Electroporation of neurons and growth cones in Aplysia californica

Journal of Neuroscience Methods 151 (2006) 114–120

Electroporation of neurons and growth cones in Aplysia californica Peter Lovell a , Sami H. Jezzini a,b , Leonid L. Moroz a,b,∗ b

a The Whitney Laboratory for Marine Bioscience, University of Florida, 9005 Ocean Shore Blvd., St. Augustine, FL 32080, USA Department of Neuroscience and McKnight Brain Institute, University of Florida, P.O. Box 100244, Gainesville, FL 32610-0244, USA

Received 13 May 2005; received in revised form 24 June 2005; accepted 24 June 2005

Abstract Specific labeling of individual neurons and neuronal processes is virtually an everyday task for neuroscientists. Many traditional ways for delivery of intracellular dyes have limitations in terms of speed, efficiency and reproducibility. Electroporation is a fast, reliable and efficient method to deliver microscopic amounts of polar and charged molecules into neurons and their compartments such as individual neurites and growth cones. Here, we present a simple and highly effective procedure for intracellular labeling of individual Aplysia neurons both in intact ganglia and in cell culture. Pleural mechanoreceptor neurons have been used as illustrative examples to demonstrate applicability of direct and local labeling of the smallest individual neurites (<2 ␮m) and single growth cones. Specifically, a 3-s train of 1.0 V hyperpolarizing pulses at 50 Hz effectively filled discrete neurites in contact with the tip of the micropipette with no dye transfer visible to other, non-contacted neurites. Application of this localized dye labeling technique to single neurites reveals a surprisingly complex morphology for patterns of axonal branching in culture. The protocol can be easily applied to a variety of models in neuroscience including accessible nervous systems of invertebrate animals. © 2005 Elsevier B.V. All rights reserved. Keywords: Electroporation; Mollusk; Growth cones

1. Introduction Electroporation is a common method used to insert polar and charged molecules, such as nucleic acids, peptides, proteins, and dyes across the impermeable lipid cell membrane into cells. This is accomplished by applying a voltage across the cell membrane in excess of the dielectric breakdown voltage to produce pores through which compounds of interest can pass. These pores open fast, have a diameter dependent on voltage, and close slowly (Ho and Mittal, 1996; Teruel and Meyer, 1997). In traditional bulk electroporation, large numbers of cells in suspension are subjected to a uniform high voltage electric field generated by plate electrodes (Neumann et al., 1999) allowing compounds in the external solution to enter the cell. While this method works well for bacterial and mammalian cell lines, it does not allow the introduction of compounds of interest into single identified cells either in vivo or in adherent cell culture conditions. ∗

Corresponding author. Tel.: +904 461 4020; fax: +904 461 4052. E-mail address: [email protected] (L.L. Moroz).

0165-0270/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2005.06.030

More recently, a number of techniques have been developed that can be used to focally electroporate into relatively few cells, or in some cases a single cell, in the intact brain or in adherent cell culture. First, Teruel et al. (1999) used field stimulation along with a device to minimize the volume of media surrounding cultured cells thus compartmentalizing the dish into electroporated and non-electroporated areas. This worked well, however, it did not allow true access to individual cells or subcellular domains. A second technique was pioneered, whereby fine carbon fiber microelectrodes produced spatially focused electric fields in a cell culture dish (Lundqvist et al., 1998). This technique selectively introduced charged dyes and plasmids into cultured hippocampal progenitor cells while excluding others in the dish. It succeeded further by filling an individual process of a progenitor cell. Although both of the above techniques have proven useful, they both suffer the same limitation in that material enters the cell directly from the extracellular media which typically has a large volume. Furthermore, they are both spatially restricted such that neither method allows electroporation of individual cells in the intact brain, nor electroporation of

