Department of Biological Sciences, Columbia University, New York, NY 10027, USA
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Abstract |
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Introduction |
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Traditionally, spines have been assumed to be relatively stable structures and, as a consequence of this belief, most ultrastructural work has relied on the classification of spines according to their morphology. This idea was challenged in 1982, when Francis Crick proposed that spines could move (twitch) in response to synaptic stimulation and that this motility was actin based (Crick, 1982). A similar idea had been proposed in a previous study from Siekevitz's laboratory, in which actin was identified at the postsynaptic density using both biochemical and immunochemical methods (Blomberg et al., 1977
). Confirming, at least in part, these suggestions, it has recently been shown that spines in dissociated cultures (Fischer et al., 1998
) and in brain slices (Dunaevsky et al., 1999) are capable of rapid motility. Thus, spine morphology is dynamic on a time scale of seconds and significant changes in shape occur even among relatively mature spines in animals well past the developmental period of dendritogenesis [postnatal day (P) 20+ mice] (Dunaevsky et al., 1999). These novel data have also shown that spine motility is intrinsic to the neuron and is controlled by actin. It therefore has become important to understand in detail which pathways control actin polymerization and depolymerization in spines because these pathways could be responsible for the continuous remodeling of the morphology of dendritic spines.
The role of small GTPases in controlling actin cytoskeletal reorganization and cell morphology is well established (Hall, 1994). In the CNS, GTPases from the Rho family (Rho, Rac and Cdc42) have been shown to be expressed in hippocampal pyramidal neurons and dentate granule cells, the neocortex and other regions (Olenik et al., 1997
). In addition, small GTPases are likely to be distributed in dendrites of pyramidal neurons, since the effector of Rho, Rho-kinase, exists in the dendrites (Hashimoto et al., 1999
). Rho GTPases have been shown to play an important role in the dendritic remodeling of Xenopus retinal ganglion cells (Ruchhoeft et al., 1999
) and of cultured cortical neurons (Threadgill et al., 1997
). Also, in transgenic mice expressing constitutively active human Rac1, the dendritic spines of Purkinje cells were increased in number and reduced in size (Luo et al., 1996
). These data suggest that small GTPases from the Rho family regulate dendroand spinogenesis, although it is not yet known if they are involved in the maintenance of extant dendritic spines. To investigate this issue, and to better understand the cellular mechanisms responsible for the maintenance of the morphology of spines, we have manipulated the expression of several members of the Rho network in cortical and hippocampal pyramidal neurons in cultured brain slices from the mouse. Combining two-photon microscopy and biolistic co-transfection with enhanced green fluorescent protein (EGFP) and dominant negative or constitutively active variants of several members of this pathway, we have detected a variety of morphological rearrangements in spines and dendrites from pyramidal neurons. Based on our data we propose a model for the effect of this pathway on spine morphology.
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Materials and Methods |
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All experiments were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NIH publication no. 86-23, revised 1987). Cortical and hippocampal brain slices were prepared from postnatal day P0P3 C57BL mice, using a tissue chopper in sterile conditions. Slices were mounted on 0.4 mm culture inserts (Millipore, Bedford, MA, PICM ORG 50) and incubated (5% CO2, 37°C) with culture medium, composed of 50 ml basal Eagle's medium (Gibco, Gaithersburg, MD), 25 ml Hank's balanced salt solution (Gibco), 25 ml horse serum (Hyclone, Logan, UT), 0.65 g dextrose, 1.0 ml HEPES (Gibco) and 1.0 ml 100X Pen-strep (Gibco).
