bit antisera (14); Vance Lemmon, Case Western Reserve University, Cleveland, NgCAM chick-specific monoclonal (15); and Ian Trowbridge, Salk Institute, La Jolla, CA, TfR cDNA (16) and TfR human-specific monoclonal (17). Monoclonal antibodies against LDLR (RPN537) were purchased from Amersham Pharmacia; monoclonal antibodies against TfR (B3/25) were obtained from Boehringer Mannheim.
Cell Culture and Viral Infection. Primary cultures of dissociated neurons from embryonic day 18 rat hippocampi were prepared essentially as described (18). Replication-defective herpes simplex viruses and adenoviruses were used to express exogenous proteins (1, 19). Viruses were titered to infect 1–10% of the neurons in culture.
Immunostaining. To detect virally expressed proteins present on the cell surface, living cultures were incubated with the primary antibody diluted in culture medium for 5–7 min at 37°C, quickly rinsed in phosphate-buffered saline, and then fixed. Primary antibodies bound to antigen were detected with the appropriate fluorescently labeled secondary antibodies after permeabilization and blocking of nonspecific background. For quantitation of the fluorescence signal, images of labeled cells (specimen images) were acquired by using either a Photometries (Tucson, AZ) CH250 camera (12 bit; 1,315×1,017 pixels) and a Zeiss Axiophot [25×Plan Apo objective; numerical aperture (N.A.) 1.2] or a Princeton Instruments (Monmouth Junction, NJ) Micromax (12 bit, 1,300×1,030 pixels) and a Leica DM-RXA (20×Plan Apo, N.A. 0.5). Infected cells were chosen by examining random fields at approximately 2-mm intervals across the coverslip. A labeled cell whose processes traversed the field was selected for analysis, so long as its processes did not overlap those of other labeled cells; cells with fewer than three identifiable dendrites were excluded. To limit possible photobleaching during the process of cell selection, total exposure time was kept to less than 10 sec. In control experiments, this level of exposure was found to cause less than a 3% reduction in fluorescence intensity. Exposure time was adjusted so that maximum pixel value was at least half saturation. After acquiring the specimen image, a dark current image generated by an equivalent exposure with the camera shutter closed was subtracted, and a shading correction based on an image of a uniformly fluorescent field was applied to compensate for uneven illumination of the field. Finally, a threshold was set to eliminate nonspecific background staining of axons and dendrites of uninfected cells respectively, and the total fluorescence in the axonal and dendritic domain was determined. A process was considered an axon if it was at least twice the length of any of its other processes. The other processes were considered dendrites. Fluorescence in the cell body was excluded from the analysis.
Live Imaging. Cells on coverslips were sealed into a heated chamber (Warner Instruments, Hamden, CT) in phenol red-free Hanks’ balanced salt solution buffered with 10 mM Hepes (pH 7.4) and supplemented with 0.6% glucose. Vesicle transport was imaged by capturing frames continuously for 30 sec (600-msec exposures) with a Micromax cooled charge-coupled device camera and a 63×Plan Apo, N.A. 1.32 objective on a Leica DM-RXA. For quantitative analysis, transport events were detected by first extracting difference images of sequential frames followed by analysis by using the kymograph drop-in function of the METAMORPH IMAGING SOFTWARE (Universal Imaging, Downingtown, PA). Briefly, lines were drawn along the axis of individual neurites, and the kymograph function was used to find the brightest pixel along a 10-pixel line perpendicular to the axis of the neurite. These values were then plotted for each frame, with time on the x axis and position along the neurite on the y axis. Thus, moving vesicles appeared as diagonal lines whose slopes were a measure of rate and direction of transport (with positive slope corresponding to anterograde transport). The number of transport events in the axon and at least three of the dendrites were determined for 3–12 cells at each time point.
Changes in the Polarization of Membrane Proteins During Development. To assess when during development membrane proteins acquire their characteristic polarized distribution, we expressed representative axonal and dendritic membrane proteins at times ranging from 1 to 14 days in culture and assessed their polarization on the cell surface by live-cell immunostaining. We selected the TfR and the LDLR as dendritic markers and NgCAM as an axonal marker. The sorting of these proteins in mature hippocampal neurons has been well characterized (19, 20). As an example of an unsorted protein, we chose a construct of the pIgR whose dendritic sorting signal had been deleted [pIgR665–668 (19, 20)]. We also assessed the polarization of L1, an endogenous axonal protein.
We first examined cells at stage 2 of development, before neurites have been specified as axons or dendrites. If the polarization of membrane proteins preceded axonal specification, one might expect axonal markers to be concentrated in a single neurite, whereas dendritic markers might be present in all of the neurites except one. When the distribution of these proteins was assessed in stage 2 neurites, we often found that some neurites exhibited more staining than others, but we never observed a cell with only a single neurite that excluded dendritic markers or that had a high concentration of axonal markers. This lack of polarity was particularly evident when cells were simultaneously infected with viruses expressing axonal and dendritic markers (Fig. 1 a). The staining for LDLR and NgCAM was most intense in the growth cones, with some growth cones staining more brightly than others. However, rather than exhibiting the complementary distribution one would expect for proteins polarized to opposite domains, the two markers tended to have a similar distribution in stage 2 cells: growth cones that were brightly stained with the dendritic marker were often brightly stained with the axonal marker as well. Differences in the intensity of staining among different growth cones may reflect the dynamics of their growth; at this stage of development, neurites undergo alternating periods of extension and retraction (21).
At stage 3 of development, both axonal and dendritic markers were polarized, although not to the extent seen in mature neurons. For example, Fig. 1 b illustrates a stage 3 neuron expressing both LDLR and NgCAM. NgCAM was present throughout the cell body and axon, with particularly intense staining in the distal axon. Little staining was present in the dendrites. LDLR was present in the dendrites and proximal axon, but little or no staining was present in the distal axon. On average, we found that 90% of the neuritic cell surface staining for NgCAM was axonal, whereas 81% of the LDLR staining was dendritic. In contrast, staining for the unpolarized protein, pIgR665–668, was about equally divided between dendrites and axon (46% dendritic).
Fig. 1 c summarizes the changes in the distribution of axonal and dendritic marker proteins that occur during the first 2 weeks in culture. The polarity of both axonal and dendritic markers increased during the first few days in culture, reaching mature levels by about day 5. Over time, a slightly greater percentage of the unpolarized protein, pIgR665–668, became associated with the axon, presumably reflecting a relative increase in the size of the axonal arbor.
The Distribution of Cell Surface and Intracellular TfR-GFP and NgCAM-GFP. During the first 2 days in culture, there were significant differences among stage 3 cells in the extent to which dendritic