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individual neurites in a geometrically complex configuration as is often seen in cell culture. To enable selective electroporation of individual neurons in brain slice cultures or dissociated cell cultures, modified electrophysiological methods have been developed. Nolkrantz et al. (2001) used a single, tapered, fused-silica, electrolyte-filled capillary to fill cultured NG108-15 cells or hippocampal neurons in organotypic slice culture with charged dyes. Even though they demonstrate filling of individual cells and processes, the large tip size of their capillary (30 ␮m i.d.) and the distance the capillary tip is held from the cell to be filled limit the spatial resolution of this method. For more precise electroporation, modified patchclamp techniques have been recently used to introduce dyes or transfect neurons in slice cultures (Rathenberg et al., 2003), in vivo (Haas et al., 2001), or cultured cell lines (Rae and Levis, 2002). With electrode tip sizes in the range of 0.5–3.0 ␮m, this final method allows for the most precise spatial resolution for electroporation. Building from these modified electrophysiological methods, there are urgent needs to develop reliable protocols for local labeling of individual neurites and growth cones in invertebrate models with accessible nervous systems. Commonly used intracellular pressure and iontophoretic dye injection have limited applications to deliver fluorescent molecules into small cells or cell compartments in reproducible and high-throughput manner. Here, we present a simple and highly effective procedure for intracellular labeling of individual Aplysia neurons both in intact ganglia and in cell culture. In the present study, we use a single-cell electroporator to rapidly, efficiently, and precisely introduce charged dyes into single identified neurons of the marine mollusc, Aplysia californica. In the intact CNS, we were able to reliably and rapidly fill sensory neurons with fluorescent dyes for morphological analysis following electrophysiological tests, a feat difficult to achieve with conventional microinjection or iontophoretic techniques. Following this, we demonstrate electroporation of charged dyes into individual pleural sensory neurons in cell culture. This was achieved either via electroporation into pleural sensory cell somata, or extrasomatically into individual neurites and single growth cones. Filling of the neurites revealed a surprisingly complex morphology for these neurons in culture.

2. Methods 2.1. Electroporation and imaging of intact ganglia Aplysia weighing 150–250 g were collected in the wild by Marinus Scientific, Long Beach, California. Animals were anesthetized by injection of 50% (volume/body weight) isotonic MgCl2 (337 mM) prior to surgical removal of the CNS. Ganglia were placed in a solution of 50% isotonic MgCl2 and 50% artificial sea water (ASW: 460 mM NaCl, 10 mM

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KCl, 55 mM MgCl2 , 11 mM CaCl2 , 10 mM HEPES, pH 7.6) to help reduce contraction of the connective tissue sheath overlying the neurons. The sheath was then removed with fine forceps and scissors to expose neuronal somata. Once the sheath was removed the solution was changed to ASW and the ganglia were allowed to equilibrate for 60 min at 4 ◦ C before electroporation. 10 M
glass electrodes were pulled on a Flaming Brown Micropipette puller (model P-87, Sutter Instrument Co.) using thin-walled 1.0 mm o.d. filamented glass capillaries (TW100F-3, World Precision Instruments). Electrode tips (∼0.5 ␮m in diameter) were filled with 0.5 ␮L of either 4% Lucifer yellow (Sigma L-0259) in 0.1% LiCl or 10 mM Alexa Fluor 594 hydrazide (Molecular Probes A-10442) in 200 mM KCL, and backfilled with 200 mM potassium acetate (KC2 H3 O2 ). Electroporation was accomplished using an Axoporator 800a (Axon Instruments) to generate desired voltages at the electrode tip. A 3 s square wave stimulus train of −5 V in amplitude with a 20 ms interpulse interval and 15% duty cycle was used for both types of dye. The Axoporator 800a specific parameters were: amplitude −5 V, frequency 50 Hz, width 3 ms, train 3 s, DC offset −1.0 V. Living ganglia were imaged under a fluorescence equipped Olympus SZX12 binocular microscope. A GFP longpass filter cube (Olympus 5-SX610: EX460-490, EM510) was used to visualize Lucifer yellow fills and a green filter cube (Olympus 5-SX615: EX460-560, EM590) for Alexa Fluor 594 hydrazide. Images were acquired digitally with a Nikon DS-L1 camera. 2.2. Neuronal isolation and cell culture Adult Aplysia approximately 40–60 g (NIH Aplysia Resource Facility, Miami, FL) were anaesthetized by injection of isotonic MgCl2 and pinned down in a dissection tray. Ganglia were removed via a dorsal midline incision, enzyme treated for 1 h at 34 ◦ C in 1% protease IX (Sigma P-6141) in sterile filtered natural sea water, briefly rinsed in 4 ◦ C sterile sea water, then pinned down in defined media (DM: 1% (w/v) L-15 Leibovitz medium [Sigma L4386] in sterile sea water with 20 ␮g/ml gentamicin sulfate [Sigma G-3632]) containing an additional 10 mM d-glucose (Sigma G-7528). The sheaths of connective tissue surrounding the ganglia were mechanically removed with fine forceps. Large motor neuron somata on the dorsal side of the abdominal ganglion were visually identified with a dissecting microscope and then removed from the ganglia via suction applied by a gilmont syringe (Gilmont, GS 110, Barrington, IL.) attached to a sigmacoated (Sigma SL-2) glass capillary pipette (WPI 1B150-6) held with a micromanipulator (WPI KITE-R). The glass pipettes were constructed with smooth fire polished tips that had inner opening diameters approximately 10 ␮m larger than the cell body to be isolated. Cells were individually removed and transferred to falcon 3001 culture dishes with attached poly-l-lysine (Sigma P-1399) coated glass coverslips and maintained in

