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INTRODUCTION |
In live neuronal cultures, especially those cocultured with other cell types, it is difficult to visualize and identify neurites and terminal process arising from individual neurons. To overcome these problems, investigators have tried a number of approaches including: plating neurons at low density intracellular injection of fluorescent dye, and labeling neurons and neural processes with fluorescently tagged tetanus toxin. These various techniques are limited by reduced neuronal survival, neuronal toxicity, photobleaching, and the inability to selectively label individual neurons, respectively. To overcome these difficulties, we have generated replication-defective adenovirus that encodes Aequorea green fluorescent protein (GFP). In these experiments, we used GFP to both visualize live neurons, neurites, and terminals in dorsal root ganglion (DRG) epithelial cocultures and as a marker gene for the coexpression of serotonin type 3 (5HT3) receptor in these neurons.
GFP offers numerous advantages as a marker gene such as: continued developmental expression, lack of reported toxicity (Chalfie et al. 1994
), complete neuronal labeling, and selective identification of genetically altered cells. Previous studies have examined coexpression of GFP and other genes in nonneuronal cells. For instance, in cultured human kidney cells transfected with a GFP-N-methyl-D-aspartate receptor type 1 (NMDAR1) chimera, the degree of green fluorescence was correlated with both NMDAR1 receptor density and the amplitude of electrophysiological responses to applications of glutamate (Marshall et al. 1995
). In Caenorhabditis elegans, GFP-tagged functional Tax-4 was expressed in neurons (Komatsu et al. 1996
). Similar studies, however, have not been done in mammalian neurons. To examine whether replication-defective adenovirus encoding GFP would label DRG neurons in coculture and not affect their electrophysiology, DRG-epithelial cocultures were infected with this construct. We examined GFP expression and the electrophysiology of both labeled and unlabeled neurons. In addition, we examined the potential for coexpression of GFP and a second gene not normally expressed in this neuronal population, in this case the 5HT3 receptor (Maricq et al. 1991
). Electrophysiological studies allowed us to verify that only fluorescent neurons expressed functional 5HT3 receptor.
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METHODS |
Generation of adenovirus containing GFP and serotonin double cassettes
GFP, mGFP4 (gift from Dr. J. Haseloff), and serotonin receptor were inserted separately and together into the pXCJL vector (gift from Dr. W. Mazur, Baylor College of Medicine) that we modified to contain the Rous sarcoma virus long terminal repeat and the bovine growth hormone polyadenylation signal (BGH). Adenovirus containing GFP alone or GFP and serotonin receptor inserts were generated by cotransfecting equal molar concentrations of either of these plasmids and pBHG11 (Microbix) into 293 cells using the liposomal (DOTAP), according to manufacturer's protocol (Boehringer Mannheim). Liposomes were added to the wells of three 24-well plates in which the 293 cells had reached ~70% confluence. Monolayers of 293 cells were incubated at 37°C overnight and refed. Successful recombinations produce infectious virions that induce the lysis of 293 cells. This cytopathic effect (CPE) can be monitored visually, total cell lysis in the well occurs within 14 days. Viral DNA isolated from 100 to 200 µl of supernatant from the wells displaying CPE was examined by polymerase chain reaction (PCR) to determine if virions contain the cDNA of interest (Zhang et al. 1993
). Virus containing DNA inserts were plaque purified twice and grown on 293 cells to produce large amounts of adenovirus. Tissue culture supernatant containing adenovirus was concentrated by centrifugation over cesium chloride using the method of Graham and Prevec (1991).
