In vivo nanosecond laser axotomy: cavitation ... - OSA Publishing

In vivo nanosecond laser axotomy: cavitation dynamics and vesicle transport G. Nageswara Rao, Sucheta S. Kulkarni, Sandhya P. Koushika and Kaustubh R. Rau National Centre for Biological Sciences–TIFR, Bangalore 560065, INDIA. koushika@ncbs.res.in, kaustubh@ncbs.res.in

Abstract: Nanosecond laser pulses (λ = 355 nm) were used to cut mechanosensory neurons in Caenorhabditis elegans and motorneurons in Drosophila melanogaster larvae. A pulse energy range of 0.8–1.2 μ J and < 20 pulses in single shot mode were sufficient to generate axonal cuts. Viability post-surgery was >95% for C. elegans and 60% for Drosophila. Cavitation bubble dynamics generated due to laser-induced plasma formation were observed in vivo by time-resolved imaging in both organisms. Bubble oscillations were severely damped in vivo and cavitation dynamics were complete within 100 ns in C. elegans and 800 ns in Drosophila. We report the use of this system to study axonal transport for the first time and discuss advantages of nanosecond lasers compared to femtosecond sources for such procedures. © 2008 Optical Society of America OCIS codes: (140.3440) Laser-induced breakdown; (170.1020) Ablation of tissue; (170.6920) Time-resolved imaging.

References and links 1. M. F. Yanik, H. Cinar, H. N. Cinar, A. D. Chisholm, Y. Jin and A. Ben-Yakar, “Functional regeneration after laser axotomy,” Nature 432, 882 (2004). 2. S. H. Chung, D. A. Clark, C. V. Gabel, E. Mazur and A. D. Samuel,“The role of the AFD neuron in C. elegans thermotaxis analyzed using femtosecond laser ablation,” BMC Neuroscience 7, DOI:10.1186/1471-2202-7-30 (2006). 3. Z. Wu, A. Ghosh-Roy, M. F. Yanik, J. Z. Zhang, Y. Jin and A. D. Chisholm, “Caenorhabditis elegans neuronal regeneration is influenced by life stage, ephrin signaling and synaptic branching,” PNAS 104, 15132-15137 (2007). 4. P. Weiss and H. B. Hiscoe,“Experiments on the mechanism of nerve growth,” J. Exp. Zool. 107 315-395 (1948). 5. R. V. Barkus, O. Klyachko, D. Horiuchi B. J. Dickson and W. M. Saxton, “Identification of an Axonal Kinesin-3 Motor for Fast Anterograde Vesicle Transport that Facilitates Retrograde Transport of Neuropeptides,” Mol. Biol. Cell 19, 274-283 (2008). 6. H. Erez, G. Malkinson, M. Prager-Khoutorsky, C. I. De Zeeuw, C. C. Hoogenraad and M. E. Spira, “Formation of microtubule-based traps controls the sorting and concentration of vesicles to restricted sites of regenerating neurons after axotomy,” J. Cell Biol. 176, 497-507 (2007). 7. M. S. Hutson and X. Ma, “Plasma and cavitation dynamics during pulsed laser microsurgery in vivo,” Phys. Rev. Lett. 99, 158104 (2007). 8. T. R. Mahoney, Q. Liu, T. Itoh, S. Luo, G. Hadwiger, R. Vincent, Z-W. Wang, M. Fukuda and M. L. Nonet, “Regulation of synaptic transmission by RAB-3 and RAB-27 in Caenorhabditis elegans,” Mol. Biol. Cell 17, 2617-2625 (2006). 9. M. L. Nonet, “Visualization of synaptic specializations in live C. elegans with synaptic vesicle protein-GFP fusions,” J. Neurosci. Methods 89, 33-40 (1999).

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Received 4 Mar 2008; revised 1 Jun 2008; accepted 2 Jun 2008; published 20 Jun 2008