Plasmids and Reagents
Rac1N17 and RhoAV14 were expressed in the pEXV vector (Ridley and Hall, 1992; Ridley et al., 1992
) and were myc-tagged. Rac1V12 (Ridley et al., 1992
) was subcloned into pSRa (Takabe et al., 1988
) at the EcoRI site. Cdc42HsV12 and Cdc42HsN17 (Kozma et al., 1995
) were expressed using the pCMV expression vector and were also myc-tagged. C3 transferase was expressed using the RcCMV vector. All genes were cloned from human libraries. Finally, Clostridium difficile toxin B was obtained from Drs Hoffman and Aktories (Freiburg, Germany) and was dissolved to 30150 ng/ml from stocks at an initial concentration of 500 ng/µl in 50 mM TrisHCl (pH 7.5), 500 mM NaCl and 20% glycerol.
Particle-mediated Gene Transfer
Cortical and hippocampal slice cultures were transfected using the Helios Gene Gun System (Bio-Rad, Hercules, CA). Plasmids were purified on a QIAGEN (Valencia, CA) column and precipitated onto gold microcarrier particles (1 µm diameter) according to the Helios Gene Gun instruction manual. For co-transfection, the DNA loading ratio was 8 µg/mg gold for each construct. For control experiments with EGFP, the ratio was also 8 µg/mg gold. Cultured slices were transfected at 619 days in vitro (DIV). Images were taken 24 days after transfection.
Two-photon Microscopy
Imaging was carried out with a custom-made two-photon laser scanning microscope consisting of a modified Fluoview (Olympus, Melville, NY) confocal microscope and a Ti:sapphire laser providing 130 fs pulses at 75 MHz at wavelengths of 740850 nm (Mira, Coherent, Santa Clara, CA) pumped by a solid-state source (Verdi, Coherent). A x40, 0.8 NA water immersion objective (IR1, Olympus) was used. Fluorescence was detected using photomultiplier tubes (HC125-02, Hamamatsu, Japan) in external, whole-area detection mode, and images were acquired using Fluoview (Olympus) software. Images of spines were acquired at the highest digital zoom (x10), resulting in a nominal spatial resolution of 20 pixels/µm. Pyramidal neurons were selected from CA13 in the hippocampus or extragranular layers of the primary visual cortex. Spines were chosen randomly from all areas of neurons, including basal, apical and oblique dendrites. For each image, several (615) focal planes ~1 µm apart were scanned; these were later projected into a single image. Image processing and analysis was done with custom-written macros using NIH-Image and Macintosh computers. Measurements are reported as mean ± SEM.
Immunocytochemistry
Imaged slices were fixed with 4% paraformaldehyde overnight. Slices were incubated in 1% Triton X in phosphate-buffered saline (PBS) for 30 min and blocked by 20% bovine serum albumin in PBS. Slices were then incubated with primary antibody for 4 h (anti-myc IgG, Covance, Richmond, CA) and then with secondary antibody for 4 h (Cy3-conjugated anti-mouse IgG, Jackson Immunoresearch Laboratories, Inc., West Grove, PA). Images were taken with a Olympus BX50WI microscope and SIT camera (C2400, Hamamatsu).
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Results |
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Members of the Rho family of small GTPases comprise a subgroup of the Ras superfamily of GTPases (Van Aelst and D'Souza-Schorey, 1997; Hall, 1998
). These proteins function as molecular switches that cycle between an inactive GDP-bound form and an active GTP-bound form. Activation of the GTPases is catalyzed by guanine nucleotide exchange factors (GEFs) which exchange GDP for GTP in response to various signals. Inactivation is catalyzed by GTPase-activating proteins (GAPs) and nucleotide dissociation inhibitors (GDIs). GAPs catalyze the hydrolysis of the bound GTP, while GDIs sequester the GTPases in the cytosol and prevent nucleotide exchange and interaction with GEFs.