Tissue culture preparation
DRG cells were obtained and cultured using standard dissection and incubation techniques (Fields et al. 1978
; Wood 1976
). Newborn Sprague Dawley rat pups were anesthetized, decapitated, and their dorsal root ganglion removed and placed immediately into chilled media (Hanks solution with 1 mg/ml collagenase). After dissection, Trypsin (100-300 µl/ml) and DNase (0.5 mg/ml) were added to the media, and cells were incubated for 20 min with gentle agitation every 5 min. Cells then were centrifuged and washed twice with Hanks solution. The Hanks solution then was replaced with Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and triturated to separate cells. The cells were again washed with DMEM + 10% FBS and incubated for 1 h at 37°C and preplated twice to remove as many fibroblasts and Schwann cells as possible. Cells were counted and 50 ng/ml of nerve growth factor (NGF) added to the media. The cells were plated in incubation media composed of DMEM supplemented with penstrep (1%), N2 (Sigma I-1884, 5 µg/ml), bovine serum albumin (50 µg/ml), and NGF (50 ng/ml) at a final density of ~600 cells/dish in the center of 30 mm dishes that were precoated with Matrigel. Cells were thereafter incubated at 37°C.
Adenovirus containing the GFP construct alone was added to culture dishes (50 pfu/cell) 3 days after plating. Adenovirus containing both the GFP and 5-HT3 constructs was added to another group of culture dishes (50 pfu/cell) 9 days after plating.
Treatment groups
Time-matched populations of DRG neurons were studied. Characterization of cells exposed to adenovirus containing the GFP construct alone involved examining three different groups of cells: neurons from control dishes not exposed to adenovirus (control), neurons exposed to the adenovirus but that were not transfected and therefore remained nonfluorescent (Group GFP
), and successfully transfected cells exhibiting GFP-fluorescence (GFP+).
The neurons in dishes exposed to adenovirus containing the combined GFP-5-HT3 construct were divided into two groups: fluorescent and nonfluorescent.
Imaging
Neurons were visualized with a Nikon Diaphot 300 epifluorescent microscope fitted with Hoffman optics. The microscope was mounted on a moveable platform controlled by a DCI 8000 position controller. This setup offered the ability to locate numerous neurons within a dish and store their x-y coordinates for repeat observation over the life of the cultures. Images were captured using a Hamamatsu C2400 High Performance SIT camera. Image processing was achieved using MetaMorph (Universal Imaging Corporation) software. Cell body diameters were measured using calibration and measurement options available in the MetaMorph software.
Electrophysiology
Whole cell patch-clamp recordings were performed 5-14 days after exposure to the adenovirus or 5-15 days postculture in the case of nonvirus exposed controls. All recordings were performed at room temperature (21-23°C) in an extracellular solutioncomposed of the following components (in mM): 126 NaCl, 2.5 KCl, 10 glucose, 2 CaCl2·2H2O, 2 MgCl2, 1.25 NaH2PO4, 20N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid (HEPES); pH 7.3. The patch electrode solution was composed of the following constituents (in mM): 5 KCl, 130 K-gluconate, 2 MgCl2, 10 HEPES, 10 ethylene glycol-bis(
-aminoethyl ether)-N,N,N
,N
-tetraacetic acid, and 1 CaCl2. Patch electrodes were fabricated from KG-33 glass capillary tubes with 1.5-mm OD and 1.12-mm ID containing 0.10-mm inner filaments (Garner Glass). Electrodes were pulled using a Narashige PP-83 puller using settings that yielded impedances of 3-6 M
when filled with the patch solution. Electrodes were mounted on carriers that were advanced using piezoelectric drives (Burleigh), allowing fine position control. An Axon Instruments Axopatch 200A patch-clamp amplifier was used for recording whole cell currents and voltages and to deliver current pulses to patched cells. Membrane voltage and current signals from the Axopatch 200A were filtered (3 kHz high frequency cut off using Frequency Devices Model 900 Bessel filter), then digitized in two separate channels at 44 kHz and stored on VCR tape using a Neurocorder DR-890 (Neurodata). Off-line data analysis was performed using WCP V1.2 electrophysiology software (Strathclyde Electrophysiology Software).
