Journal of Neuroscience Methods 152 (2006) 8390

Isolation of neuronal substructures and precise neural microdissection using a nanocutting device
Wesley C. Chang a, , Christopher G. Keller b , David W. Sretavan a
a

Departments of Ophthalmology and Physiology, Program in Neuroscience, Bioengineering Graduate Program, University of California, 10 Koret Way, K110, Box 0730, San Francisco, CA 94143, USA b MEMS Precision Instruments, Richmond, CA, USA Received 27 July 2005; accepted 18 August 2005

Abstract We describe a set of microfabricated nanocutting devices with a cutting edge of less than 20 nm radius of curvature that enables high precision microdissection and subcellular isolation of neuronal structures. With these devices, it is possible to isolate functional substructures from neurons in culture such as segments of axons and dendrites, dendritic spines and Nodes of Ranvier. By ne-tuning the mechanical compliance of these devices, they can also act as alternatives to costly laser capture microdissection workstations for harvesting specic neuronal populations from tissue sections for analysis. The small size of the device (1 mm2 100 m) allows convenient insertion into researcher specic experimental set-ups. Its ease of use and possibility for batch fabrication makes this a highly effective and versatile tool for tissue microdissection and the microanalysis of neuronal function. 2005 Elsevier B.V. All rights reserved.
Keywords: Nanoknife; Neuron; Transection; Axonal injury; Dendritic spines; Nodes of Ranvier; Growth cones; Micro-electro-mechanical systems (MEMS); Laser capture microdissection

1. Introduction In recent years, signicant progress has been made in uncovering the genetic and cellular basis of a wide range of processes in development, plasticity, behavior and diseases of the nervous system. Given the highly compartmentalized nature of neurons and the complexity of neuronal circuitry, a deeper quantitative understanding will require the convenient and precise isolation of functionally important neuronal subunits for microanalysis. Experimental work with CNS and PNS entities such as dendritic spines and Nodes of Ranvier would be enhanced by the availability of a set of small, easy-to-use laboratory tools that provide reliable microscale isolation of specic neuronal substructures for physiological, imaging and molecular studies. Our understanding of neuronal physiology in health and disease will also benet from economical and readily available means of neuronal dissection from tissue sections. Studies of axonal and dendritic function currently rely on glass micropipettes to severe processes from cultured neuCorresponding author. Tel.: +1 415 476 4135. E-mail addresses: changw@vision.ucsf.edu, wcchang@itsa.ucsf.edu (W.C. Chang). 0165-0270/$ see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2005.08.020

rons or tissue explants in vitro (Araki et al., 2004; Dotti and Banker, 1987; Eberwine et al., 2001; Ju et al., 2004). While this method can cut many axon segments simultaneously in bulk for biochemical studies, cutting by micropipettes involves mechanical shearing and stretching that may lead to unintended cellular and molecular changes. More importantly, this method of shearing cannot be used to isolate axon and dendritic segments smaller than tens of microns in length or be used within a densely populated and complex axonal or dendritic eld. Laser ablation systems offer more precise control (Emmert-Buck et al., 1996; Schutze et al., 1998) but are costly and require large dedicated workstations that are often difcult to integrate into researcher-specic experimental set-ups. Laser cutting is also usually achieved through the localized heating and vaporization of biological tissues and thus studies examining real-time molecular function in small subcellular compartments using this approach may require careful interpretation. Here we describe a set of small, versatile microfabricated nanocutting devices that have high precision capabilities such as the isolation of single dendritic spines and Nodes of Ranvier. In addition, these devices can effectively dissect entire neuronspecic layers within tissue sections and thus serve as low