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50% DM 50% filtered haemolymph for 24–48 h at room temperature. Pleural sensory neurons were first transferred as a cluster onto a non-adhesive surface (uncoated plastic falcon 3001 culture dish), triturated with the sigmacoated pipette to separate individual sensory neurons, then transferred individually to the final culture conditions as above.

2.3. Electroporation and imaging of cultured neurons Electroporation of charged dyes into cultured Aplysia neurons was accomplished using protocols outlined in Section 2.1 with a few technical modifications. Dye filled electrodes held in a Eppendorf InjectMan NI2 micromanipulator were maneuvered to come into contact with either neuronal somata or processes of cultured neurons. Alexa Fluor 594 hydrazide and Lucifer yellow were electroporated into the cells using lower voltage hyperpolarizing pulses than used for intact ganglia (−1.0 V compared to −5.0 V) and 0.0 V DC offset. One train of pulses was usually sufficient to completely load the neuron, however, multiple trains were used on occasion when electroporating into the soma with little detrimental effect on visible anatomy. During the electroporation, the culture media was exchanged with low Ca2+ ASW (in mM: NaCl 436.8, KCl 9, MgCl2 22.9, MgSO4 25.5, NaHCO3 2.1) to minimize potentially damaging Ca2+ influx into the cell (Gallant and Galbraith, 1997). Regular culture media was returned to the dish after electroporation. After the filled electrode was attached to the manipulator and placed in the dish near the cell, complete dye filling of cells could be easily accomplished in less than 10 s. Dye filled cultured Aplysia neurons were imaged on a Nikon TE2000-E inverted microscope using Nomarski DIC optics and epi-fluorescence. Images were captured by a Retiga EXi (Q-imaging) digital camera controlled by a PC computer running Metamorph (version 6.2r4, Universal Imaging Corp.) software.

3. Results 3.1. Electroporation of charged dyes into individual Aplysia neurons in the intact ganglia Individual pleural sensory neurons could be rapidly and efficiently filled by electroporation using glass microelectrodes (Fig. 1). Multiple neurons could be labeled using a single dye filled electrode (Fig. 1). The 6 Lucifer yellow injected neurons in Fig. 1B on the left were all injected using one dye filled microelectrode, as were the 10 Alexa Fluor 594 hydrazide injected neurons on the right. All of the labeled neurons in Fig. 1 were filled within a span of 15 min. A single 3 s voltage pulse train was adequate to fill smaller neurons while larger cells were filled using three or four trains. Alexa Flour 594 was easily observed entering neurons under the dissecting microscope with regular non-fluorescent illumination (Fig. 1). Sometimes Lucifer yellow was also visible under regular illumination as injected neurons took on a yellow appearance (not shown). Filled axons became visible under fluorescence in the pedal pleural connective within 20 min of injection. 3.2. Electroporation of charged dyes into cultured Aplysia neurons To determine whether electroporation could be used to selectively fill individual neurons in cell culture with charged fluorescent dyes, we placed pleural sensory neurons or abdominal motor neurons into primary culture conditions and electroporated their somata with either Lucifer yellow or Alexa fluor 594 hydrazide. We found that gently touching the tip of a ∼10 M
glass electrode to the surface of the somata and applying 3 s hyperpolarizing trains of −1.0 to −1.5 V at 50 Hz rapidly filled the neuronal cell body with dye with near 100% efficiency. From the cell body, both dyes were seen to diffuse out into all neurites associated with that neuron thus completely filling the cell. Most importantly, dye