Neurons with <1-G
seals were not studied further. In voltage-clamp mode, series resistance was measured and ranged from 8 to 23 M
. Series resistance was compensated >50% in all cells. After the whole cell current transient was eliminated, the whole cell capacitance was read off of the Axopatch 200A control panel meter. After series resistance, compensation was adjusted and the recording was switched to current-clamp mode (I = 0) and the resting potential recorded. Neurons with resting potentials more depolarized than
45 mV were not studied any further. Neurons were monitored for several minutes to check for stability and to determine if spontaneous spiking occurred. Current then was adjusted to a level such that a membrane potential of
70 mV was reached. A series of 50-ms square-wave current pulses were then delivered to elicit action potentials (AP) and yield information as to AP thresholds, AP amplitudes and AP duration at half amplitude. The first pulse in each series was 250 pA and each successive pulse (delivered at 5-s intervals) was increased by 50 pA until the final pulse, which was 1,000 pA in amplitude. For some cells, a similar series of 3-ms current pulses also was delivered to assess the degree of afterhyperpolarization (AHP) exhibited. AP durations were defined as the difference between the time points at which the falling and rising phases of the spike reached 50% of the spike amplitude.
Neurons that were exposed to the GFP+serotonin (5-HT) construct were treated similarly except that instead of a series of current pulses, spritzes of 10
3 M 5-HT were delivered using a Narishige IM-200 pressure spritzer while the neurons were held at
70 mV in voltage clamp. Methylene blue, which alone had no effect on DRG electrophysiology, was added to the spritzing solution (which consisted of 10
3 M 5-HT freshly dissolved in the extracellular solution). Application of 5-HT was confirmed by observing a cloud of ejected blue solution that clearly reached the DRG neuron.
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RESULTS |
Expression of green fluorescent protein by dorsal root ganglion neurons
To examine the potential of labelling live neurons with GFP, we treated DRG neuronal cultures with adenovirus encoding mGFP4s/t (mGFP4/Ad). This modified form of GFP has been mutated to eliminate the potential recognition of a cryptic intron (Haseloff and Amos 1995
; Zolotukhin et al. 1996
). We also have increased the brightness of GFP by changing the serine in position 65 to a threonine (Heim et al. 1995
). Green fluorescence of DRG neurons treated with 50 pfu/cell mGFP4/Ad was first detected in the soma 2 days after infection, spreading into the neurites over longer periods of time. After 7-10 days postinfection, an extensive network of neurites from individual neurons could be discriminated by GFP fluorescence (Fig. 1). Interestingly, in all cultures only a small number of nonneuronal cells appeared to show GFP fluorescence, although numerous nonneuronal cells were present in these cultures (Fig. 1). Neurons labeled with GFP could be examined routinely under fluorescent microscopy with no apparent loss in the number or intensity of fluorescent neurons. Actually, over time GFP fluorescence appeared to be brighter, most likely due to accumulation of GFP within the neurons and their processes.

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| FIG. 1.
Photomicrographs of live neurons showing green fluorescent protein (GFP) fluorescence after transfection with mGFP4/Ad. A: within 10 days posttransfection, GFP-fluorescence can be identified throughout neuronal cell bodies and neurites. B: higher magnification of a different neuron, 12 days posttransfection shows GFP-positive neurites. C: phase image of B demonstrates that the majority of nonneuronal cells ( ) remain GFP-negative. D: neuronal processes of GFP-positive neurons could be identified as far as several millimeters from cell body where they were found to terminate.
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Not all DRG neurons appeared to be transduced by mGFP4/Ad. To determine the percent of neurons expressing GFP and the effect this expression had on neuronal survival, we counted the number of GFP-positive neurons and compared it with the number of GFP-negative neurons in randomly selected fields. In these cultures, on average, 10% of the neurons were labeled by GFP, with variations of 5-15% between individual cultures when examined 6-17 days after transfection (Fig. 2A). Neurons expressing GFP also appeared to represent a homogeneous population of DRG neurons as determined by measurements of neuronal cell body diameters (Fig. 2B). Under our culture conditions, the mean diameter of control, GFP-positive and -negative neurons were, respectively, 24.4, 25.8, and 23.4 mm (Fig. 2B, Table 1). There was no statistical difference among these three groups.

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| FIG. 2.
