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Journal of Neuroscience Methods 93 (1999) 37–48

A versatile microporation technique for the transfection ofcultured CNS neurons

Mary N. Teruel a, Thomas A. Blanpied b, Kang Shen a, George J. Augustine b,Tobias Meyer a,*

a Department of Cell Biology, Nanaline Duke Bldg. Rm. 346, Box 3709, Duke Uni6ersity Medical Center, Durham, NC 27710, USAb Department of Neurobiology, Duke Uni6ersity Medical Center, Durham, NC 27710, USA

Received 3 March 1999; received in revised form 14 July 1999; accepted 17 July 1999

Abstract

The application of molecular techniques to cultured central nervous system (CNS) neurons has been limited by a lack of simpleand efficient methods to introduce macromolecules into their cytosol. We have developed an electroporation technique thatefficiently transfers RNA, DNA and other large membrane-impermeant molecules into adherent hippocampal neurons. Micropo-ration allowed the use of either in vitro transcribed RNA or cDNA to transfect neurons. While RNA transfection yielded a higherpercentage of transfected neurons and produced quantitative co-expression of two proteins, DNA transfection yielded higherlevels of protein expression. Dextran-based calcium indicators also were efficiently delivered into the cytosol. Microporatedneurons appear to survive poration quite well, as indicated by their morphological integrity, electrical excitability, ability toproduce action potential-evoked calcium signals, and intact synaptic transmission. Furthermore, green fluorescent protein(GFP)-tagged marker proteins were expressed and correctly localized to the cytosol, plasma membrane, or endoplasmic reticulum.The microporation method is efficient, convenient, and inexpensive: macromolecules can be introduced into most adherentneurons in a 3 mm2 surface area while requiring as little as 1 ml of the material to be introduced. We conclude that themicroporation of macromolecules is a versatile approach to investigate signaling, secretion, and other processes in CNS neurons.© 1999 Elsevier Science B.V. All rights reserved.

Keywords: Electroporation; Hippocampal neurons; Quantitative co-transfection; RNA; YFP; CFP; GFP

www.elsevier.com/locate/jneumeth

1. Introduction

The study of signaling processes in intact cells re-quires the combination of molecular biological andphysiological methods. Such processes have beendifficult to study in neurons, in part because neuronsare not readily amenable to methods such as transfec-tion. Although several techniques have been developedfor transfecting neurons, these techniques are time con-suming, more difficult, and less effective in comparisonto the many means available for transfection of celllines. For instance, the construction of viruses such asvaccinia virus (Pettit et al., 1995) or adenovirus(Moriyoshi et al., 1996) is laborious and expensive.

Further, these viruses often have long-term toxic ac-tions. Microinjection (Kaang et al., 1993) or particle-mediated methods (Lo et al., 1994) produce very lowyields when used in cultured central nervous system(CNS) neurons. Lipofection (Holt et al., 1990) or cal-cium-phosphate precipitation (Xia et al., 1996), thoughin many laboratories adequate for DNA transfection,cannot be used to supply RNA or other reagents.

We have attempted to circumvent these problems bydeveloping a method based on electroporation, thetransient permeabilization of the plasma membraneduring application of an electric field. Electroporationis a well established technique for transfecting sus-pended cells such as bacteria, yeast, and cell lines.Recent application of this method to adherent, non-neuronal cells (Teruel and Meyer, 1997) suggests thepossibility that electroporation might be used also oncultured neurons. Further, electroporation creates tran-

* Corresponding author. Tel.: +1-919-681-8072; fax: +1-919-681-7978.

E-mail address: [email protected] (T. Meyer)

0165-0270/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 5 -0270 (99 )00112 -0

M.N. Teruel et al. / Journal of Neuroscience Methods 93 (1999) 37–4838

sient pores that establish direct paths from the extracel-lular space to the cytosol, which provides a means todeliver reagents other than DNA. In this respect, elec-troporation offers a broader range of applications thantransfection techniques such as lipofection or calcium-phosphate precipitation, which are restricted to DNA.For example, electroporation has proven capable ofintroducing RNA, fluorescent dyes, peptides andproteins into cell lines (Subramanian and Meyer, 1997;Oancea et al., 1998; Shen and Meyer, 1998).

Here we describe a microvolume electroporationdevice (termed a ‘microporator’) derived from the ap-paratus described by Teruel and Meyer (1997) andshow that this apparatus is effective in introducingRNA, DNA and dextran molecules into hippocampalneurons in primary culture. Futhermore, we show thatby using the microporation technique with RNA,proteins can be quantitatively co-expressed in neurons.Our studies suggest that the microporation of reagentsinto cultured neurons is a powerful approach to studysignal transduction, secretion, and other cellular pro-cesses in neurons.