Fig. 1. Individual sensory neurons labeled by electroporation of negatively charged fluorescent dyes Lucifer yellow and Alexa fluor 594. (A) The left pleural ganglion of Aplysia as it appears under white light. Neurons of the sensory cluster that are labeled with the red Alex fluor dye are clearly visible. (B) An overlay of fluorescent images of the same pleural ganglion taken on green (Lucifer yellow) and red (Alexa 594) channels. Axons projecting to the pedal ganglion became visible within 20 min. Plp, pleuro-pedal connective; Cp, Cerebropedal connective. Scale bars ∼100 ␮m. The overlay was done using Corell photo-paint 11.

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from the two cells, one filled with Alexa fluor 594 the other with Lucifer yellow, physically overlap one another in many areas, including a relatively extended length of close apposition (closed arrow heads), however, no transfer of either dye between the two cells is apparent. Furthermore, processes from a third, unfilled pleural sensory neuron, extend up from the lower edge of the figure and make contact with both the Alexa fluor 594 and the Lucifer yellow filled neurons (open dashed arrows). No dye transfer to these unfilled neurites is apparent in either case. 3.3. Electroporation of charged dyes into individual neuronal processes of abdominal neurons Fig. 2. Aplysia pleural sensory neurons in cell culture labeled by electroporation of fluorescent dyes into the cell body. (A) Digital overlay of two fluorescent channels and DIC image reveals complete neuronal dye filling following singe cell electroporation of the two sensory cell somata. Dye coupling between the cell filled with Lucifer yellow (green) and the cell filled with Alexa Fluor 594 hydrazide (red) was not apparent even though there were extensive areas of contact between their neurites (solid arrows). Furthermore, transfer of dye to neurites of a third unfilled neuron did not occur (dashed arrows). Scale bar = 50 ␮m. (B) Low magnification overlay of the same neurons as in (A). Scale bar = 100 ␮m.

electroporated into a single neuron did not leak out into the external media nor did it enter other neurons even though they were in close physical proximity. This can be seen clearly in Fig. 2 in which two pleural sensory neurons growing in close proximity were filled with the different dyes. Neurites

Following the successful electroporation of charged dyes into the somata of cultured Aplysia neurons and the subsequent diffusion of the dyes out into all attached processes, we attempted to use the same technique to introduce these dyes into discrete neurites of individual cells at locations far removed from the cell body. We first started single neurite electroporation on cultured random abdominal motor neurons due the large size of their neurites (up to 50 ␮m in diameter) as compared to neurites from sensory cells (up to roughly 5–10 ␮m in diameter). The tip of the electroporation electrode was maneuvered so that it gently pressed against the surface of single neurites growing in highly branched configurations (Fig. 3A) and subjected to a single pulse train of 3 s at −1.0 V and 50 Hz. Surprisingly, even though the trunk of the highly branched neurites appeared to be com-

Fig. 3. Aplysia abdominal ganglion motor neurons in cell culture labeled via extrasomatic electroporation of Alexa fluor 594 hydrazide into distal neurites. (A) Electroporation of fluorescent dye into a singe process in a dense net of neurites growing from a single cell reveals a complex morphology in which dye is confined to a discrete longitudinally oriented sub-domain of the neurite. Scale bar = 50 ␮m. (B) At higher magnification, very fine dye filled processes could be seen embedded within a larger neurite. Scale bar = 20 ␮m. (C) In some preparations of actively growing neurons, dye filled neurites appear to terminate abruptly part way along an underlying unfilled neurite. These large dye filled terminals are potentially growth cones of the extending labeled neurite as it grows along a neuritic substrate. Scale bar = 50 ␮m.