Dorsal root ganglion (DRG) neuron characteristics. A: plot showing percentage of neurons that became GFP-positive at various times after exposure to adenovirus with GFP construct. GFP-positive neurons were those that exhibited definite fluorescence as compared with those in same culture dish that were exposed to adenovirus with GFP construct and did not express GFP. Mean percentage of GFP-positive neurons is displayed ( ). Percentage of neurons with GFP fluorescence did not significantly correlate with days postexposure (r = 0.32). B: comparison of mean DRG cell body diameters for GFP-positive and -negative neurons in control (nonviral exposed) group, adenoviral GFP construct exposed group, and group exposed to adenovirus containing constructs for both GFP and5-HT3 receptor. There are no significant differences in mean cell body diameters between any of 3 groups (2-tailed t-test).
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Electrophysiological properties of DRG neurons transfected with mGFP4/Ad
To determine if transfection of DRG neurons with adenovirus encoding GFP changed the electrophysiological properties of neurons, we examined neurons that were grown in virus free conditions (control), in the presence of virus and either GFP negative (GFP
) or GFP positive (GFP+). The electrophysiological parameters measured or calculated included: whole cell resistance, resting potential, AP threshold, AP duration, AP amplitude, and whole cell capacitance. There were no statistically significant differences between the untreated control (C) DRG neuron group and the GFP+ or GFP
virus-treated groups. Representative images of the DRG neurons and their evoked AP that were recorded are shown for all three groups in Fig. 3. The majority of cells (17 out of 20) exhibited a dip in the downward phase of the action potential. AHP was present equally in all three groups studied and occurred in 9/20 cells, ranging from
2.3 to
11.3 mV. Exposure to maximal fluorescent excitation during the course of the recordings, which were
4 h, did not produce any signs of cellular deterioration.

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| FIG. 3.
Bright field, matching epifluorescent images, and action potential recordings from noninfected control (A), GFP-positive (B), and GFP-negative (C) DRG neurons. Action potentials in response to 50-ms voltage pulses were recorded in current-clamp mode with membrane potential initially held at 70 mV. Calibration bars on photomicrographs 10 µm. Calibration bars for waveforms are 20 mV (vertical) and 5 ms (horizontal).
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Functional coexpression of mGFP4 and 5-HT3 receptor
To examine the functional coexpression of GFP and a neurotransmitter receptor by DRG neurons, we generated a second adenoviral construct that encoded both mGFP4 and 5-HT3 receptor in separate expression cassettes. This adenovirus then was added to neurons, and after 7 days, DRG cultures were examined for fluorescence and both GFP
and GFP+ neurons were examined electrophysiologically for the presence of functional 5-HT3 receptor. As described in METHODS, 10
3 M serotonin was applied. GFP+ neurons (n = 4) all exhibited a strong inward current (1,714 ± 540; mean ± SE) in response to 5-HT (Fig. 4). Whereas, of the GFP
neurons (n = 6) all failed to exhibit any detectable response to administration of 5-HT (Fig. 4).

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| FIG. 4.
Bright field (left), matching epifluorescent images, and current recordings from a control neuron (A) and a neuron expressing both GFP and 5-HT3 receptor (B). Application of 10 3 M serotonin ( ) produces an inward current only in GFP-positive neurons. Recordings were performed in voltage-clamp mode with membrane potential held at 70 mV. Calibration bars for images are 20 µm. Calibration bars for waveforms are 100 pA (vertical) and 2 s (horizontal).
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DISCUSSION |
In this study, we show that DRG neurons transfected with mGFP4/Ad remain viable and healthy in culture for
17 days after infection. Transfected neurons displayed GFP fluorescence throughout their soma and neurite processes several days after infection, with the fluorescence, extending into the neurite terminal processes. The expression and fluorescence of GFP did not appear to effect normal electrophysiological properties of the neurons. In addition, we demonstrate that GFP provides an excellent viable marker toidentify cells that have been genetically modified.