2. Materials and methods

2.1. Preparation of GFP-fusion constructs

To evaluate the efficacy of transfection, we examinedexpression of the green fluorescent protein (GFP) or itsderivitives, cyan fluorescent protein (CFP) or yellowfluorescent protein (YFP). The cDNAs for these GFPvariants was kindly provided by Roger Tsien (Univer-sity of California, San Diego). The cloning and in vitrotranscription of elastase–GFP (ER–GFP) is describedin Subramanian and Meyer (1997). The GFP used inthese studies was an enhanced GFP (EGFP) with mam-malian codon usage (Haas et al., 1996), green-shiftedfluorescence (Ser65Thr; Heim and Tsien, 1996), andimproved folding (Phe64Leu; Cormack et al., 1996).The EGFP coding sequence was amplified in a poly-merase chain reaction (PCR) and then cloned into acustom-modified in vitro translation vector, pSHiro3,which was optimized for in vitro transcription in mam-malian cells (Yokoe and Meyer, 1996). This vectorcontained 5% and 3% b-globin untranslated regions and aKozak sequence. PM–GFP were made by inserting aten amino acid sequence at the N-terminal end withconserved myristoylation and palmitoylation sequencesfrom Lyn (MGCIKSKRKD). A similar ten amino acidsequence from the same family member, Fyn, wasshown to be sufficient for plasma membrane localiza-tion through myristoylation and palmitoylation (Resh,1994). DNA sequencing was performed for all con-structs to exclude PCR error.

In vitro transcription and RNA processing of GFPand PM–GFP were performed according to the proce-dure described by Yokoe and Meyer (1996). Briefly, theGFP fusion constructs cloned in pSHiro3 were lin-earized with EcoRI, whose restriction site is down-stream of the 3% untranslated region, and in vitrotranscription was performed with SP6 RNA polymeraseusing a mMESSAGE mMACHINE commercial kit(Ambion, Austin, TX) according to the manufacturer’sprotocol. The reaction was terminated by addition of10 mM EDTA, and the RNA was purified by anRNeasy column (Qiagen, Chatsworth, CA). Polyadeny-lation (addition of a poly A tail) was carried out at37°C for 30 min in a 50-ml reaction mixture containing40 mM Tris–HCl, pH 8.0, 10 mM MgCl2, 2.5 mMMnCl2, 250 mM NaCl, 0.25 mg/ml RNA, 250 mMATP, and 5 units of poly (A) polymerase (Life Tech-nologies, Gaithersburg, MD). EDTA 20 mM was usedto terminate this reaction, and the polyadenylatedmRNA was purified using an RNeasy column. Theeluent (purified mRNA) was dried and dissolved at 1mg/ml in electroporation buffer (125 mM NaCl, 5 mMKCl, 1.5 mM MgCl2, 10 mM glucose, and 20 mMHEPES, pH 7.4).

For DNA transfection experiments, the cDNA wasdissolved at 1–2 mg/ml in the same electroporationbuffer used for the RNA electroporation protocol de-scribed above.

2.2. Cell culture and preparation

Hippocampal neurons were cultured using modifica-tions of the procedures described in Bekkers andStevens (1991) and Ryan and Smith (1995). RegionCA1 was dissected from the hippocampus of 2–4-day-old Sprague-Dawley rats and dissociated with papainor trypsin. Cells were plated onto coverslips coated witheither Matrigel (Collaborative Biomedical Products,Bedford, MA) or 500 kDa MW poly-D-lysine (Sigma).To minimize the number of cells and amount ofmedium required, cells were plated in microwellsformed by using a silicone sealant (Dow-Corning) toseal cloning cylinders (8 mm i.d., Bellco Glass,Vineland, NJ) to the coverslip. Cells were cultured for6–15 days before use. Culture medium consisted ofminimal essential media (Gibco), 5% fetal bovine serum(FBS; Gibco), B-27 (Gibco) and Mito+ serum exten-der (Collaborative Biomedical) at their suggested con-centrations, 3.6 g/l glucose, Pen/Strep (Gibco,5000/5000 U), and 500 mM MEM sodium pyruvate(Gibco). The cells were typically plated at densities of20 000–40 000 per cloning cylinder. After 24 h in cul-ture, the plating media was replaced with mediumwhich also contained the antimitotic combination of 80mM 5-fluoro-2-deoxyuridine (Sigma) and 200 mMuridine (Sigma). The cells were incubated at 37°C in a

M.N. Teruel et al. / Journal of Neuroscience Methods 93 (1999) 37–48 39

5% CO2 humidified chamber and were fed by exchang-ing 50% of the medium twice per week.