The Rho family includes at least 14 members. A summary of the Rho pathway is outlined in Figure 1. The better characterized of these proteins are Cdc42, Rac1 and 2, and RhoA, B and C. These GTPases have been implicated in a diverse range of processes,including regulation of cell morphology, activation of signal transduction pathways, and regulation of cell proliferation and differentiation (Bagrodia et al., 1995
; Coso et al., 1995
; Minden et al., 1995
; Zhang et al., 1995
; Brown et al., 1996
; Van Aelst and D'Souza-Schorey, 1997
; Hall, 1998
). The Rho family members were originally identified as proteins that regulate the organization of the actin cytoskeleton in fibroblasts. Micro-injection of Cdc42Hs into fibroblasts and a variety of other cell types causes the transient induction of filopodiathin, actin-rich protrusions at the cell surface. This is followed by the formation of thin sheets of actin at the cell surface known as membrane ruffles or lamellipodia. While the induction of filopodia is caused by Cdc42Hs activation, the induction of lamellipodia is probably due to the ability of Cdc42Hs to activate Rac. Thus, co-expression of Cdc42Hs with a dominant negative Rac mutant results in the sustained induction of filopodia without the subsequent induction of lamellipodia. Consistent with this, microinjection of activated Rac leads to the induction of lamellipodia, but not filopodia (Kozma et al., 1995
; Nobes and Hall, 1995
). Microinjection of a third GTPase, RhoA, into fibroblasts, leads to an increased formation of actin bundles that traverse the cell, known as stress fibers, as well as focal adhesions, where cells attach to the extracellular matrix. (Ridley and Hall, 1992
). Cdc42, Rac and RhoA were originally proposed to act in a linear cascade, whereby Cdc42 activates Rac, which in turn activates RhoA (Nobes and Hall, 1995
). Recent work, however, has suggested that, rather than activating Rho, in some cells Cdc42Hs and Rac may actually antagonize the effects of Rho, thus leading to a reduction in stress fiber formation and contractility (Dutartre et al., 1996
; Manser et al., 1997
; Burridge, 1999
; van Leeuwen et al., 1999
).
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For most of our experiments we co-transfected neurons with two constructs: an EGFP driven by a CMV promoter, and a second construct consisting of a small GTPase in a mammalian expression vector. The use of EGFP enabled us to image with detail the morphology of the dendrites and spines of transfected neurons, while at the same time manipulating individual members of the cascade with the second construct.
To ensure that the EGFP transfection was not affecting dendritic or spine morphology, we first examined neurons transfected only with EGFP. In each slice, dozens of cells were fluorescently labeled 2 days after transfection. After 4 days, the intensity of fluorescence decreased, although the time course of EGFP expression varied from cell to cell, even among neurons of the same cell type. EGFP-labeled cells could be classified as pyramidal and non-pyramidal. Hippocampal and cortical pyramidal cells had a long apical dendrite and a skirt of basal dendrites (Figure 2) (Feldman, 1984
). Dendritic trees were studded with spines, at average densities of 0.7 ± 0.07 spines/µm (n = 226 spines from six cells). Spines ranged in length from 0.5 to 3 µm (average = 1.3 ± 0.08 µm, n = 226 spines from six cells) and from 0.5 to 2 µm in diameter. Structures smaller than 0.5 µm might not be detected by our microscope, and thus these measurements underestimate their true densities. We therefore concluded that EGFP transfection did not alter the morphology of the neurons.
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To evaluate the general effect of the Rho cascade on the maintenance of spine morphology, we imaged hippocampal and neocortical pyramidal neurons transfected with GFP after treatment with toxin B, which specifically inactivates all three members of the Rho cascade but not the proteins of the Ras cascade (Just et al., 1995). Bath application of toxin B at concentrations of 30150 ng/ml for 24 h affected the morphology of dendrites and spines in many cells (Figure 3
). The effects on some neurons were very dramatic: instead of normal dendritic morphologies, these cells had an abundance of thin, long dendrites that extended either from the soma or from the main dendritic trunks (12/33 neurons; Figure 3A
). The asymmetry in somata and main dendritic trunk indicated that these neurons must have originally belonged to the pyramidal cell class. These small dendrites did not taper appreciably and occasionally produced small branches of similar diameter (Figure 3B
). There were either no clearly distinguishable spines in those dendrites or very small structures that could have been very small, stubby spines. Because of this we were unable to quantify the density of spines in these neurons.