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10. T. Lee,and L. Luo, “Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis,” Neuron 22, 451-61 (1999). 11. A. Mahr and H. Aberle, “The expression pattern of the Drosophila vesicular glutamate transporter: A marker protein for motoneurons and glutamatergic centers in the brain, Gene Expr. Patterns 6, 299-309 (2006). 12. A. V. Cherian and K. R. Rau, “Pulsed laser-induced damage in rat corneas: time-resolved imaging of physical effects and acute biological response,” J. Biomed. Opt. 13, 024009 (2008). 13. M. F. Yanik, H. Cinar, H. N. Cinar, A. Gibby, A. D. Chisholm, Y. Jin and A. Ben-Yakar, “Nerve regeneration in Caenorhabditis elegans after femtosecond laser axotomy,” IEEE J. Quantum Electron. 12, 1283-1291 (2006). 14. V. Venugopalan, A. Guerra III, K. Nahen and A. Vogel, “Role of Laser-Induced Plasma Formation in Pulsed Cellular Microsurgery and Micromanipulation,” Phys. Rev. Lett. 88, 078103 (2002). 15. U.K. Tirlapur and K. K¨onig, “Targeted transfection by femtosecond laser,” Nature 418, 290-291 (2002). 16. W. Watanabe and N. Arakawa, “Femtosecond laser disruption of subcellular organelles in a living cell,” Opt. Express 12, 4203-4213 (2004). 17. A. Heisterkamp, I. Zaharieva Maxwell, E. Mazur, J. M. Underwood, J. A. Nickerson, S. Kumar and D. E. Ingber, “Pulse energy dependence of subcellular dissection by femtosecond laser pulses,” Opt. Express 13, 3690-3696 (2005). 18. V. Magidson, J. Lonˇcarek, P. Hergert, C. L. Reider and A. Khodjakov,“Laser Microsurgery in the GFP Era: A Cell Biologist’s Perspective,” Methods Cell Biol. 82, 239-266, (2007). 19. W. Grill, P. G¨onczy, E. H. K. Stelzer and A. A. Hyman “Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo,” Nature 409, 630-633 (2001). 20. M.S. Hutson, Y. Tokutake, M.-S. Chang, J.W. Bloor, S. Venakides, D.P. Kiehart, G.S. Edwards “Forces for morphogenesis investigated with laser-microsurgery and quantitative modeling,” Science 300, 145-149 (2003). 21. F. Bourgeois and A. Ben-Yakar, “Femtosecond laser nanoaxotomy properties and their effect on axonal recovery in C. elegans,” Opt. Express 15, 8521-8531 (2007). 22. J. J. Fernandes and H. Keshishian “Nerve-muscle interactions during flight muscle development in Drosophila,” Development, 125, 1769-1779 (1998). 23. K. R. Rau, P. A. Quinto-Su, A. Hellman and V. Venugopalan, “Pulsed laser microbeam-induced cell lysis: timeresolved imaging and analysis of hydrodynamic effects,” Biophys. J. 91, 317-329 (2006). 24. A. Vogel, M. R. C. Capon, M. N. Asiyo-Vogel, and R. Birngruber, “Intraocular photodisruption with picosecond and nanosecond laser pulses: Tissue effects in cornea, lens, and retina,” Invest. Ophthalmol. Visual Sci. 35, 3032-3044 (1994). 25. A. Vogel, N. Linz, S. Freidank and G. Paltauf, “Femtosecond-Laser-Induced Nanocavitation in Water: Implications for Optical Breakdown Threshold and Cell Surgery,” Phys. Rev. Lett. 100, 038102 (2008).

1. Introduction Recent reports on using femtosecond (fs) lasers for conducting neuronal axotomy in C. elegans have generated a lot of excitement in the field of neuroscience [1, 2, 3]. The ability to selectively ablate an individual axon in a genetically tractable organism makes it an extremely powerful system to study several aspects of neuronal function with an important one being regeneration. Another fundamental cellular process, viz. transport of cargo, is essential in long polarized cells like neurons since protein synthesis occurs in the cell body and synaptic transmission at the end of the axon. Long term assays conducted over several hours to study transport were introduced in and were till recently limited to larger organisms such as mice [4]. These are usually carried out by tying a suture to compress long nerves such as the sciatic nerve and examining cargo accumulation at later time-points. Attempts to develop similar transport assays in smaller genetic model organisms such as Drosophila have met with some success [5]. Post axotomy imaging of transport in Aplysia neurons in culture has revealed that transport changes occur rapidly and a microtubule-dependent vesicle trap is set up that optimizes growth cone formation [6]. Since growth cone formation is an important step in axon regrowth it is critical to develop assays in vivo that allow determination of changes to transport during this process. We report the development of a transport assay in C. elegans using laser axotomy to physically block transport. The nanosecond (ns) laser used in our studies reproduced earlier results using fs systems for neuronal axotomy. By imaging accumulations of fluorescently labeled synaptic vesicle proteins at the site of axonal microsurgery we can characterize anterograde and retrograde transport. This assay is simple in procedure and provides quick results (1 hour) and could #93418 - $15.00 USD

