Small GTPases Rac and Rho in the maintenance of dendritic spines and branches in hippocampal pyramidal neurons - PubMed (original) (raw)

Small GTPases Rac and Rho in the maintenance of dendritic spines and branches in hippocampal pyramidal neurons

A Y Nakayama et al. J Neurosci. 2000.

Abstract

The shape of dendritic trees and the density of dendritic spines can undergo significant changes during the life of a neuron. We report here the function of the small GTPases Rac and Rho in the maintenance of dendritic structures. Maturing pyramidal neurons in rat hippocampal slice culture were biolistically transfected with dominant GTPase mutants. We found that expression of dominant-negative Rac1 results in a progressive elimination of dendritic spines, whereas hyperactivation of RhoA causes a drastic simplification of dendritic branch patterns that is dependent on the activity of a downstream kinase ROCK. Our results suggest that Rac and Rho play distinct functions in regulating dendritic spines and branches and are vital for the maintenance and reorganization of dendritic structures in maturing neurons.

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Figures

Fig. 1.

Fig. 1.

Rac1 and RhoA are expressed in developing hippocampus. A, Dark-field image of a coronally sectioned P8 rat hippocampus hybridized with an antisense S35-riboprobe against Rac1, showing distribution of Rac1 mRNA in the dentate gyrus and CA1 and CA3 pyramidal cell layers.B, Dark-field image of antisense riboprobe showing distribution of RhoA mRNA. C, Dark-field image of the Rac1 sense riboprobe showing background staining. _D,_Bright-field image of the same hippocampus shown in _C_with the CA1, CA3, and dentate gyrus (DG) labeled. Scale bar, 2 mm.

Fig. 2.

Fig. 2.

Development of hippocampal pyramidal neurons in cultured slices. A–C, Representative images of transfected hippocampal neurons show the developmental stage at the onset of experiments in our standard preparation. For these images only, transfection was performed at 1 DIV, and slices were fixed 24 hr later. A, Low magnification is shown of a hippocampal slice biolistically transfected with mCD8 (red), with brackets defining the DAPI-labeled (blue) pyramidal cell layers of CA1 and CA3.Pink labeling (from overlapping_red_ and blue signals) represents transfected cells, including a granule cell in the dentate gyrus (arrowhead) and a pyramidal neuron overlapped by glia in the CA1 cell layer (arrow). B, A CA3 pyramidal neuron (composite confocal image using a 16× objective) is shown. The immunocytochemical detection of mCD8 reveals the structure of both apical and basal dendrites, as well as the axon (arrow). C, The spines on the apical dendrites of the neuron pictured in B (composite confocal image using a 100× objective) are shown.Arrows point to spines with characteristic head and neck morphology, whereas arrowheads show filopodial protrusions. D, Double labeling of mCD8 and PSD-95: GFP proteins in apical dendrites (composite confocal images using a 40× objective with a digital zoom factor of 3) is shown. mCD8 labeling is in red (D, D′′), and PSD-95:GFP labeling (PSD-95:GFP) is in_green_ (D′, D′′).Arrows indicate spines with a head (PSD-95:GFP positive), whereas arrowheads indicate filopodial protrusions (PSD-95:GFP negative). E, Dendritic branch segments gradually increase on both apical and basal dendrites with successive days in culture (apical, n = 10, 13, 8, 10; basal, n = 10, 14, 7, 9 for 2, 4, 6, 9 DIV, respectively). F, Spine density increases over successive days in culture, whereas filopodial protrusions decrease. Spines and filopodia were counted from stacked confocal images collected at 40× with a zoom of three, from a stereotyped region of the dendrite as defined in Materials and Methods (apical,n = 10, 19, 17, 12; basal, n = 11, 14, 17, 11 for 2, 4, 6, 9 DIV, respectively). Scale bars:A, 1 mm; B, 50 μm; C, D, 10 μm.

Fig. 3.

Fig. 3.

Dominant-negative Rac1 expression results in a progressive reduction of the dendritic spine density and mild changes in the dendritic-branching pattern. _A,_Transfected myc-tagged wild-type Rac1 protein is distributed along the dendrites and in dendritic spines (green, anti-myc; red, anti-mCD8; composite confocal image using 40× objective; digital zoom factor of 3). _B,Representative images are shown of apical dendrites that were transfected with the marker mCD8 alone (top) or with mCD8 and myc-tagged Rac1N17 (bottom) for 1, 2, or 3 d (composite confocal images of mCD8 staining using 100× objective).C, Apical dendrites from neighboring pyramidal neurons are shown 3 d after expressing mCD8 alone (red) or expressing both mCD8 (red) and myc-tagged Rac1N17 (green) and therefore appearing_yellow (composite confocal image using a 40× objective with a digital zoom factor of 3). Although it appears that Rac1N17 dendrites have thicker dendrites in this image, quantification of the average diameter of dendrites within every image used to measure dendritic spine density does not reveal any significant difference (paired t test, apical, p = 0.19; Rac1N17 = 1.50 ± 0.14 μm; control = 1.22 ± 0.51 μm; n = 18, 9, respectively; basal,p = 0.12; Rac1N17 = 0.83 ± 0.1 μm; control = 1.05 ± 0.08 μm; n = 9, 8, respectively). D, Rac1N17 expression progressively reduces the number of spines on apical and basal dendrites (for 1, 2, 3 d, apical mCD8, n = 16, 19, 10; apical Rac1N17, n = 19, 11, 24; basal mCD8,n = 15, 14, 10; basal Rac1N17,n = 18, 11, 17, respectively). _E,_Quantification of dendritic branch segments after 3 d of Rac1N17 expression is shown (n = 17, 23, 18, 21 for apical mCD8, apical Rac1N17, basal mCD8, basal Rac1N17, respectively).F, Sholl profiles of the basal dendrites of Rac1N17-expressing and control CA1 pyramidal neurons (3 d after transfection) illustrate the slight change in dendritic-branching pattern with Rac1N17 expression (n = 14, 15 for basal mCD8, Rac1N17, respectively). Post hoc t tests reveal that the only significant difference occurs at 50 μm (paired t test, **p < 0.01). Scale bars: A, B, 5 μm; C, 10 μm.