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Fig. 1. Design, fabrication and mechanical properties of the nanocutting device. (A) Scanning electron microscope (SEM) image of serpentine exure suspension frame fabricated from single crystal polysilicon. Scale = 200 m. (B) SEM image of silicon-nitride nanoknife. The nanoknife is an elongated pyramidal-shaped structure with the top edge serving as the knife or cutting edge. The dimensions of the nanoknife are dictated by the lithographic masks used during the fabrication process and can be modied as needed. The regions marked by the asterisks represent artifacts from the fabrication process and do not interfere with device function. Scale = 20 m. (C) SEM image of a cross section of the knife showing the radius of curvature at the cutting edge. The silicon-nitride deposited by low pressure chemical vapor deposition into the etched silicon mold produced a sharp cutting edge of 20 nm in radius of curvature. Scale = 100 nm. (D) SEM image of the nanocutting device consisting of a nanoknife assembled within a suspension exure. Scale = 200 m. (E) Schematic diagrams showing (in cross-sections) the fabrication steps for the nanoknife. See text for details. (F) Schematic diagrams showing (in cross-sections) the fabrication steps for the suspension exures. See text for details. (G) Results of nite element modeling analysis show that the torsional deection in the elbows of the serpentine exures as well as exure length govern the overall stiffness of the nanocutting device during the primary cut stroke. The color-coding indicates the magnitude of local principle strain in the structure, with scale ranging from 0 (blue) to a maximum strain of 7 106 (red) (note: strain is dimensionless, L/L) for a 1 N load. (The vertical displacement indicated by the arrow has been accentuated for clarity.) The number of turns in the serpentine exure can be increased or decreased to produce devices with different compliances that are each best suited for isolation of specic types of tissues (see also Table 1). (For interpretation of the references to color in this gure legend, the reader is referred to the web version of the article.)

cost alternatives to conventional laser capture microdissection systems. The cutting device is created by attaching a nanoknife to a silicon microsuspension that provides compliant mechanical support. Its small overall size allows convenient insertion into existing experimental workstations. In this article, we present the design and fabrication of these nanocutting devices, describe their tunable mechanical properties and demonstrate their ability for simple yet highly precise microdissection in a number of systems of interest to the neuroscience community. 1.1. Device description The device is an assembly of two components produced from silicon-based microfabrication: a micromechanical suspension (Fig. 1A) and a nanoknife (Fig. 1B). The mechanical suspension is a planar, contiguous silicon structure consisting of a 1 mm2 frame and a pair of serpentine exures that permit multiaxial motion for the centrally placed nanoknife. The knife itself is an optically transparent elongated pyramidal structure, made of silicon-nitride, with the top edge of the structure serving as an ultrasharp cutting edge. The radius of curvature of the cutting edge is approximately 20 nm (Fig. 1C) as determined by scanning electron microscopy (SEM) and is thus on the order of the

diameter of a single microtubule or the width of a synaptic cleft (Kandel et al., 2000; Scott et al., 2003). The cutting edge can be fabricated in lengths from one to several hundred microns depending on intended use. When assembled, the nanoknife occupies a central position within the supporting suspension frame, with the pyramidal apex, which forms the knife-edge, protruding out perpendicularly from the plane of the device (Fig. 1D). During use, the assembled device is simply mounted at one end of a 10 cm long shaft, which in turn is held and precisely positioned by a standard micromanipulator. The micromanipulator also delivers the cutting stroke. No additional equipment is
Table 1 Compliance of serpentine exure with varied number of turns No. of turns 0 1 2 3 4 5 Compliance ( N/ m) >15.4 15.4 7.7 5.1 3.9 3.1 Tissues Tissue microdissection Tissue microdissection Axons (myelinated) Nodes of Ranvier Axons (unmyelinated) Dendrites Dendritic spines