To make autapses (Bekkers and Stevens, 1991; Tongand Jahr, 1994), coverslips were coated with 0.15%agarose (Sigma), allowed to dry for 1 h, then sprayedwith a fine mist of poly-D-lysine (0.5 mg/ml) and colla-gen (0.5 mg/ml). Cells were prepared as above andplated at 2000–5000 cm−2. The culture medium wasexchanged 24 h after plating, but antimitotic agentswere not added and the cells were not fed thereafter.Only neurons that were completely isolated from otherneurons (typically on glial islands 200–400 mm in di-ameter) were used for electrical recordings.

To perform the Trypan Blue viable/non-viable cellassay, a 1:1 mixture of extracellular buffer and TrypanBlue (Sigma) was applied for 5 min to neurons that hadbeen microporated 8 h earlier. Only the neurons thatlooked morphologically intact and that excluded theTrypan Blue were counted as viable.

2.3. Optical measurements and microscopy

Confocal fluorescence and differential interferencecontrast imaging were performed with a self-built mi-croscope that used software and hardware modifiedfrom a design developed by Stephen Smith (StanfordUniversity). Four hundred and forty two nanometers(HeCd laser) and 514 nm (argon-ion laser) excitationand appropriate emission filters were used for the imag-ing of CFP and YFP, respectively. Ca2+ responsesevoked by electrical field stimulation were measured onan inverted Nikon Diaphot microscope coupled to anOdyssey confocal imaging system (Noran Instruments,Middleton, WI). During imaging, the culturing mediawas replaced with an extracellular buffer consisting of135 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1.5 mMMgCl2, 20 mM glucose, and 20 mM HEPES (bufferedto pH 7.4). All experiments were performed at roomtemperature (�25°C).

[Ca2+] was monitored using the calcium indicatorCalcium Green-1 dextran (70 000 MW, MolecularProbes, Eugene, OR). The following equation was usedto convert the relative peak amplitude of fluorescenceto an apparent free intracellular Ca2+ concentration:[Ca2+]=Kd[F(t)−Fmin]/[Fmax−F(t)], where F(t) is themeasured background-corrected intensity of CalciumGreen-1 dextran fluorescence, Kd=240 nM and Fmax

was measured as the fluorescence intensity after addi-tion of ionomycin (�1 mM) in the presence of extracel-lular [Ca2+] of 1.5 mM. Fmin was measured on aspectrofluorometer (Fluoromax, SPEX Industries,Edison, NJ) to be 35% of Fmax.

2.4. Whole-cell patch-clamp recording

For patch clamp recording, cells were bathed in asolution containing 150 mM NaCl, 3 mM KCl, 2 mM

CaCl2, 2 mM MgCl2, 20 mM glucose, and 10 mMHEPES. The patch pipette solution was filled with anintracellular solution containing 100 mM gluconic acid,5 mM MgCl2, 5 mM NaATP, 0.3 mM NaGTP, 5 mMEGTA, and 40 mM HEPES, with the pH adjusted to7.3 with CsOH. Currents were recorded using an EPC-9amplifier. Series resistance was monitored and compen-sated ]80%, and ranged from 5 to 20 MV. A secondpatch pipette filled with the extracellular solution (�2MV) was used for extracellular stimulation of nearbyneurons.

2.5. Field stimulation

Calcium transients were generated using the fieldstimulation electrodes described in Jacobs and Meyer(1997). This device consisted of two parallel platinumwires (3 mm apart) that were lowered until they nearlycontacted the coverslip covered with neurons. Com-puter-controlled current pulses yielding fields of �10V/cm were applied in bursts, 1 s in duration andconsisting of 30 1-ms pulses (i.e. 30 Hz).

3. Results

3.1. The microporator

Fig. 1A shows a schematic view of the small-volumeelectroporation device (microporator) that was used inour experiments. This device was modified from thedesign of Teruel and Meyer (1997). The foot of themicroporator is placed onto a coverslip of neurons,creating a restricted space above the neurons. Reagentsto be introduced into porated neurons are injected intothe small space through a hole in the center of the footstructure (Fig. 1B). The volume of this space is keptsmall to restrict flow of electrical current and also toreduce the amount of reagent that is needed for trans-fection. In this version of the microporator, the distanceunder the foot structure was increased from 100 mm to300 mm to reduce shear forces on the neurons duringthe application of the reagent and to increase the spaceavailable for the cells.