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Fig. 4. Individual neurites of cultured Aplysia pleural sensory neurons sequentially filled by extrasomatic single-cell electroporation. (A) Following a first round of electroporation, a single fine process from a pleural sensory neuron was filled with Alexa fluor 594 while a large growth cone (*) growing from the same cell body remained unfilled. (B) Moving the glass micropipette into contact with this growth cone and subjecting the cell to second round of extrasomatic electroporation successfully filled this growth cone with dye. Scale bar = 50 ␮m in each.

posed of a single process when viewed under Nomarski DIC optics, dye consistently filled only a discrete region of the neurite. Similarly, when electroporated into less branched neurites (Fig. 3B), dye flowed through thin longitudinally oriented lines in the neurite instead of completely filling the neurite. This morphology remained consistent over time (up to 6 h) with no apparent lateral spread of dye within the neurites. Taken together, this means that instead of being a single thick process, most Aplysia neural processes in vitro are actually composed of many thin separate processes fasiculated together. Subsequent time lapse video analysis supports this data in that growth cones of new neurites are commonly seen to extend along the surface of existing neurites before branching away at some distal point. In Fig. 3C, extrasomatic dye filling revealed a thin fluorescent process extending along a wide unfilled neurite. The dye filled process appears to terminate in large varicosities on the surface of the underlying unfilled process (open arrows Fig. 3C). Since this cell was still actively growing, these dye filled varicosities are likely growth cones at the tip of the neurite as it extends using the unfilled neurite as a substrate. 3.4. Electroporation of charged dyes into individual processes of cultured Aplysia sensory neurons Sensory neurons are some of the smallest neurons in Aplysia (Walters et al., 2004). The fine processes of these neurons have proven difficult to manipulate and, to our knowledge, microinjection into individual sensory neurites has never been successfully accomplished. In the present study, we placed individual pleural sensory neurons in cell culture and used electroporation to introduce charged dyes. Alexa fluor 594 was first electroporated into a fine process extending from the soma of a cultured pleural sensory neuron (Fig. 4A). The large unfilled growth cone to the left of the fluorescent process is attached to the same cell. If the neuron

had been filled using somatic electroporation, all processes from this cell would have been filled simultaneously, however, since the process was filled extrasomatically, the growth cone contains no dye. Subsequent filling of this growth cone (Fig. 4B) was accomplished by a second round of electroporation in which the tip of the electrode was moved such that it came into gentle contact with the growth cone. This clearly demonstrates that, using electroporation, it is possible to introduce charged molecules into specific neurites of cultured sensory neurons, and growth cones, and that it is possible to use multiple rounds of electroporation into different parts of the same cell.

4. Discussion In the present study, we used microelectrodes to electroporate charged dyes into individual neurons both in the intact CNS and in primary cell culture. We were able to selectively introduce molecules into discrete extrasomatic compartments of cultured neurons, including neurites and individual growth cones of pleural sensory neurons. We found that dye labeling by electroporation was less physically invasive than microinjection enabling us to routinely label very fine processes without visibly damaging them. Fast and precise labeling of individual Aplysia neurons in intact ganglia was easily accomplished within minutes or even seconds using the Axoporator 800a. A single 3-s train of hyperpolarizing pulses resulted in dye fills that were qualitatively similar in brightness to fills typically obtained after 30–45 min using classical iontophoretic injection (Magoski and Bulloch, 1997). This time saving alone will greatly facilitate morphological analysis of neurons in living preparations as compared to more conventional techniques. Lucifer yellow is often used to label identified Aplysia cells after electrophysiological studies. Ganglia can then be