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Fig. 2. Examples of axons and dendrites isolated using the nanocutting device. (A) Axon eld in culture prior to a cutting sequence. Arrow points to an embryonic mouse retinal ganglion cell axon. The dark rectangular-shaped structure represents the outline of the nanoknife held above the plane of the axon. (B) The nanoknife has been lowered into position. The axon is visible through the transparent knife. Scale A, B = 100 m. (C) The knife is lowered an additional amount to execute cutting. (D) The cut (arrow) in the axon is visible after removal of the nanoknife. Scale C, D = 20 m. (E, F) Photos of an additional axon before and after cutting (arrow). Scale = 20 m. (G) A pair of axons that was simultaneously cut using a nanoknife with a 200 m long cutting edge (arrows). Scale = 20 m. (H) DIC image of an E16 hippocampal neuron from a GFP transgenic mouse with extensive dendritic arborization after eight days in culture. The boxed region is shown in higher magnication in IK. Scale = 20 m. (I) Fluorescence image of a portion of the dendritic arbor. (J) DIC image of dendritic arbor after cutting. One dendrite within the complex eld has been isolated (arrow). (K) Fluorescent image of the image shown in J. Scale IK= 10 m. (L) Examples of cuts in unmyelinated (arrow) and myelinated (arrowhead) sciatic nerve axon from an adult mouse. (M) Cut in adult mouse myelinated sciatic nerve axon. Scale L, M = 10 m. (N) A pair of adult sciatic nerve axons from a GFP transgenic mouse. (O) Same axons as in N. The axon on the right has been cut and retains bright GFP uorescence. The top half of the cut axon was removed for greater visibility. Scale N, O = 10 m.

necessary and the small thin planar geometry of the device allows it to be easily inserted, used and then removed from commonly used laboratory set-ups for electrophysiological, cell culture, or imaging studies.

1.2. Microfabrication Both device components are fabricated using fabrication techniques originally developed for the manufacture of

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silicon-based microelectronics. The nanoknife and the suspension frame are fabricated separately. The nanoknife is fabricated by rst creating a mold in single crystal silicon by the anisotropic etching of single crystal silicon using a lithographically patterned window (Fig. 1E). This highly directional etching occurs nearly along crystal planes and produces a pyramidal trench with a molecularly thin bottom edge. This faceted trench serves as the mold for the nanoknife. The length of its bottom edge corresponds to the length of the knife-edge and can be altered by changing the size (length minus width) of the original etch window. A 1 m thick lm of silicon-nitride is then conformally deposited by low pressure chemical vapor deposition (LPCVD) into the silicon mold, precisely replicating the mold geometries, including the sharp edge. The silicon mold is removed by etching away the backside of the single crystal silicon substrate to expose the thin, optically transparent but mechanically stiff pyramidal silicon-nitride lm that serves as the outer skin of the nanoknife. The supporting frame and suspension is fabricated as a contiguous piece in single crystal silicon (Fig. 1F). The twodimensional footprint of the structure, including the shape of the frame and exures, is patterned via photolithography onto the surface of the silicon substrate. This pattern is then etched into the substrate, and the etched component is released. The shape, size and conguration of the frame and suspension can be explicitly patterned and thus its mechanical properties can be explicitly tuned and device footprints substantially smaller than 1 mm2 can be fabricated. 1.3. Compliance tuning Since a major design goal is to produce a single set of versatile devices capable of severing and isolating subcellular compartments as well as neuronal microdissection in tissue sections, it is necessary to match the range of cutting forces delivered by the nanocutting devices to the mechanical properties of the target tissues to ensure effective cutting while limiting wear on the nanoknife and prolong device longevity. In its current conguration, compliance of the cutting device is governed mainly by the mechanical properties of the serpentine exures. During the cutting stroke, the primary motion of the nanoknife is perpendicular to the major plane of the device, and device stiffness in this axis is dictated by the torsional deection in the elbows of the serpentine exures (Fig. 1G). Finite element modeling of suspensions with different numbers of switchbacks in the serpentine exure was used to calculate increases in device stiffness with progres-