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Effect of Cdc42 on Spine Morphology
To test whether Cdc42 played a role in the maintenance of spine morphology, we first examined the effect of expressing a constitutively active form of Cdc42 (Cdc42HsV12) in pyramidal neurons (Figure 4). Protein expression was confirmed by immunocytochemistry against myc tags, which showed that Cdc42V12 was distributed throughout the neurons (Figure 4A
). Some pyramidal cells (3/5) had normal apical and basal dendrites and spines (Figure 4A,B
); however, in other neurons (2/5), some dendrites were abnormally short, as if they were retracting. The tips of these shorter dendrites were swollen (Figure 4C
). On affected dendrites spines were stubby (Figure 4C
). Nevertheless, pooling together all cells, Cdc42HsV12-expressing neurons had normal values of spine density and length (0.64 ± 0.12 spines/µm, 1.12 ± 0.10 µm spine length; n = 163 spines from five cells).
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Effect of Rac on Spine Morphology
We next examined whether Rac played a role in the maintenance of spine morphology. As originally reported by Luo et al. (Luo et al., 1996), we found that expression of a constitutively active form of Rac (Rac1V12) had a major effect on spine density and morphology (Figure 6
). Every fluorescent neuron had abnormal spines. Most neurons had dendrites covered by lamellipodia-like veils (Figure 6A,B
). Some of these veils could be discerned to be composed of a multitude of very small, thin spines (Figure 6C
), so it is possible that all veils were actually not continuous structures but were instead composed of hundreds of small spines. This possibility is supported by data from Rac1V12-transgenic mice, where similar structures were found by electron microscopy to be composed of mini spines (Luo et al., 1996
). Because of the small size of these spines, we could not quantify their density, although they appeared to be much higher than normal. These data show that Rac can promote the appearance of spines.
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Effect of Rho on Spine Morphology
We next focused our attention on the influence of Rho on spine maintenance. We first tested this by expressing RhoAV14, a constitutively active form. We detected major effects on spine density and morphology (Figure 7). In some neurons, spines were either present at very low densities or absent (Figure 7A,B
; 4/9 cells; 0.15 ± 0.08 spines/µm, n = 38). These cells had clear apical and basal dendrites, which indicated that in spite of being non-spiny neurons they belonged to the pyramidal cell class. The morphologies of the few spines present were also abnormal, the spines having short necks (0.99 ± 0.05 µm, n = 38). At the same time, in 5/9 neurons dendrites had spines at normal densities (0.70 ± 0.08 spines/µm, n = 349) and sizes (1.12 ± 0.12 µm, n = 444). We concluded from these results that Rho expression can repress the maintenance and elongation of spines.
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Discussion |
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In this study we make use of particle-mediated gene transfer (Arnold et al., 1994; Lo et al., 1994
), or biolistics, to systematically introduce mutant forms of several proteins in a genetic cascade into neurons in cultured brain slices. As our results and previous work demonstrate (Arnold et al., 1994
; Lo et al., 1994
), this approach makes genetic studies in brain slices feasible. Part of its usefulness for analysis of the effect of individual genes in neuronal development and function is that this method obviates the need to make transgenic or knockout mice to investigate questions where the phenotype can be appropriately assayed in brain slices. In particular, as we show here, the combination of biolistics with two-photon microscopy (Denk et al., 1994
) and GFP expression (Chalfie et al., 1994
) enables long-term morphological or functional studies of living neurons after transfection with mutant forms of regulatory proteins. Similar studies could be carried out to characterize genetically or to identify candidate genes involved in the regulation of developmental or physiological processes of neurons.
