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Chapter 14

Use of Cell-Stretch System to Examine the Characteristics of Mechanosensor Channels: Axonal Growth/Neuroregeneration Studies

Koji Shibasaki

Abstract

Temperature-sensitive TRP (so-called “thermoTRP”) channels are well recognized for their contributions to sensory transduction, responding to a wide variety of stimuli including temperature, nociceptive stimuli, touch, and osmolarity. However, the precise roles for the thermoTRP channels during development have not been determined. To explore the functional importance of thermoTRP channels during neural devel-opment, the temporal expression was determined in embryonic mice. Interestingly, TRPV2 expression was detected in spinal motor neurons in addition to the DRG from E10.5, and was localized in axon shafts and growth cones, suggesting that the channel is important for axon outgrowth regulation. We revealed that endogenous TRPV2 was activated in a membrane-stretch-dependent manner in developing neurons by knocking down the TRPV2 function with dominant negative TRPV2 and TRPV2-speci fi c shRNA, and signi fi cantly promoted axon outgrowth. In this section, the author introduces experimental methods to investigate the mehcanosensor functions of TRPV2 in axonal outgrowth or regeneration.

Key words: Mechanosensor , TRPV2 , Electroporation , In ovo , DRG , Motor neuron , Culture

Mechanisms of axonal outgrowth still have many mysteries, although many chemoattractive and chemorepulsive molecules related to axonal outgrowth were identi fi ed, and their intracellular signaling was examined. It is a speci fi c characteristic that neurons can grow to the length of more than 1 m in humans ( 1, 2 ) . This mechanism of elongation has been called “passive stretching” ( 2 ) . From embryonic stages, the passive stretching-dependent axonal outgrowth begins. As our body grows, the distances between

1. Introduction

Arpad Szallasi and Tamás Bíró (eds.), TRP Channels in Drug Discovery: Volume II, Methods in Pharmacology and Toxicology, DOI 10.1007/978-1-62703-095-3_14, © Springer Science+Business Media, LLC 2012

232 K. Shibasaki

neuronal cell bodies and growth cones gradually increase, thereby exerting tensile forces on the axons.

In some of in vitro studies, the growth cones of cultured sen-sory axons were attached to glass needles to examine their response to forces ( 3, 4 ) . Axons could be stretched up to approximately 100 μ m over a few hours ( 2 ) . Moreover, arti fi cial external forces induce axonal outgrowth. A group developed a unique chamber system in which neurons are cultured on two initially contiguous platforms that are pulled apart by a stepped motor ( 5, 6 ) in order to improve axonal regeneration following injury. The axons plated onto the platforms can be elongated by this system. This system provides ten times faster speed than typical growth-cone-mediated axonal outgrowth rates ( 7 ) . In addition to the above observations, using orthopedic leg-lengthening procedures in adult rats, it was found that applied forces in vivo could double inter-nodal dis-tances. Notably, acute stretching resulting in high tension, as it occurs clinically when large nerve gaps are directly joined, impairs axonal regeneration ( 8, 9 ) .

Recently, we reported that TRPV2 was a mechanosensor chan-nel which contributed to axonal outgrowth in a membrane stretch-dependent manner ( 10 ) , consistent with previous report described the sensor function of TRPV2 against hypotonic stimulus ( 11 ) . Taken together, these results indicate that forces are powerful stimulators of axonal outgrowth through TRPV2 activation. In addition to those reports, we also recently reported that activation of TRPV2 through mechanical stimulus by intestinal movement regulated intestinal motility ( 12 ) . In this section, the author intro-duces good systems to investigate the mehcanosensor functions in axonal outgrowth or regeneration.

1. ICR strain mice were utilized. Embryos were considered as E0.5 at noon on the day at which vaginal plugs were observed.

2. Fertilized chicken eggs were purchased from Gen Corporation (Gifu, Japan), and the eggs were cultured for 3 days at 38.5°C until they become embryos at the Hamburger and Hamilton stage (HH) 10–14.

3. All animal care and procedures were performed according to NIH, NIPS (National Institute for Physiological Sciences) and Gunma University guidelines.