sively fewer switchbacks (Table 1). From experimentation, we determined that nanocutting devices with high compliance are optimal for cutting small structures such as single unmyelinated axons, while devices with lower compliance (higher stiffness) are required for precise cellular microdissection from xed tissue sections. 1.4. Device performance: isolation of axons and dendrites We found the nanocutting devices to be highly effective and easy to use for isolating both axonal and dendritic processes from neurons in vitro. Axons from retinal ganglion cells (Fig. 2AG) and dendrites of hippocampal neurons (Fig. 2HK) were immediately severed upon contact with the nanoknife. There was minimal lateral movement and shearing of the cellular processes, resulting in clean and sharp ends. Given the small size of the nanoknife and its optical transparency, it was possible to reach into a complex dendritic eld under microscope guidance to sever a specic process, leaving adjacent structures untouched (Fig. 2HK). Unmyelinated and myelinated axons from acutely isolated adult mouse sciatic nerves (Fig. 2L, M) and optic nerves (not shown) were also tested. The nanocutting device produced uniform and repeatable cutting of both types of axons, serving as a very precise microscale surgical scalpel. The high tensile strength of silicon-nitride (Ciarlo, 2002), together with a suspension design that limited the forces impinging on the nanoknife-edge, produced a robust nanocutting device. The knives can be coated with 3% bovine serum albumin or peruoropolyether (Ausimont Fomblin) to minimize tissue sticking. (The peruoropolyether lubricant forms a molecularly thin layer to cover the knife surface but does not dissolve in aqueous solutions.) We have used a single device repeatedly in more than two hundred cutting cycles without any observable degradation in performance. The nanocutting devices do not require special handling or storage and we have successfully used devices that have been stored in air at room temperature for up to two years. Silicon and silicon-nitride based devices can also be autoclaved for use in sterile conditions. Furthermore, if necessary, the knife, along with its entire support structure, are strong enough to withstand gentle wiping and are resistant to a variety of cleaning solvents. Axons and dendrites obtained from GFP transgenic mice were cut and tested for membrane resealing. Cells are known to possess an intrinsic calcium-dependent self-repair mechanism and areas of membrane damage are repaired via vesicle exocytosis that is similar to neurotransmitter vesicles release (McNeil

Fig. 3. Isolation of neuronal subcompartments and tissue microdissection of neuronal subpopulations. (A) Brighteld image showing myelinated sciatic nerve axons from an adult mouse. The arrow points to a Node of Ranvier of interest. (B) Brighteld image of same axons as in A. The Node of Ranvier has been isolated using the nanocutting device. The 5 m long isolated segment consists of the nodal region and a portion of the paranodal region. (C) Immunolocalization of sodium channels in the Node of Ranvier (arrow) shown in A. (D) Fluorescence image of B showing sodium channels within the isolated Node of Ranvier. Scale AD = 20 m. (E) Brighteld image showing a dendritic spine of interest (arrowhead) on a rat hippocampal neuron after eight days in culture. (F) The same dendritic spine after removal of its distal half. The remaining proximal stump is indicated by the arrow. Scale E, F = 10 m. (G) Brighteld image showing dendritic spines (arrowheads) on a hippocampal dendrite. (H) Both dendritic spines have been cut leaving the distal spine regions as isolated compartments (arrows). Scale G, H = 10 m. (I) Ten micron thick cryostat section of the retina from an adult mouse. The cryostat section is attached to a plastic membrane for microdissection. Arrow indicates location of retinal ganglion cell layer. (J) The same retinal cryostat section shown in I after microdissection using the nanocutting device. (K) The dissected tissue containing the retinal ganglion cell layer (arrow). Scale IK = 100 m.

