± 4.8 μm, n = 30 and 8.1 ± 4.4 μm, n = 14, respectively). Likewise, the length of neurite elongation was decreased in the presence of 10 μM nocodazole (depolymerizer of microtubules) (5.4 ± 2.5 μm, n = 16). Furthermore, treatment with the actin polymerization inhibitor latrunculin B (10 μM) resulted in decreases in elongation length (7.8 ± 3.6 μm, n = 25). Although treatment with 10 μM cytochalasin D (inhibitor of actin polymerization, see Supplementary Fig. S10) did not significantly affect elongation length (9.3 ± 4.6 μm, n = 19), double treatment with colchicine and either cytochalasin D or latrunculin B strongly inhibited neurite elongation (3.0 ± 6.2 μm, n = 11, or 1.4 ± 1.8 μm, n = 19, respectively). The distinct effects produced by cytochalasin D and latrunculin B are attributable to the different effects of the reagents on actin. Cytochalasin D caps the barbed ends of F-actin, and inhibits both the polymerization/depolymerization and the direct coupling between F-actins. Besides, cytochalasin D has a potential to sever F-actin33. Therefore, treatment of cytochalasin D fragmented F-actin but did not reduce the amount of F-actin (Supplementary Fig. S10). On the other hand, latrunculin B binds G-actin, inhibits the polymerization and promotes the depolymerization34. Actually, the amount of rhodamine phalloidin-labelled F-actin was significantly decreased in the presence of latrunculin B (Supplementary Fig. S10). Meanwhile, treatment with 25 μM ciliobrevin D (inhibitor of dynein), 25 μM blebbistatin (a direct inhibitor of myosin II activity), and 20 μM ML-7 [an indirect inhibitor of myosin II activity via inhibition of myosin light chain kinase (MLCK)] diminished neurite elongation (0.2 ± 0.5 μm, n = 16, 7.0 ± 3.2 μm, n = 20, and 6.7 ± 3.9 μm, n = 17, respectively). Conversely, 10 μM Y-27632 [an indirect inhibitor of myosin II activity via inhibition of rho-kinase (ROCK)] had no effect on the elongation length compared to the control cells (10.7 ± 3.6 μm, n = 15). These results support the following conclusions: 1) neurite outgrowth induced by microheating is predominantly the result of enhanced microtubule and actin dynamics, and 2) interactions between the cytoskeletal networks and molecular motors are essential for rapid neurite elongation.

Bottom Line: This high-speed, persistent elongation of neurites was suppressed by inhibitors of both microtubule and actin polymerization, indicating that the thermosensitive dynamics of these cytoskeletons play crucial roles in this heat-induced neurite outgrowth.Furthermore, we showed that microheating induced the regrowth of injured neurites and the interconnection of neurites.These results demonstrate the efficacy of optical microheating methods for the construction of arbitrary neural networks.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.

ABSTRACT
Optical microheating is a powerful non-invasive method for manipulating biological functions such as gene expression, muscle contraction, and cell excitation. Here, we demonstrate its potential usage for regulating neurite outgrowth. We found that optical microheating with a water-absorbable 1,455-nm laser beam triggers directional and explosive neurite outgrowth and branching in rat hippocampal neurons. The focused laser beam under a microscope rapidly increases the local temperature from 36 °C to 41 °C (stabilized within 2 s), resulting in the elongation of neurites by more than 10 μm within 1 min. This high-speed, persistent elongation of neurites was suppressed by inhibitors of both microtubule and actin polymerization, indicating that the thermosensitive dynamics of these cytoskeletons play crucial roles in this heat-induced neurite outgrowth. Furthermore, we showed that microheating induced the regrowth of injured neurites and the interconnection of neurites. These results demonstrate the efficacy of optical microheating methods for the construction of arbitrary neural networks.

No MeSH data available.


Related in: MedlinePlus