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be used to study several aspects of transport in neurons, some of which we discuss later. We also studied the physical nature of the ablation process using time-resolved imaging to capture cavitation bubble dynamics in C. elegans and Drosophila larvae. Time-resolved imaging of the cavitation bubble size provides a direct measure of the amount of damage during axotomy procedures and can also provide estimates of the mechanical forces generated during the process. We compare cavitation dynamics from our study with those reported in a recent study on laser microsurgery in Drosophila embryos using 355 nm nanosecond laser pulses [7]. We also discuss the advantages of nanosecond lasers as compared to femtosecond lasers for such procedures. 2. Materials and methods The 3rd harmonic output (λ = 355 nm, τ p = 6 ns) of a Nd:YAG laser (Spitlight 600, Innolas, Munich, Germany) was introduced through the back port of an inverted microscope (Olympus iX71) and reflected by a dichroic mirror (Chroma 532/355 - zet532nbdc) into the objective back aperture. The microscope has two back ports allowing separate paths for fluorescence excitation using a standard mercury arc lamp and ablation with the Nd:YAG laser. Transgenic C. elegans jsIs821 and jsIs37 expressing GFP::RAB-3 [8] and synaptobrevin::GFP (SNB-1::GFP) [9] respectively in mechanosensory neurons were used in our experiments. Drosophila OK371-Gal4; UAS-membrane GFP targeted to motor neurons were also used [10, 11]. C. elegans L4 larvae were anesthetized using 0.13% (w/v) sodium azide on an agar pad, while Drosophila 1 st instar larvae were immobilized using 4 ◦ C cooling. Positioning the animal on the coverslip was random for both C. elegans and Drosophila. In C. elegans, focused posterior lateral mechanosensory (PLM) axons are 1 to 3 μ m from the coverslip. In Drosophila, we targeted axon bundles 40 to 50 μ m away from the ventral ganglion with these bundles being 5 to 10 μ m from the coverslip. Axotomy was carried out with a 100×, 1.3 NA objective. Fluorescently labelled axons were brought into focus and irradiated, with the laser being operated in single shot mode. The operator manually delivered single pulses to the desired site till it was visually confirmed that the axon was cut. In all surgeries reported less than 20 pulses (typically 10 to 15) were required for generating a cut. Pulse energy used was 0.8 μ J for C. elegans and 1.2 μ J for Drosophila. Laser pulse energy was measured at the objective back aperture by removing the objective from the turret and allowing the beam to illuminate an energy detector (J3-05, Molectron Inc.). The manufacturer specified transmission of our objective at 355 nm is 40%. All pulse energies reported were calculated after factoring in the objective transmissivity. The probability of plasma formation in vivo was ≈30% at 0.8 μ J. Time-resolved images of the ablation process were collected using a setup previously optimized in our lab [12]. In brief, the sample was illuminated with a broadband nanosecond pulse at a specified time delay with respect to the ablation laser pulse. The illumination pulse was generated by pumping a dye solution (LDS 698, Exciton Inc., 0.1 μ M in methanol) with the 2nd (λ = 532 nm) harmonic output of the Nd:YAG laser. The fluorescent emission of the dye was coupled into a fiber optic line with the distal end of the fiber being focussed onto the sample. The length of the fiber optic line decided the delay between the ablation pulse and the illumination pulse arriving at the sample. Images were collected on an intensified CCD (Andor iStar) with an exposure duration of 5 ns and gain of 120. Multiple images were collected at the same time delay to determine average bubble sizes. For each image the laser focus was moved to a new position on the targeted axon, so that residual bubbles from a previous measurement would not affect the image. Contrast and brightness were manually adjusted for all figures using the levels menu in Adobe Photoshop. Areas of GFP::RAB-3 accumulations quantified in Fig. 7 were measured in ImageJ using images of cut and uncut axons captured with the same exposure time and #93418 - $15.00 USD

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camera gain. Significance was tested using the student t-test with unequal variance. 3. Results 3.1. Nanosecond laser axotomy In Fig. 1 we present results of axonal microsurgery using nanosecond laser pulses in C. elegans larvae. A larva in which the PLM neuron was targeted is shown before ((a),(c),(e)) and after ((b),(d),(f)) the surgery. The damaged region or neuronal cut is shown magnified in the inset in (b), (d) and (f). In all cases we observe a break in the axon at the site of laser focus. These particular axotomies have been chosen to exhibit the range of damage that the laser microbeam produced. For instance, in 1(b) and 1(d) we could observe clear breaks in the axon extending over 3-5 μ m. In 1(f) however the visible break is barely discernable being < 1 μ m and the only other evidence for axonal cutting is the displacement of the proximal end due to cavitation forces generated by plasma formation. The average extent of damage in these axons was measured to be 1.95 ± 1.81 μ m (n = 8). The large variation in extent of damage occurs due to inclusion of all cut axons including those that move significantly due to the cavitation forces.