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fixed and further analyzed by immunohistochemistry or in situ hybridization (Jezzini et al., 2005) to determine proteins or genes expressed in the identified neuron. This approach is limited to a few neurons per ganglion when using classical iontophoretic injection because of the large amount of time required to fill each one. Using electroporation one could routinely label several dozen identified neurons in a single preparation. Electroporation into a discrete neuronal process in culture has been previously reported using either carbon fiber microelectrodes (Lundqvist et al., 1998) or electrolyte-filled capillaries (Nolkrantz et al., 2001). In these studies, fluorescent dyes were seen to enter short single processes just distal to where the neurite leaves the soma and diffuse rapidly back into the cell body. While extremely useful, carbon fiber electrode or capillary derived methods of electroporation lack spatial resolution since the compound to be electroporated is located in the external media and thus can enter the entire cross-sectional area of the targeted neurite. Labeling individual neurites in a fascicle would be problematic using such techniques. Using a single dye filled electrode for electroporation provides for better spatial resolution and enables access to discrete extrasomatic domains. Extrasomatic electroporation of dyes into cultured Aplysia neurons revealed a complex morphology in which thick neurites often consist of a fascicle of finer neurites. Previously, the only method for visualizing this structural feature of cultured neurons has been electron microscopy (Schacher, 1988). Using electroporation, discrete neurites as small as ∼2 ␮m in diameter embedded within larger bundles can easily be filled with dye. This enables rapid morphological analysis of live neurites under the fluorescent compound microscope. Pleural sensory neurons are among the most commonly studied neuron types in Aplysia and play a central role in many Aplysia models of synaptic plasticity, learning and memory mechanisms (Kandel, 2001). These neurons form glutamatergic synapses on motor neurons (Dale and Kandel, 1993; Levenson et al., 2000) and are often bifurcated with branches showing local protein synthesis dependent differential phenotypes following various learning paradigms (Bailey et al., 2000; Casadio et al., 1999; Martin et al., 1997). With this in mind, various labs have long sought physical access to individual extrasomatic areas to study local RNA trafficking and regional translation, however the small size of the pleural sensory neurites make microinjection virtually impossible. The spatial precision of electroporation allows relatively easy intracellular access to discrete pleural sensory neurites. Furthermore, it is possible to subject the neurites to temporally separate multiple rounds of electroporation over the course of an experiment. In summary, single-cell electroporation is a powerful technique that is unique in providing fast and easy intracellular access to spatially discrete parts of individual identified neurons. In the present study, we have succeeded in introducing negatively charged dyes into individual neurites of cultured

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Aplysia neurons. While this provides an excellent tool for morphological analysis, the future potential of single-cell electroporation as a means to manipulate neurons at the molecular level is limitless. Electroporation has long been the technique of choice for introducing foreign DNA into bacteria (Neumann et al., 1999) and has recently been used to transfect individual vertebrate neurons both in vivo (Haas et al., 2001) and in vitro (Haas et al., 2001; Rathenberg et al., 2003; Teruel et al., 1999). Along with the introduction of foreign DNA, electroporation is one of the best and least invasive methods to disrupt targeted transcripts by intracellular introduction of antisense RNA, or dsRNA. Furthermore, with the emerging importance of extrasomatic RNA targeting, trafficking, and protein synthesis in various neuronal models (Kindler and Monshausen, 2002; Rehbein et al., 2002; Schacher et al., 1999; Spencer et al., 2000), the access to these discrete sites afforded by electroporation provides an extremely useful tool for a wide variety of purposes.

Acknowledgements This work was supported by NIH and NSF grants, and in part by Packard and McKnight Brain Research Foundation grants to L.L.M.