1. Membrane stretch was applied by a computer-controlled step-ping motor machine (STB-150, STREX, Japan) as previously described ( 13 ) . In vitro and in ovo electroporations were per-formed by a pulse generator, ECM830 (BTX).

2. Materials

2.1. Animals

2.2. Equipment

23314 Use of Cell-Stretch System to Examine the Characteristics…

1. A standard bath solution containing 140 mM NaCl, 5 mM KCl, 2 mM MgCl 2 , 2 mM CaCl 2 , 10 mM HEPES, and 10 mM glu-cose, pH 7.4, was used for whole cell patch-clamp recordings. The standard bath solution for the patch-clamp experiments was the same as that used in fl uorescence measurements.

2. Pipette solution for whole-cell recordings contained 140 mM CsCl, 0.5 mM EGTA, 2 mM Mg-ATP, 2 mM K 2 -GTP, and 10 mM HEPES, pH 7.4.

Embryonic mouse DRG or motor neurons were prepared using a modi fi ed protocol originally designed for cultivation of the mouse hippocampus ( 14 ) .

1. DRGs or the ventral half of spinal cords was dissected from E12.5 embryos and dissociated using mechanical trituration.

2. Cells were plated on poly- d -lysine-coated coverslips (15 mm round, Assistant, Germany) at a fi nal density of 3–5 × 10 5 cells/coverslip in Neurobasal Medium (Invitrogen, Carlsbad, CA) with B27 supplement (Invitrogen), NGF (10 ng/mL, Sigma, St. Louis, MO), NT-3 (10 ng/mL, Calbiochem, La Jolla, CA), and penicillin/streptomycin (1:250, Invitrogen, Carlsbad, CA).

3. After 12 h, coverslips were immersed in fresh Neurobasal Medium with B27 supplement, NGF and NT-3. To prevent overgrowth of glia and fi broblasts, cultures were treated with cytosine arabinoside (5 μ M; Calbiochem). Embryonic DRG explant cultures were also performed from E12.5 embryos. Dissected DRGs were put on poly- d -lysine-coated coverslips in Neurobasal Medium with B27 supplement, NGF and NT-3. After 12 h, coverslips were immersed in fresh Neurobasal Medium with B27 supplement, NGF and NT-3 (with cytosine arabinoside (5 μ M)). To examine the effect of low calcium on TRPV2-dependent axon outgrowth, we uti-lized low Ca 2+ DMEM (0.15 mM low Ca 2+ ) or regular DMEM (2 mM normal Ca 2+ ) with 10% fetal bovine serum, NGF, NT-3, and penicillin/streptomycin instead of above culture medium.

1. Membrane stretch was applied by a machine (STB-150) as described above.

2. Dissociated DRG or ventral spinal cord cells were transferred onto a 4-cm 2 silicon chamber (Fig. 1a , b) coated with 50 μ g/mL fi bronectin at a density of 3 × 10 4 cells/cm 2 .

2.3. Solutions

3. Methods

3.1. Cultivation of Dissociated Embryonic DRG/Motor Neuron Cells and Embryonic DRG Explants

3.2. Application of Cyclic Stretch

234 K. Shibasaki

3. After 2 days, the silicon chamber was attached to a stretching apparatus that was driven by a computer-controlled stepping motor (Fig. 2a ). Using this system, quantitative and uniform stretch (+2.8% length for 15 s) was applied to the cells upon Ca 2+ -imaging experiments by Fura-2 (Fig. 2b, c ).

In vitro electroporation was performed by a modi fi ed protocol as previously described ( 15, 16 ) .

1. Embryonic DRGs or the ventral half of spinal cords (at E12.5) were dissociated using mechanical trituration from anesthe-tized embryos by chilling on ice. DRGs and spinal cords were transferred into DNA solutions (5 μ g/ μ L) in PBS containing 0.1% fast green as a tracer were transferred to the electropora-tion chamber with dissociated DRG cells.