For this work we concentrated our efforts on examining the role that the Rho family of small GTPases play in the maintenance of spine morphology. Although we have detected some effects on dendritic rearrangements (see Figures 3 and 4C), we imaged neurons at relatively short times after transfection, conditions which may not be optimal to detect in brain slices the pronounced dendritic rearrangements seen in cultured neurons (Threadgill et al., 1997
). Nevertheless, these shorter periods in our experiments seem adequate to explore the actions of the Rho proteins in spine morphogenesis. This is based both on the variety of abnormal spine phenotypes encountered and on the very fast morphological rearrangements seen in spines, which can happen in less than a minute (Fischer et al., 1998
; Dunaevsky et al., 1999a
).
The effects of C3 transferase demonstrate that Rho must be present in the imaged dendrites. Since we cannot confirm the expression of RacN17 in our neurons, we cannot demonstrate with our data that Rac1 is present in dendrites. We have previously encountered difficulties in expressing RacN17 in HeLa and NIH3T3 cells (A. Minden, unpublished experiments). Nevertheless, we think that Rac1 is likely to be present in the dendrites of the imaged neurons because endogenous Rac1 is known to be expressed in hippocampal pyramidal neurons (Olenik et al., 1997) and because RacN17 has effects on the dendrites from cortical pyramidal neurons (Threadgill et al., 1997
).
Our study was carried out to assay whether there are major effects of the Rho pathway on spine maintenance. We have not detected any major difference in data from neocortex and hippocampus, so we have consequently pooled our data and conclusions to address the basic regulation of spine morphology in pyramidal neurons. Also, in this study, morphological effects on spines were assayed at the light microscopy level and there are instances, as in the RacV12 data, where the structures were at the limit of our resolution. A combination of GFP transfection with immunoelectron microscopy will allow a proper assessment of the spine morphological phenotypes (Dunaevsky et al., 1999b).
Proposed Model: Antagonistic Effects of Rac and Rho
We have detected a variety of effects of modulating the Rho signaling network on the density and morphology of cortical and hippocampal pyramidal neurons (Table 1; Figure 9
). Briefly, RacV12 (constitutively active) increases spine density while RhoAV14 (constitutively active) reduces it. Also, RacV12 reduces spine size, whereas RhoAV14 reduces spine length and C3 transferase (a Rho blocker) increases spine length. Finally, blocking the entire pathway with toxin B produces different effects on different neurons, with changes both in spine density and morphology. Because of these results, we conclude that the Rho pathway is involved in the maintenance and rearrangement of spine morphology in pyramidal neurons. Our data thus extend to pyramidal cells the published work on effects of RacV12 on Purkinje cells (Luo et al., 1996
). Since our experiments were done on developed neurons with extant spines, our conclusions also apply to the maintenance of spine morphology.
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We hypothesize that a similar antagonism occurs in dendritic spines. The different effects that Rac and Rho have on both spine density and spine size could be due to two molecular mechanisms (Figure 10). We propose that Rac and Rho have opposite effects on a common target (factor X, or nucleating factor) that is present at the dendrite and is involved in nucleating the position of the future spine. Our data suggest that Rac may positively regulate factor X, while Rho has a negative regulatory role. Since Rac and Rho generally do not share the same direct targets, factor X would most likely be an indirect target. This would be analogous to myosin light chain, which is regulated indirectly by Rac and Rho via PAK and Rho kinase, respectively. A second factor (factor Y; elongation factor) would be active at the spine itself and would be responsible for its length and size (Figure 10
). This factor may be inhibited only by Rho, because the reduction of spine size observed with RacV12 might be a consequence of the high number of spines. Factor Y could potentially be a direct Rho target, such as Rho kinase, or may be an indirect target of the Rho pathway. Future experiments will explore the identity of these putative target molecules and the mechanisms by which they regulate spine density and morphology.
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Notes |
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Address correspondence to Ayumu Tashiro, Department of Biological Sciences, Columbia University, 1212 Amsterdam Avenue, Box 2436, New York, NY 10027, USA. Email: at226{at}columbia.edu.
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