References Bailey CH, Giustetto M, Zhu H, Chen M, Kandel ER. A novel function for serotonin-mediated short-term facilitation in Aplysia: conversion of a transient, cell-wide homosynaptic hebbian plasticity into a persistent, protein synthesis-independent synapse-specific enhancement. Proc Natl Acad Sci USA 2000;97:11581–6. Casadio A, Martin KC, Giustetto M, Zhu H, Chen M, Bartsch D, et al. A transient, neuron-wide form of CREB-mediated long-term facilitation can be stabilized at specific synapses by local protein synthesis. Cell 1999;99:221–37. Dale N, Kandel ER. l-Glutamate may be the fast excitatory transmitter of Aplysia sensory neurons. Proc Natl Acad Sci USA 1993;90: 7163–7. Gallant PE, Galbraith JA. Axonal structure and function after axolemmal leakage in the squid giant axon. J Neurotrauma 1997;14:811–22. Haas K, Sin WC, Javaherian A, Li Z, Cline HT. Single-cell electroporation for gene transfer in vivo. Neuron 2001;29:583–91. Ho SY, Mittal GS. Electroporation of cell membranes: a review. Crit Rev Biotechnol 1996;16:349–62. Jezzini SH, Bodnarova M, Moroz LL. Two-colour in situ hybridization in the CNS of Aplysia californica. J Neurosci Methods 2005 (Epub). Kandel ER. The molecular biology of memory storage: a dialogue between genes and synapses. Science 2001;294:1030–8. Kindler S, Monshausen M. Candidate RNA-binding proteins regulating extrasomatic mRNA targeting and translation in mammalian neurons. Mol Neurobiol 2002;25:149–65. Levenson J, Sherry DM, Dryer L, Chin J, Byrne JH, Eskin A. Localization of glutamate and glutamate transporters in the sensory neurons of Aplysia. J Comp Neurol 2000;423:121–31. Lundqvist JA, Sahlin F, Aberg MAI, Stromberg A, Eriksson PS, Orwar O. Altering the biochemical state of individual cultured cells and organelles with ultramicroelectrodes. Proc Natl Acad Sci USA 1998;95:10356–60.

120

P. Lovell et al. / Journal of Neuroscience Methods 151 (2006) 114–120

Magoski NS, Bulloch AG. Localization, physiology, and modulation of a molluskan dopaminergic synapse. J Neurobiol 1997;33:247–64. Martin KC, Casadio A, Zhu H, Yaping E, Rose JC, Chen M, et al. Synapse-specific, long-term facilitation of Aplysia sensory to motor synapses: a function for local protein synthesis in memory storage. Cell 1997;91:927–38. Neumann E, Kakorin S, Toensing K. Fundamentals of electroporative delivery of drugs and genes. Bioelectrochem Bioenerg 1999;48:3–16. Nolkrantz K, Farre C, Brederlau A, Karlsson RI, Brennan C, Eriksson PS, et al. Electroporation of single cells and tissues with an electrolytefilled capillary. Anal Chem 2001;73:4469–77. Rae JL, Levis RA. Single-cell electroporation. Pflugers Arch 2002;443:664–70. Rathenberg J, Nevian T, Witzemann V. High-efficiency transfection of individual neurons using modified electrophysiology techniques. J Neurosci Methods 2003;126:91–8. Rehbein M, Wege K, Buck F, Schweizer M, Richter D, Kindler S. Molecular characterization of MARTA1, a protein interacting with the dendritic targeting element of MAP2 mRNAs. J Neurochem 2002;82:1039–46.

Schacher S. Identified Aplysia neurons maintained in dissociated cell culture: regeneration, synapse formation and synaptic plasticity. In: Beadle DJ, Lees G, Kater SB, editors. Cell culture approaches to invertebrate neuroscience; 1988. p. 57. Schacher S, Wu F, Panyko JD, Sun ZY, Wang D. Expression and branchspecific export of mRNA are regulated by synapse formation and interaction with specific postsynaptic targets. J Neurosci 1999;19:6338–47. Spencer GE, Syed NI, van Kesteren E, Lukowiak K, Geraerts WP, van Minnen J. Synthesis and functional integration of a neurotransmitter receptor in isolated invertebrate axons. J Neurobiol 2000;44:72–81. Teruel MN, Blanpied TA, Shen K, Augustine GJ, Meyer T. A versatile microporation technique for the transfection of cultured CNS neurons. J Neurosci Methods 1999;93:37–48. Teruel MN, Meyer T. Electroporation-induced formation of individual calcium entry sites in the cell body and processes of adherent cells. Biophys J 1997;73:1785–96. Walters ET, Bodnarova M, Billy AJ, Dulin MF, Diaz-Rios M, Miller MW, et al. Somatotopic organization and functional properties of mechanosensory neurons expressing sensorin-A mRNA in Aplysia californica. J Comp Neurol 2004;471:219–40.