The electroporation protocols described in Terueland Meyer (1997) were also modified for application toneurons. Before electroporation, the medium in thecloning cylinder was carefully removed and stored, andthe cells were then placed in electroporation buffercontaining a glutamate receptor blocker, 6-cyano-7-ni-troquioxaline-2,3-dione (CNQX; 10 mM). This drugwas added to prevent potential excitotoxicity caused byglutamatergic synaptic transmission activated duringporation. After electroporation, this buffer was re-moved, and the cells were once again placed in culturemedium from the cloning cylinder in which they had


been cultured. The cells were then returned to theincubator for 1 h or more to recover. Good transfectionresults were also obtained when the neurons were left inthe same medium they were cultured in during pora-tion, recovery, and imaging and thus were not subjectedto possible differences in medium/buffer osmolarity.

3.2. Transfection of hoppocampal neurons bymicroporation

We first determined whether the microporator couldbe used to transfect cultured neurons with mRNA. Weachieved transfection of cultured hippocampal neuronswith RNA via the four-step process shown in Fig. 2A.Capped and polyadenylated (A) RNA can be used toexpress proteins in mammalian cell lines (Yokoe andMeyer, 1996), so we first synthesized modified RNA by

in vitro transcription in the presence of a methyl cap-analog. Second, poly-(A) polymerase was added toobtain a 3% poly(A) tail. While this step is not necessaryfor protein expression in Xenopus lae6is oocytes, inmammalian cells it increases the amount of expressedproteins at least 3-fold (H. Yokoe and E. Oancea,personal communication). Third, RNA was introducedinto neurons by injecting 1 ml of RNA (�1 mg/ml) intothe microporation region. For neurons 8 days old oryounger, three voltage pulses—each 220 V/cm, lasting30 ms and of opposite polarity—were given at 20 sintervals. For neurons older than 8 days, only twovoltage pulses were used. Fourth, an incubation periodof 2–8 h was allowed for synthesis of protein from themRNA.

To assess the efficacy of transfection, we looked atthe expression of green fluorescent protein (GFP) inneurons grown 9–15 days in culture. Following intro-

Fig. 1. Diagram of a microporator for use on cultured neurons, modified from an earlier device described by Teruel and Meyer (1997). (A)Schematic view of the microporator. (B) The most important change in this device is a 300-mm space under the microporator foot, whichminimizes shear forces on the sheet of neurons during sample application. The foot is guided by linear bearings onto a coverslip with neurons inculture, and the sample solutionreagent to be porated is applied through a microliter injector in the center of the foot. A 1-ml reagent sample issufficient to porate neurons within an area of 3 mm2.


Fig. 2. RNA transfection of cultured hippocampal neurons. (A) Schematic representation of the RNA transfection technique. First, capped RNAis synthesized by in vitro transcription. Second, a 3% poly(A) tail is added to the RNA by in vitro addition of poly(A) polymerase. Third, themodified RNA is concentrated and then porated into adherent neurons using the microporator device. Fourth, the cells are left to synthesize thetransfected protein for a period of at least 2 h. (B) Hippocampal neuron transfected with RNA encoding EGFP. A differential interferencecontrast image of a microporated neuron is shown on the left, and a confocal fluorescence section through the same neuron is shown on the right.(C) Transfection of neurons and glial cells in co-cultures. The arrows are pointing to examples of transfected glial cells. (D, E) Lowermagnification images showing the high fraction of neurons transfected by this method. In many regions, greater than 50% of the neurons aretransfected. The scale bar is 20 mm in all images.Fig. 3. DNA transfection of cultured hippocampal neurons. (A) A confocal fluorescence section through hippocampal neuron transfected withDNA encoding EGFP. (B) Epifluorescence image of neurons microporated with DNA after 3 days in culture, imaged 2 weeks later.


duction of RNA by poration, large numbers of neuronsand glial cells expressed GFP. Differential interferencecontrast and fluorescent images of a transfectedhippocampal neuron are shown in Fig. 2B. This cellhad a typical neuronal morphology while expressinglarge amounts of GFP (Fig. 2B, right). Likewise, trans-fected glial cells seemed unperturbed by the porationprocess (Fig. 2C). By comparing the intracellularfluorescence intensity of GFP to that of fluorescein-conjugated dextran (Yokoe and Meyer, 1996; Terueland Meyer, 1997), we estimated the maximal intracellu-lar concentration of GFP expressed following RNAtransfection to be \3 mM. The transfected area ap-peared to cover approximately 3 mm2 following pora-tion with a 1 ml sample of RNA, but larger volumes ofreagents could be applied to electroporate larger num-bers of neurons. In the areas on the coverslip exposedto RNA, more than 90% of the intact neurons weretransfected (Fig. 2D, E). Under most conditions, wefound that the fraction of cells transfected was greatestprior to 10 days in culture.