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and Steinhardt, 2003; Steinhardt et al., 1994; Zeng et al., 1995). Resealing has also been described for invertebrate axons, PC12 cell axons and developing DRG axons in culture injured by glass micropipettes (Detrait et al., 2000; Godell et al., 1997; Howard et al., 1999). We have tested the nanocutting devices on adult tissues obtained from transgenic animals that express soluble cytoplasmic GFP under the -actin promoter (Okabe et al., 1997). The results showed that adult mammalian CNS and PNS axons as well as CNS dendrites also have the capacity for selfrepair and readily retained GFP after cutting. (Axon resealing shown in Fig. 2N, O). In a recent study of local protein synthesis in isolated dendritic segments (Ju et al., 2004), it was reported that only 25% of dendrites severed using glass micropipettes remained sufciently structurally intact for experimental use. We have found that greater than 95% of hippocampal dendritic segments isolated by the nanoknife remained structurally intact with no blebbing or diminution of GFP uorescence. The highly consistent sharp cuts produced by the nanocutting device may make this instrument a good choice for studies of physiological processes in isolated axonal and dendritic compartments. 1.5. Nodes of Ranvier and dendritic spines In order to determine whether the nanocutting device can operate at an even smaller scale, we tested its performance in isolating functional substrucures of interest such as Nodes of Ranvier in peripheral nerve axons and dendritic spines from hippocampal neurons. Individual myelinated axons from the sciatic nerves of adult mice were isolated (Fig. 3A) and the sodium channels that mark the Nodes of Ranvier were visualized by antibody staining (Fig. 3C). Individual Nodes of Ranvier could be isolated using the nanocutting device resulting in a small segment containing sodium channels together with a portion of the paranodal region on either side (Fig. 3B, D). The ability to specically isolate an individual compartment responsible for saltatory action potential conduction may further our understanding of channel protein trafcking, molecular mechanisms of myelination and the pathology of demyelinating diseases. Dendritic spines contain the postsynaptic apparatus of most excitatory CNS synapses and are thought to be the site of synaptic plasticity (Hering and Sheng, 2001; Kasai et al., 2003; Tsay and Yuste, 2004). The dendritic spines of hippocampal neurons maintained in culture for eight days were cut using the nanocutting device. It was relatively easy to specically target a particular dendritic spine for isolation. Dendritic spines can be severed in half, leaving a proximal stump (Fig. 3E, F), or their distal ends can be isolated as separate compartments (Fig. 3G, H). The high degree of precision by which important neuronal substructures, such as single dendritic spines, could be isolated may enable new studies of plasticity, learning and memory at a much ner level of spatial resolution. 1.6. An alternative to laser capture microdissection Studies of regional specicity in the nervous system have been greatly aided by the ability to microdissect desired neuronal subpopulations or white matter regions from frozen tissue

sections for molecular and biochemical analysis. Currently, this type of tissue harvesting is typically performed using dedicated laser capture microdissection systems. Such systems have the disadvantage of being relatively high cost, large, standalone workstations that are not widely disseminated. By tting nanoknives with suitably low compliance suspensions, nanocutting devices can serve as small, low cost instruments that enable the precise dissection of subregions of interest from frozen cryostat sections, comparable to what is achieved using a dedicated commercial laser capture microdissection system. Fig. 3IK shows the dissection of the retinal ganglion cell layer (arrow) from a 10 m thick cryostat tissue section from an adult mouse retina. The tissue section was transferred from the cryostat onto a commercially available microscope slide covered with plastic membrane (Leica). Microdissection was performed on a standard inverted laboratory microscope simply by using a nanocutting device mounted on a micromanipulator and with the cutting path manually traced by movement of the microscope stage. The dissected piece (Fig. 3K) consisted of a 25 m wide strip of retinal tissue containing the retinal ganglion cell layer (arrow) along with a section of the plastic membrane. Although only rectangular-shaped pieces have been isolated from tissue sections thus far, the use of knives with shorter knife edges (1 m) should make it possible to trace and cut out any arbitrary shaped region from tissue sections. The ability of simple nanocutting devices to perform tissue microdissection potentially make them a widely disseminated, low cost alternative to conventional laser capture microdissection systems. 1.7. Studies of de novo growth cone formation Given its ability for repeatable and precise cutting action without the need for destructive tissue ablation, the nanocutting device can be a useful investigative tool for studies of axonal responses to injury and growth cone regeneration. The phenomenon of new growth cone formation from a cut axon stump has previously been studied in vitro in invertebrates and DRG neurons, where glass micropipettes were the instrument of choice (Godell et al., 1997; Howard et al., 1999; Ziv and Spira, 1998). Following injury to the mammalian nervous system in vivo, injured axons in the PNS, and to a more limited extent axons in the CNS, will form a new growth cone, exhibit other forms of axon terminal remodeling, and attempt to re-extend (Allcutt et al., 1984; Cajal, 1928; Howard et al., 1999; Kulbatski et al., 2004; Selles-Navarro et al., 2001; Zeng et al., 1995). The molecular mechanisms that control how mammalian axons or dendrites recover from injury to issue a new growth cone are of substantial interest for understanding the cell biology of neurotrauma and process regeneration. These molecular mechanisms have been difcult to address without a precise and repeatable method that can selectively injure individual axons or dendrites for study. Fig. 4 shows results from an experiment in which a nanocutting device was used to sever a specic dendrite within the complex dendritic arbor of a hippocampal neuron in culture. The cut dendritic process resealed (Fig. 4B) and within minutes of injury, a new actively motile growth cone regenerated and grew in place of the original dendritic process (Fig. 4CE). The