2. Five square pulses (33 mV) of 50-ms duration with 950-ms intervals were applied by a pulse generator, ECM830 (BTX).

3.3. In Vitro and In Ovo Electroporation

3.3.1. In Vitro Electroporation

Fig. 1. In vitro cell-stretch system. ( a , b ) Elastic silicone chambers and their dimensions. Two pieces of cover glass ( rect-angle ) are attached to the bottom of the silicone chamber with an adhesive agent and a 1 mm width slit (from glass edge to edge) is made in the center of the chamber so that only the slit area can be elongated upon extension. ( c ) DRG neurons were cultured on the silicone chamber (the gray 18 mm × 18 mm square place in ( b )) after EGFP cDNA was electroporated. Many soma and axons were visualized by EGFP expression.

23514 Use of Cell-Stretch System to Examine the Characteristics…

In ovo electroporation was performed as described previously ( 17 ) .

1. Chicken eggs were windowed, small amounts of EGFP-reporter plasmids were injected in the neural tube lumen in chick embryos at the HH 10–14 with 0.05% fast green (Fig. 3a ), and the injected DNA was unilaterally pulse-electroporated (35 mV, 5 times, 50 ms duration with 950 ms intervals).

2. The window was sealed with adhesive tape and the eggs were returned to the incubator (38.5°C) for further incubation. The bodies of embryos were dissected after 1 day, and the EGFP-leveled axon length was measured and quanti fi ed (Fig. 3b ).

Fura2 fl uorescence was measured by Fura2-AM (Molecular Probes, Carlsbad, CA) in a standard bath solution as described above. The 340:380 nm ratio was recorded. Whole-cell recording data were sampled at 10 kHz and fi ltered at 5 kHz for analysis (Axon 200B ampli fi er with pCLAMP software, Axon Instruments, Foster City, CA).

3.3.2. In Ovo Electroporation

3.4. Fluorescent Measurements and Electrophysiology

Fig. 2. In vitro cell-stretch system. ( a ) After 48 h of cell culture, the silicone chamber is set in two arms of the extension device on the Ca 2+ -imaging microscope. An arrow in ( c ) indicates the direction of extension. ( b) HEK293 cells expressing TRPV2 were exposed to membrane stretch (102.8% extension) for 15 s by the STREX machine during Ca 2+ -imaging. The red signals in the most left picture represent the TRPV2 transfected cells revealed by Ds-Red co-expression. Fura-2 ratio traces by symbols are from the cells indicated by the same symbols in the pseudocolor image. Ca 2+ in fl ux was observed only in the trasfected cells ( red cells ) by 102.8% stretch. ( c ) The representative traces are shown in the graph (both trans-fected and non-transfetced cells). These fi gures are cited and modi fi ed by a previous report ( 10 ).

236 K. Shibasaki

1. The silicon chambers, which were used for membrane stretch experiments, must be coated by fi bronectin.

2. The silicon chambers can be used repeatedly after wash and pasteurized.

3. Power of uniform stretch was determined by distance of each slide glass (see Fig. 2a ) in bottom of the silicon chamber.

4. For in ovo electroporation, you must make fi ne glass pipettes. The pipette resistance should be over 15 Mohm (if you can check by patch-clamp).

4. Notes

Fig. 3. In ovo electroporation revealed that TRPV2 was involved in axonal outgrowth through its activation by membrane stretch. ( a ) Schematic drawing of in ovo electroporation method. Egg shells were broken by scissors, and the chick embryos were visualized. DNA solution (5 mg/mL) was injected into the neural tube by glass pipette (the author used mouth glass pipet-like ES cell injection for KO mice generation). After DNA injection (you can check by the color of Fastgreen), platinum electrodes were placed both sides of the embryo. Then, fi ve square pulses (33 mV) of 50-ms duration with 950-ms intervals were applied by a pulse generator, ECM830 (BTX). The egg shells were sealed by tapes, and the eggs were incubated for 24 h at 38.5°C. ( b ) Representative images of motor neurons, which were identi fi ed by neuro fi lament expres-sion ( red ) in chick embryos; a control spinal cord tissue expressing EGFP, a tissue expressing wild type TRPV2 ( WT-V2) and a tissue expressing dominant negative TRPV2 (DN-V2). Arrowheads indicate commissure axons. All plasmid DNAs were incorporated by electroporation in ovo at HH 10–14 stages. After 1 day, chick embryos were fi xed and tissue sections were prepared. WT-V2 expression signi fi cantly enhanced axon outgrowth compared with EGFP ( dashed square ), but DN-TRPV2 expression signi fi cantly reduced axon outgrowth compared with EGFP ( dashed square ). Scale bar, 1 mm. These fi gures are cited and modi fi ed by a previous report ( 10 ).