Because preparation of DNA is simpler and lessexpensive than preparing RNA, we also tested theability of the microporator to transfect hippocampalneurons with DNA. We achieved DNA transfectionusing the same procedure as for RNA, except that 1 mlof 2 mg/ml plasmid DNA encoding GFP was applied.Fig. 3A, B show neurons transfected with DNA. Theseneurons were typical in their morphology and brightlyfluorescent as a result of GFP expression. While thefraction of transfected neurons was always lower whenDNA was used instead of RNA (�1–30%), the trans-fected neurons expressed GFP at high concentrations.When quantified using the method described above, weestimated the concentration of expressed GFP to be\10 mM in many cells following DNA transfection.

The time course of protein expression differed for thetwo transfection techniques. GFP was observed as littleas 2 h following RNA transfection, typically reached amaximum within 8–12 h, and declined thereafter. Thistransient expression presumably is due to the rapiddegradation of mRNA within the cell. Following DNAtransfection, GFP was rarely observed within 4 h, butthe concentration continued to increase over the courseof at least 24 h and was maintained for many days orweeks in some neurons (Fig. 3B). The slower onset ofexpression following DNA transfection presumablyarises from delays in transcription of the DNA intoRNA. The longer duration of expression suggests thatthe DNA persists within the cell for at least severaldays.

For most experiments, we used 1 mg/ml RNA or 2mg/ml DNA and found that these concentrations pro-vided high rates of transfection and high levels ofexpression. In general, changing the concentration ofRNA changed the average expression level in trans-

fected cells, but did not affect the fraction of cells thatwere transfected. On the other hand, decreasing theDNA concentration to 0.1 mg/ml or below reduced thenumber of cells transfected; however, even at 0.1 mg/mlDNA, a few neurons were often transfected and someof these appeared to express large amounts of GFP.

The number of adherent cells could be reduced byeither injection of the sample or application of theelectric field. However, damage was minimized by useof the protocols and device described here, and withcare, few neurons were immediately killed or detachedfrom the coverslip. To measure the proportion of neu-rons that survived the procedure, we microporatedneurons grown 6 days in culture and counted the viableneurons 8 h later by Trypan Blue exclusion. When theneurons were very healthy before electroporation morethan 50% of the neurons survived. Together, theseresults demonstrate that neurons in primary culture canbe efficiently transfected via electroporation. Further-more, cells successfully transfected using this methodremain healthy, displaying normal morphology and thecapability for normal transcription and translation.

3.3. Quantitati6e co-expression of YFP and CFP byRNA transfection

We next asked whether electroporation could trans-fect cultured neurons with more than one construct at atime. To accomplish this, we synthesized RNAs thatencoded two variants of GFP: cyan fluorescent protein(CFP) and yellow fluorescent protein (YFP). These twoproteins differ in their spectral properties, permittingsimultaneous and independent detection of the expres-sion of each (Heim and Tsien, 1996). The RNAs encod-ing each of these proteins included a ten amino acidlong targeting sequence that targeted the proteins to theplasma membrane (see below). The expression of theseproteins was assessed by imaging with a confocal mi-croscope able to excite and collect emission from eachprotein separately. With this setup, when CFP alonewas expressed in neurons (Fig. 4A), fluorescence waseasily detected in the CFP channel, but fluorescencewas less than 5% as bright in the YFP channel. Con-versely, expressed YFP was detected in the YFP chan-nel but not in the CFP channel (Fig. 4B). Thus, thesetup achieved parallel confocal imaging of CFP andYFP and was therefore able to assess coexpression ofthe two fluorescent proteins.