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Fig. 4. De novo dendritic growth cone formation in hippocampal neurons. Panels represent images of a portion of the dendritic eld of a GFP transgenic mouse hippocampal neuron after eight days in culture. (A) The arrow points to the site on the dendrite where the cut will be made. (B) Immediately after cut. (C) New growth cone formation after 8 min. (D) Further elaboration of the growth cone at 10 min. (E) At 15 min. Scale AE = 10 m.

nanocutting device thus provides an easy to produce and highly repeatable model of single axon or dendrite injury. The viability of the parent neuron and the ability of the cut stump to regrow also demonstrate the suitability of the nanocutting devices for use in biological studies. Given the simplicity of device design, it should be possible to combine a nanocutting device with pharmacological and molecular perturbation protocols to identify signaling pathways and cytoskeletal elements required for this adaptive response to injury. 2. Discussion In this study, we describe a small, easy to use nanocutting device that is ideally suited for the precise isolation of neuronal substructures and neuronal subpopulations from tissue sections. In addition to useful features such as its optical transparency and the lack of autouorescence, its small footprint allows easy insertion into existing imaging and physiological experimental workstations, immediately endowing these workstations with additional utility at the microscale. Although the nanocutting devices can be used repeatedly and can be sterilized if needed, its manufacturing process allows large numbers of devices to be produced simultaneously and these devices can almost be used as disposable labware. The design of the device is focused on two parameters that are important for any cutting tool. One is the sharpness of the cutting edge, and the other is the mechanism that ensures sufcient cutting forces are delivered for the task at hand without compromising the integrity of the knife. These issues are addressed by taking advantage of the excellent mechanical properties of silicon-based materials and the ability of photolithographic processing to form highly precise features such as a nanoscale cutting edge and the mechanically compliant suspension exures. Although the current generation of nanocutting devices described here can serve a wide range of function by merely being attached to a micromanipulator and using the micromanipulator to deliver the cutting stroke, future versions of the device will incorporate an on-board force generation (actuation) mechanism to perform cutting. With this improvement, a micromanipulator is required only to position the device and is then removed. The use of an on-board self actuation mechanism prevents external vibration from interfering with cutting and will fully realize the microscale precision intrinsic to these fabricated devices.

A reliable and highly precise method to isolate specic axonal and dendritic compartments from a single identied neuron may nd application in a wide range of studies. This includes work addressing issues of mRNA transport and translation in single axons or dendrites (Eberwine et al., 2001; Kanai et al., 2004; Willemsen et al., 2004), and protein transport and synthesis in axons, dendrites and growth cones (Campbell and Holt, 2001; Hirokawa and Takemura, 2004; Sutton et al., 2004). Additional research questions include the molecular mechanisms of axonal and dendritic fate determination and factors that govern the critical period during which axonal and dendritic fates remain plastic (Bradke and Dotti, 2000; Dotti and Banker, 1987). The nanocutting devices may also nd utility in areas of potential clinical signicance. Axonal segments that have been severed from their cell bodies undergo a degenerative process called Wallerian degeneration. The biological mechanisms for this process appears to involve the ubiquitin-proteasome system (Ehlers, 2004; Zhai et al., 2003), but the complete details remain unknown. The ability to specically trigger Wallerian degeneration in an identied axon could be combined with other molecular perturbation strategies to gain a deeper understanding of the mechanisms involved. We believe that this nanocutting device provides the neurobiological research community with a low cost tool that exhibits a high level of reliability and precision that will enhance the microscale analysis of neuronal function. Acknowledgements The authors wish to thank members of the Sretavan laboratory for their comments and technical assistance, and the UC Berkeley Microfabrication laboratory for use of their facilities. This research was conducted through the support of the Sandler Family Foundation, That Man May See, NIH and the Research to Prevent Blindness Foundation. W. Chang was supported by NEI postdoctoral vision training grant awarded to UCSF. References
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