23714 Use of Cell-Stretch System to Examine the Characteristics…

5. After you purchased the fertilized chicken eggs, you have to keep those at 38.5°C for 3 days. The embryos located to top of the eggs. So you must carefully consider where you would like to make windows for the manupuration.

6. If you need long culture for in ovo electroporation, you have to transfer the embryos to the other egg shells.

7. If the response is too small in Ca 2+ -imaging experiments, you should add 0.02% (v/v) pluronic F-127 (in 140 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 2 mM MgCl2, 10 mM HEPES, and 10 mM glucose, pH 7.4, adjusted with NaOH). This manupu-ration greatly enhances the response.

References

1. Smith DH (2009) Stretch growth of inte-grated axon tracts: extremes and exploitations. Prog Neurobiol 89:231–239

2. Suter DM, Miller KE (2011) The emerging role of forces in axonal elongation. Prog Neurobiol 94:91–101

3. Bray D (1984) Axonal growth in response to experimentally applied mechanical tension. Dev Biol 102:379–389

4. Lamoureux P, Buxbaum RE, Heidemann SR (1989) Direct evidence that growth cones pull. Nature 340:159–162

5. P fi ster BJ, Iwata A, Meaney DF, Smith DH (2004) Extreme stretch growth of integrated axons. J Neurosci 24:7978–7983

6. P fi ster BJ, Bonislawski DP, Smith DH, Cohen AS (2006) Stretch-grown axons retain the ability to transmit active electrical signals. FEBS Lett 580:3525–3531

7. Gordon-Weeks PR (ed) (2000) Neuronal growth cones. Cambridge University Press, Cambridge

8. Sunderland IR, Brenner MJ, Singham J, Rickman SR, Hunter DA, Mackinnon SE (2004) Effect of tension on nerve regeneration in rat sciatic nerve transection model. Ann Plast Surg 53:382–387

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stretch in developing sensory and motor neurons. J Neurosci 30:4601–4612

11. Muraki K, Iwata Y, Katanosaka Y, Ito T, Ohya S, Shigekawa M, Imaizumi Y (2003) TRPV2 is a component of osmotically sensitive cation channels in murine aortic myocytes. Circ Res 93:829–838

12. Mihara H, Boudaka A, Shibasaki K, Yamanaka A, Sugiyama T, Tominaga M (2010) Involvement of TRPV2 activation in intestinal movement through nitric oxide production in mice. J Neurosci 30:16536–16544

13. Naruse K, Yamada T, Sokabe M (1998) Involvement of SA channels in orienting response of cultured endothelial cells to cyclic stretch. Am J Physiol 274:H1532–H1538

14. Shibasaki K, Suzuki M, Mizuno A, Tominaga M (2007) Effects of body temperature on neural activity in the hippocampus: regulation of rest-ing membrane potentials by transient receptor potential vanilloid 4. J Neurosci 27:1566–1575

15. Matsuda T, Cepko CL (2004) Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci USA 101:16–22

16. Nakahira E, Kagawa T, Shimizu T, Goulding MD, Ikenaka K (2006) Direct evidence that ven-tral forebrain cells migrate to the cortex and con-tribute to the generation of cortical myelinating oligodendrocytes. Dev Biol 291:123–131

17. Itasaki N, Bel-Vialar S, Krumlauf R (1999) ‘Shocking’ developments in chick embryology: electroporation and in ovo gene expression. Nat Cell Biol 1:E203–E207