When neurons were microporated with a 1:1 mixtureof RNA for CFP and YFP, all transfected neuronsexpressed both proteins (Fig. 4C). We evaluated theefficiency of cotransfection by quantifying the fluores-cence intensity of each protein in many neurons. Asshown in Fig. 4D, the absolute intensity of expressionvaried considerably from neuron to neuron; however,within a given neuron, the two proteins were always


Fig. 4. Quantitative co-transfection by RNA microporation. (A, B) Effective separation of fluorescence from CFP and YFP following RNAtransfection of either CFP (A) or YFP (B). The excitation wavelength was 442 nm for CFP and 514 nm for YFP. Emission was collected between450 and 490 nm for CFP and above 525 nm for YFP. Proteins were targeted to the plasma membrane as described in Section 3. (C) Cotransfectionof CFP and YFP with a 1:1 mixture of their respective RNAs. (D, E) Comparison of cotransfection by 1:1 mixtures of RNA (D) or DNA (E).Fluorescence intensity was measured for each protein in identical areas of the neurons.

expressed at nearly identical levels. This observationcontrasted with the pattern of expression followingtransfection of the DNAs for the same two proteins.

Following DNA cotransfection, only some of the trans-fected neurons expressed both proteins and the relativeexpression levels of the two proteins were widely scat-


tered (Fig. 4E). Thus, although either DNA or RNAcan be used for co-transfection by microporation, thequantitative coexpression of multiple proteins can onlybe achieved by using RNA.

3.4. Microporated neurons produce action potentialsand calcium signals

Although microporated neurons are morphologicallyintact, we were interested in determining whether pora-tion affected their physiological properties. We firsttested their membrane excitability. Whole-cell patch-clamp recordings from GFP-transfected neurons re-vealed that these cells have normal action potentialsand ionic currents. For example, depolarization elicited

voltage-dependent Na+ and K+ currents that appearedqualitatively very similar to those recorded from non-transfected neurons (Fig. 5A, left). The Na+ currentwas reversibly blocked by tetrodotoxin (500 nM), apotent blocker of Na+ channels (Fig. 5A, center andright). Thus, poration does not appear to affect theexcitability of neurons.

We also used a fluorescent calcium indicator to testwhether electrical stimulation could elevate cytosoliccalcium concentration (Jacobs and Meyer, 1997). Themembrane-impermeant calcium indicator, CalciumGreen-1 dextran (70 kDa molecular weight), could beloaded into neurons via microporation (Fig. 5B). Thisshows that poration can deliver macromolecules otherthan RNA and DNA into neurons. A burst of depolar-izing stimuli induced calcium transients (Fig. 5C; N=50 responses out of 50 cells tested) that were 250–300nM in amplitude (spatially averaged value) and wereindistinguishable from those measured in non-poratedneurons (Jacobs and Meyer, 1997). Thus, microporatedneurons have intact voltage-gated calcium channels,can generate action potentials, and in response to ac-tion potentials can produce calcium signals similar tothose measured in neurons that are not microporated.

3.5. Synaptic transmission is intact in transfectedneurons

We next evaluated synaptic transmission in trans-fected neurons. For this purpose, we used whole-cellpatch-clamp methods to record excitatory postsynapticcurrents (EPSCs) in neurons microporated with RNAencoding GFP. EPSCs could be recorded in one GFP-transfected neuron when another transfected neuronwas stimulated with an extracellular electrode (Fig. 6A;N=10 responses out of ten cell pairs tested). Theseresponses could be blocked reversibly by CNQX (10mM; Fig. 6A), confirming that they arose from func-tional glutamate receptors. When transfected neuronswere directly stimulated by applying a brief depolariz-ing voltage step through the patch pipette, the cellproduced an action potential current (off scale in Fig.6B). In some cases, this current was followed by pro-longed responses with multiple peaks (Fig. 6B). Thesemultiple peaks were eliminated by CNQX, indicatingthat they likely arose from feedback excitation of thecell through a network of nearby neurons. Becauseboth the neuron from which the recording was madeand most of its neighbors had been microporated, thepresence of a synaptic response indicates that transfec-tion does not interfere with the release ofneurotransmitters.

To more closely examine presynaptic function inelectroporated neurons, we cultured neurons in isola-tion on microislands of substrate and glia. In theseconditions, the neurons form synapses upon themselves

Fig. 5. Intact electrical excitability and calcium signalling in micropo-rated neurons. (A) Voltage-dependent Na+ and K+ currentsrecorded from a microporated neuron. Voltage steps were given froma holding potential of −70 mV to +10 mV and +50 mV, respec-tively. TTX (500 nM) reversibly blocked the initial inward Na+

current. (B) Differential interference contrast and confocal fluores-cence images of a hippocampal neuron microporated with 70 kDaCalcium Green dextran. Due to its large size, this dextran analog isexcluded from the nucleus and can be used to measure cytosoliccalcium. (C) Field stimulation-induced Ca2+ responses measured asfluorescence increases in the soma of a neuron microporated withCalcium Green dextran. Transient increases in intracellular Ca2+

were triggered by each of six bursts of stimuli (at arrows), each 1 s induration and including 30 pulses.