Fig. 1. Axonal microsurgery of PLM neurons in C. elegans larva with nanosecond laser pulses. The axon is shown prior to (a,c,e) and immediately after surgery (b,d,f). Insets in (b,d,f) show magnified regions around the cut site. Clear breaks can be seen in the axon (b,d). In (f) the extent of damage is extremely small and is only noticeable as a dark band. The proximal end of the axon was also displaced due to the cavitation forces generated during the laser microsurgery. Pulse energy was 0.8 μ J with the laser being operated in single shot mode.

We found that a pulse energy of 0.8 μ J was sufficient to cut axons in C. elegans with minimal collateral damage. The laser was used in single shot mode and the operator manually fired the laser till the axon was cut. The number of pulses varied between individuals but was always < 20. In a majority of surgeries one laser pulse was sufficient to effect a complete axonal cut, with the position of the laser spot being the major determinant of success. In a few cases we also observed partial cutting of the axon effected by the first few pulses, following which a succeeding laser pulse would fully cut it. Physical movement of the cut ends due to cavitation forces could also be observed. Thus, the site of laser focus and physical movement influenced the extent of damage observed immediately after the axotomy. Successful axonal cutting resulted in a retraction of the two ends away from the site of laser irradiation. This retraction was observable within 1 hr of microsurgery and has also been noted by other researchers [3, 13]. #93418 - $15.00 USD

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We measured the extent of damage in C. elegans to be 4.65 ± 1.85 μ m (n = 17) at 1 hr after laser microsurgery. To determine collateral damage during laser axotomy, we viewed animals 4, 6 and 8 hours post-surgery under DIC and noted scarring in only 8% of the animals (n = 24). Animals undergoing laser axotomy showed viability levels > 95% after removal of anesthetic and continued to undergo development and become fertile adults. Viability in control animals (sham surgery treated) was 100% (n = 20). Post surgery, C. elegans larvae were maintained at 16 ◦ C and followed in the 1-3 hr time period to assess the time required for accumulation of vesicles and determine cellular outcomes after damage. In Fig. 2(a,b,c) axons in 3 individual larvae are shown at different times after surgery. One hour post surgery (Fig. 2(a)) we could observe robust accumulations of GFP::RAB-3, a known synaptic vesicle marker. The accumulation of GFP::RAB-3 signal results from a block at the distal and proximal cut ends. At later time points we do not see any accumulations. Instead we observed regenerative processes similar to those reported earlier for fs laser surgery in C. elegans. At 3 hours, we could observe close proximity of the distal and proximal ends (Fig. 2(b), inset). This maybe an attempted reconnection of the cut ends and was observed in 46% of the animals (n = 13). Regrowth of a new axon from the proximal end occurred in 54% of the animals (n = 13). At 9 hours, (Fig. 2(c)), regrowth was seen in all individuals (n = 4). Similar putative reconnection events have been observed by others [3, 13].

Fig. 2. Manifestions of axonal recovery at different time-points in C. elegans. In (a) vesicle accumulations are seen at 1 hour, in (b) axonal reconnection is seen at 3 hours and in (c) axonal regrowth at 9 hours. Inset in (a) shows a magnified view of the accumulation zone and inset in (b) shows the magnified reconnection zone. Arrow in (c) indicates the growing proximal end, while arrowhead indicates the distal stump.

3.2. Time-resolved imaging in vivo To determine the dynamics of the ablation process we conducted time-resolved imaging in vivo. Focused nanosecond laser pulses beyond an intensity threshold (I th > 1010 W/cm2 ) result in plasma formation during laser microsurgery [14]. The high temperature plasma generates a cavitation bubble whose expansion and collapse leads to collateral damage around the site of the laser irradiation. Our time-resolved imaging could successfully capture these fast bubble dynamics in C. elegans larvae (Fig. 3). This is the first time that the damage process has been visualized at high resolution during laser microsurgery in vivo. In C. elegans, we observed a weak plasma with a lifetime