sequence (33) into the pTSC vector containing a modified Thy-1 promoter (43). A 9.5-kb EcoRv/PvuI fragment was injected into oocytes of B6CF1 strain mice, and transgenic lines were established by standard techniques. Positive progeny were identified by PCR using GFP-specific primers and by Southern blot analysis.
Organotypic slice cultures from transgenic mice were established as described by Gahwiler et al. (44). After at least 4 weeks in culture individual slices were mounted in purposebuilt observation chambers (Life Imaging Services, Olten, Switzerland) and perfused with artificial cerebrospinal fluid or Tyrode’s solution. No difference was apparent between buffers. Time-lapse recordings were made by using a Leica IRBE inverted microscope equipped with a Nipkow disk-microlens confocal system (Life Science Resources, Cambridge, U.K.). To display time-dependent changes in printed figures, a subtraction protocol was used to sum differences between images in time-lapse recordings by using METAMORPH software (Universal Imaging, West Chester, PA). The results were displayed by using a pseudocolor look-up table with dark blue indicating lack of change and red to yellow increasing amounts of motility (42).
Comparison of Actin and MAP2 Distributions Using Spectral Variants of GFP. To asses the distribution and dynamics of microfilaments and microtubules in dendrites, we prepared eukaryotic expression vectors containing actin labeled with GFP and MAP2c labeled with YFP. To compare their properties within the same dendrite, hippocampal neurons from 18-day rat embryos were simultaneously transfected with actin-GFP and MAP2c-YFP and maintained in dispersed cell culture for at least 3 weeks. By this time most excitatory synapses are made onto dendritic spines of mature appearance that are contacted by presynaptic terminals whereas earlier immature lateral filopodia are abundant (45–49). Fig. 1 A shows a living cell in such a culture visualized by phase-contrast microscopy (Left) and with filter sets selective for GFP (Center) or YFP (Right). Even at the low magnification shown in Fig. 1 A, punctate labeling along dendrites, indicative of actin-GFP accumulation in dendritic spines, was evident (Center). By contrast the same dendrites visualized by MAP2-YFP were smooth in appearance (Right), indicating the absence of MAP2 from dendritic spines.
To study these distributions in more detail, actin-GFP and MAP2c-YFP images were taken at higher magnification and compared by assigning them contrasting colors (actin, green; MAP2c, red) and overlaying the images. Fig. 1 B shows the results of this procedure for a segment of dendrite from a doubly transfected cell in which the strong targeting of actin into spines and the contrasting restriction of MAP2 to the dendrite shaft is evident. The data shown in Fig. 1 B were taken from a time-lapse recording in which successive images were captured alternately by using the GFP or YFP filter sets. Such recordings show the same rapid dynamics of actin in dendritic spines described in previous studies (14, 42). By contrast, MAP2 showed no detectable dynamic activity over the 15 min of recording (see Movie 1, which is available as supplemental data on the PNAS web site, www.pnas.org). To represent this result in still images, six frames of actin-GFP and six frames of MAP2c-YFP, recorded alternately 30 s apart, were converted into profile outlines by using a computer routine. Each was assigned a different color and all six then were overlaid onto a single gray-scale fluorescence image from the same timelapse series. Changes in the shape of dendritic spines then are revealed by the separately colored outlines representing the successively recorded images (Fig. 1 C). By contrast, the same procedure applied to images of MAP2c shows no detectable change during the period of recording (Fig. 1 D).
This tight localization of MAP2 to dendritic microtubules was not only seen for the juvenile MAP2c splice variant but also for the high molecular weight MAP2b form that is expressed in the adult brain (50). Fig. 2 shows results for hippocampal neurons transfected with MAP2b-GFP. Like the embryonic MAP2c form, adult MAP2b is localized in dendrites but not within axons (arrow in Fig. 2 A and B). Both here and in higher magnification