Fig. 6. Intact synaptic transmission in microporated hippocampalneurons. (A) EPSCs in a GFP-transfected neuron recorded in re-sponse to stimulation of a nearby GFP-transfected neuron. Tracesbefore during and after application of CNQX (10 mM) are shown. (B)A complex, recurrent EPSC recorded in a GFP-transfected neuron inresponse to a brief depolarizing step applied through the patchpipette (0.8 ms step to 0 mV from 70 mV). (C) EPSCs during and 1s after evoking 50 action potentials at 20 Hz in an isolated, autapticneuron grown on a glial island and microporated with 70-kDafluorescein dextran. (D–F) Amplitudes of EPSCs recorded duringand after application of a train of 50 action potentials (bars). Thedashed line is the average response amplitude to low frequencystimuli (0.06 Hz) given before the train. The last stimulus was given1 s after termination of the train. Graphs illustrate responses from anisolated, autaptic neuron that had not been porated (D), responsesfrom the porated autaptic neuron shown in C (E), and responsesfrom the porated neuron shown in A (F).

(autapses) and provide a particularly favorable arrange-ment for synaptic stimulation and recording (Bekkersand Stevens, 1991). Recurrent synaptic responses weremeasured in every autaptic cell (N=7 synaptic re-sponses out of seven cells examined) that was loadedwith fluorescein–dextran via microporation. An exam-ple of such a response is shown in Fig. 6C (left).Following the initial inward current, which is caused byan action potential, was an EPSC that arose from therelease of glutamate from the presynaptic terminals ofthe neuron onto itself. This directly demonstrates thatporated neurons are capable of releasing neuro-transmitter.

We also examined synaptic depression, a form ofplasticity that is thought to arise from the depletion ofan available pool of releasable synaptic vesicles (Thies,1965; Liley and North, 1968; Stevens and Wang, 1995).In control neurons, a high-frequency train of actionpotentials caused a depression in the amplitude ofEPSCs, and EPSCs recovered within seconds after thetrain stopped (Fig. 6D). Synaptic depression appearedto be very similar in autapses porated with fluoresceindextran (Fig. 6E; N=3 of three) as well as in amulti-cell culture transfected with GFP (Fig. 6F). In allthree situations, the half-maximal decrease in EPSCamplitude occurred after approximately five action po-tentials and nearly complete recovery was observedwithin 1 s. These results indicate that the kinetics ofsynaptic depression are unaffected by microporation.This suggests that synaptic vesicle release and recycling(Rosenmund and Stevens, 1996), as well as presynapticterminal calcium homeostasis (Swandulla et al., 1991),are not affected by the microporation process. Weconclude that transfection affects neither the presynap-tic nor the postsynaptic functions of neurons.

3.6. Targeting of proteins in transfected neurons

Because neurons are highly compartmentalized cells,we were interested in determining whether proteinsexpressed by our transfection procedure were localizedto their correct targets. For this purpose, we askedwhether proteins that are destined for the plasma mem-brane or endoplasmic reticulum were correctly ex-pressed and targeted in the neurons. GFP was fused toa ten amino acid N-terminal myristoylation and palmi-toylation sequence which has been shown to localizeproteins to the plasma membrane (Resh, 1994). Whenthis PM–GFP construct was expressed in neurons, itwas clearly located on the neuronal plasma membrane(Fig. 7A, B). The branching pattern could be resolvedvery clearly in neurons expressing this protein, makingit a useful technique for visualizing the fine structure ofneurons. We also expressed an elastase–GFP fusionconstruct which localizes to the lumen of the endoplas-mic reticulum in other cell types (Subramanian andMeyer, 1997). This protein showed a tubular network


staining, reminiscent of the endoplasmic reticulum,when expressed in neurons (Fig. 7C, D). The endoplas-mic reticulum appeared to be continuous along theprocesses of some, but not all, of these transfectedneurons (Terasaki et al., 1994). This construct was alsoexpressed in glial cells (Fig. 7E). The flatter morphol-ogy of the glial cells made the continuous, as well as

isolated, structures of the endoplasmic reticulum morereadily apparent. Together, these examples show thatneurons, as well as glial cells, can be readily transfectedwith different GFP-based probes and that these probestarget to the correct location. This enables studies ofdynamic changes in the localization of cellular proteinsand organelles in intact neurons.