For these experiments, we tried several GFP cDNAs, most of which only produced very low levels of fluorescence before obtaining the mGFP4. This GFP was modified to eliminate the recognition of a cryptic intron that prevents appropriate expression in some forms of plants (Haseloff and Amos 1995
) and human cell lines (Zolotukhin et al. 1996
). We increased the fluorescent potential of the GFP four to sixfold further by changing the serine in position 65 to a threonine (Heim et al. 1995
). Together these modifications created a form of GFP that produces high levels of expression and bright fluorescence DRG neurons. In our DRG-epithelial coculture, labeling with GFP was primarily found to occur in the DRG neurons, but not in epithelial cells, Schwann cells, or fibroblasts. It is likely that these nonneuronal cell types do not have the vitronectin receptor required for internalization of the virus (Wickham et al. 1993
, 1994
). In addition, the numbers of Schwann cells and fibroblasts are decreased due to preplating and growth in serum free medium. This selective neuronal labeling is an unexpected benefit of using the adenoviral transfection vector. In our culture system, 10% of the neurons exposed to the recombinant GFP adenovirus expressed GFP. Transfection efficiency is known to be cell type and age dependent (Huard et al. 1995
). For our cultures, we used newborn DRG neurons, and, had we used embryonic DRG neurons, our transfection efficiency would have been higher (Luis Parada, personal communication).
In most respects, cells transfected with GFP appeared indistinguishable from nontransfected exposed cells and nonexposed control cells. Cell body diameters did not differ across groups, indicating that GFP transfection did not result in atrophy or toxicity of neurons. Cell resting potentials were unaffected by GFP transfection. Additionally, the threshold for triggering action potentials, action potential amplitudes, and action potential durations were unaffected by GFP transfection. In plants, high levels of GFP have been shown to interfere with regeneration, most likely due to the generation of fluorescent related free radicals (Haseloff and Amos 1995
). Although expression of high levels of GFP may reduce neurite growth, neurite growth in GFP+ neurons was quite robust with many neurites extending between 1 and 2 mm in length during a 2-wk period. Further confirmation that their was no significant difference in neurite growth can be inferred from the fact that the whole cell capacitance was not significantly different between the control and GFP+ DRG neurons.
The experiments in which DRG cells were cotransfected with GFP and 5-HT3 receptor show that the 5-HT3 receptor can be expressed functionally in our culture preparation and that GFP coexpresses with the receptor and is a good marker for its expression. All fluorescent cells that were exposed to adenovirus with both the GFP and 5-HT3 constructs demonstrated large inward currents in response to puffs of 5-HT consistent with studies of 5-HT3 currents reported using other preparations (Robertson and Bevan 1991
; Yang et al. 1992
). We considered the 5-HT3 receptor an excellent choice for these experiments because in numerous earlier recordings from DRG cells in our culture preparations inward currents were not seen in response to puffs of 5-HT. A review of the literature failed to identify any reports of 5-HT3 currents in cultures of DRG cells prepared from neonatal rats. Interestingly, 5-HT3 currents have been recorded in a fraction of acutely dissociated DRG cells from adult rats (Robertson and Bevan 1991
) and frogs (Yakushiji and Akaike 1992
). Therefore it may be that 5-HT3 receptors normally appear postnatally in development or the induction of their expression may normally require factors that are absent from our incubation media. Our success with 5-HT3 receptor expression is very encouraging in that it indicates that the adenovirus vector should prove useful for transfecting cells with a variety of constructs. This provides a tool in which the expression of various proteins, receptors, and ion channels in DRG cells can be controlled. Obviously, such an ability could prove invaluable for investigating mechanisms of sensory transduction, second messenger systems, ion channel gating, and other cellular processes.
The general approach of cotransfecting cells with GFP and proteins of interest holds tremendous potential for many areas of neuroscience. If the technique is applied to in vivo preparations, it should be possible to investigate the role of specific ion channels, receptors, and other types of proteins involved in synaptic transmission and neuronal plasticity. One could feasibly transfect pre- or postsynaptic neurons, identify viable genetically altered cells, and examine them electrophysiologically. One also could transfect cells with potential neuroprotective agents and then administer ischemia. Later one could examine such a preparation to see whether cells that fluoresced with GFP (and therefore expressed the potential protective agent) were spared. The potential applications for these techniques are numerous.