Fig. 4.

Fig. 4.

Effects of activated Rac1 expression on dendritic morphology. A–H, Representative distal (A–D) and proximal (E–H, just_above_ cell bodies) apical dendrites and spines of hippocampal pyramidal neurons, 24 hr after being transfected with mCD8 only (A, E), mCD8 and Rac1L61 (B, F), mCD8 and Rac1L61K40 (C, G), or mCD8 and Rac1L61A37 (D, H) (composite confocal images using a 40× objective with a digital zoom factor of 3).Red staining in all images represents anti-mCD8 immunoreactivity, and green staining in_B–D_ and F–H represents anti-myc immunoreactivity. All mutant Rac proteins were expressed at comparable levels and were distributed throughout the entire neuron on the basis of their myc staining (see yellow labeling in_B–D_, F–H because of overlapping_green_ signal for myc and red signal for mCD8). Asterisks represent mature spines with heads,arrowheads indicate ruffle-like structures, and_arrows_ point to long and thin filopodial-like protrusions. Scale bars, 10 μm.

Fig. 5.

Fig. 5.

Expression of activated RhoA results in dendritic simplification. A–C, Representative images of pyramidal neurons that have expressed the marker mCD8 alone (A), mCD8 and myc-tagged RhoAV14 (B), or mCD8 and myc-tagged RhoAN19 (C) for 1 d (composite confocal images using 16× objective). Insets, Myc immunostaining for RhoAV14 (B) and RhoAN19 (C).D, Tip of an apical dendrite from a pyramidal neuron expressing RhoAV14 for 2 d (composite confocal image using 40× objective, with a digital zoom factor of 3). The soma is toward the_bottom_ of the image. E, Quantification of dendritic branch segments after 1, 2, and 3 d of RhoAV14 expression. Both apical and basal dendrites exhibit a reduced number of dendritic segments (see Materials and Methods) with RhoAV14 expression compared with that of neurons expressing mCD8 alone (***p < 0.001; **p < 0.01; for 1, 2, 3 d, apical mCD8, n =14, 13, 17, and RhoAV14,n = 25, 19, 23; basal mCD8, n = 11, 14, 18, and RhoAV14, n = 25, 18, 22). _F,Quantification of dendritic branch segments after 1, 2, and 3 d of RhoAN19 expression. Neurons expressing RhoAN19 do not show a change in dendritic branch segment number (for 1, 2, 3 d, apical,n = 9, 10, 7; basal, n = 7, 11, 7, respectively). G, H, Sholl profiles for basal dendrites of CA1 neurons 2 d after expressing mCD8 alone (n = 11) or expressing RhoAV14 (G;n = 16) or RhoAN19 (H;n = 8). Scale bars: A–C and_insets, 50 μm; D, 10 μm.

Fig. 6.

Fig. 6.

Function of ROCK in the maintenance of dendritic branches. A–E, Representative images of neurons 2 d after transfection and 100 μ

m

Y-27632 treatment are shown. Neurons were transfected with mCD8 alone (A, B) or cotransfected with RhoAV14 (C, D) or ROCKΔ3 (E). E, Inset, The myc immunostaining for ROCKΔ3 is shown. Additionally, neurons in_B_ and D were treated with 100 μ

m

Y-27632 at the time of transfection (composite confocal images using 16× objective). F, Quantification of basal dendritic branch segment numbers of mCD8- and of mCD8 plus RhoAV14-expressing neurons with or without Y-27632 treatment is shown. Y-27632 treatment alone does not affect dendritic segment number (p = 0.31; control treatment,n = 23; Y-27632 treatment, n = 23). Y-27632 treatment blocks RhoAV14-associated dendritic segment reduction (***p < 0.001; RhoAV14 + control,n = 17; RhoAV14 expression + Y-27632 treatment,n = 24). G, Y-27632 application does not alter the basal dendritic spine density of neurons expressing mCD8 alone (p = 0.65; n = 29, 21 for control, Y-27632 treatment, respectively). Y-27632 treatment is capable of restoring the spine density of neurons expressing RhoAV14 close to control level (p = 0.08;n = 16 for Y-27632 treatment and RhoA V14 expression). H, Activated ROCKΔ3 expression results in significant reduction of dendritic segments compared with that in mCD8 alone (p < 0.001 for both apical and basal;n = 19, 20, respectively, for ROCKΔ3). Scale bars, 50 μm.

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