Fig. 7. Correct targeting of expressed GFP-tagged proteins. (A, B) Plasma membrane localization of a GFP construct with a consensus sequencefor palmitoylation/myristoylation. Neurons were transfected with RNA encoding a ten amino acid myristoylation and palmitoylation sequence(from Lyn) fused to the N-terminal end of GFP. Two transfected neurons with different morphologies are shown. (C, D) Localization of a GFPconstruct targeted to the endoplasmic reticulum (ER–GFP). Neurons were transfected with RNA encoding a fusion protein of elastase and GFPwhich has been previously shown to be a marker of the lumen of the endoplasmic reticulum (Subramanian and Meyer, 1997). Reticular structuresthat are highly reminiscent of the endoplasmic reticulum can be seen in the cell body and processes of hippocampal neurons. (E) Confocal sectionbelow the neuronal layer from a culture transfected with RNA for ER–GFP. A fragmented and dynamically rearranging structure of theendoplasmic reticulum in glial cells is clearly visible.


4. Discussion

Our study demonstrates that microporation is anefficient method to introduce RNA, DNA and othermacromolecules into neurons. This offers a new andversatile approach to neuronal transfection and makespossible other experimental approaches for the studyof neuronal function. By a series of tests, we haveshown that microporated neurons are functionally in-tact. First, using differential interference contrast andfluorescence imaging, we found that microporatedneurons are morphologically indistinguishable fromnon-transfected neurons. Second, the transcriptionand translation machinery of these neurons was in-tact, as demonstrated by the expression of GFP viaboth RNA and DNA transfection. Third, we foundthat neurons could still fire action potentials and hadintact calcium signaling. Fourth, porated cells re-tained intact synaptic transmission. Finally, neuronsappeared to localize expressed proteins to their appro-priate intracellular targets. Taken together, these mea-surements show that microporation of macro-molecules can be used to study synaptic transmissionand many other molecular and cellular processes inintact, functional neurons.

The ability to transfect with either RNA or DNAoffers tremendous flexibility. The principle differencebetween RNA and DNA transfection is that micropo-rated RNA has only to reach the cytosol instead ofthe nucleus for protein expression to occur. Thus,RNA transfection offers the following important ad-vantages. (1) Protein expression is rapid; severalmicromolar of intracellular GFP can be synthesizedin as little as 2 h (Yokoe and Meyer, 1996). (2) Themethod is efficient. Using RNA transfection, morethan 50% of the neurons can often be transfected.This will enable simultaneous imaging of several neu-rons or even entire neuronal networks. It also shouldbe a benefit for small-scale biochemical experiments,which usually require a large fraction of transfectedcells. (3) RNA transfection is quantitative. Differentcells transfected with a given amount of RNA expresssimilar amounts of protein. Since the concentration ofexpressed protein falls in a more narrow range withRNA transfection compared to DNA transfection(Fig. 4), expression levels can be more easily titratedby diluting the RNA before loading in the micropora-tor. (4) Co-expression of different proteins in thesame cell is readily possible. Imaging studies of thedistribution of different proteins tagged with differentGFP color variants can be achieved by mixing theirrespective RNA. Furthermore, multiple proteins thatreconstitute a signaling pathway or dominant negativeinhibitors of cell function could be co-expressed inindividual cells at defined relative concentrations. Onthe other hand, DNA is simpler and cheaper to pre-

pare than RNA, and DNA transfection results inconsiderably higher protein concentrations andlonger-lasting protein expression.

We showed that microporation can be used notonly for transfection but also to introduce dextran-based fluorescent indicators into cultured neurons. In-deed, previous studies of cell lines suggest that themicroporator can be used to introduce many differentbiologically active compounds or optical indicators,such as peptides (Stauffer et al., 1997) and cagedcompounds (Horne and Meyer, 1997). Thus, we ex-pect that microporation will enable many new experi-mental strategies to unravel the cellular and molecularmechanisms underlying neuronal function.

Acknowledgements

We thank S. Smith, Stanford University, and T.Ryan, Cornell University Medical School, for provid-ing us with support in the construction of the confo-cal microscope, and thank D. Holowka, CornellUniversity, Ithaca, NY, for suggesting the plasmamembrane localized GFP construct. We also thank E.Oancea, W. Chen, K. Subramanian and T. Staufferfor valuable discussions and experimental support.This work was supported by a Fellowship from theDavid and Lucile Packard Foundation to T.M., Na-tional Institutes of Health Grants GM-48113 andGM-51457 to T.M. and National Institutes of HealthGrant NS-17771 and a McKnight Foundation Grantto G.A.

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