A role for RhoB in synaptic plasticity and the regulation of neuronal morphology

Abstract

Actin-rich dendritic spines are the locus of excitatory synaptic transmission and plastic events such as long-term potentiation (LTP). Morphological plasticity of spines accompanies activity-dependent changes in synaptic strength. Several Rho GTPase family members are implicated in regulating neuronal and, in particular, spine structure via actin and the actin-binding protein cofilin. However, despite expression in hippocampus and cortex, its ability to modulate actin-regulatory proteins, and its induction during aging, RhoB has been relatively neglected. We previously demonstrated that LTP is associated with specific RhoB activation. Here, we further examined its role in synaptic function using mice with genetic deletion of the RhoB GTPase (RhoB(-/-) mice). Normal basal synaptic transmission accompanied reduced paired-pulse facilitation and post-tetanic potentiation in the hippocampus of RhoB(-/-) mice. Early phase LTP was significantly reduced in RhoB(-/-) animals, whereas the later phase was unaffected. In wild-type mice (RhoB(+/+)), Western blot analysis of potentiated hippocampus showed significant increases in phosphorylated cofilin relative to nonpotentiated slices, which were dramatically impaired in RhoB(-/-) slices. There was also a deficit in phosphorylated Lim kinase levels in the hippocampus from RhoB(-/-) mice. Morphological analysis suggested that lack of RhoB resulted in increased dendritic branching and decreased spine number. Furthermore, an increase in the proportion of stubby relative to thin spines was observed. Moreover, spines demonstrated increased length along with increased head and neck widths. These data implicate RhoB in cofilin regulation and dendritic and spine morphology, highlighting its importance in synaptic plasticity at a structural and functional level.

Figure 1.

Effect of RhoB gene deletion on Rho GTPase levels and gross hippocampal morphology. A, Western blot images illustrating that the complete absence of the RhoB protein in brain tissue from RhoB^−/− (−/−) mice compared with RhoB^+/+ (+/+) littermates has no effect on other Rho GTPases, including Rac1 and CDC42. B, No difference in the levels of active RhoA (RhoA-GTP) or total RhoA in hippocampal tissue from RhoB^−/− and RhoB ^+/+ littermate controls was observed. C, Comparable coronal sections (20 μm, frozen) from males were processed for Nissl staining. Note that the structures of the hippocampus are similar in both RhoB^−/− and RhoB ^+/+ mice. Scale bar, 500 μm. Numbers represent size markers (kilodaltons).

Figure 2.

Electrophysiological properties of hippocampal CA1 pyramidal neurons of the RhoB^−/− mouse. A, Basal synaptic transmission was unaffected in the CA1 region of RhoB^+/+ (+/+) hippocampal slices (black squares) when compared with RhoB^−/− (−/−) slices (open circles) as determined by their input/output responses. B, Paired-pulse facilitation was reduced at an interstimulus interval (ISI) of 20 ms in RhoB^−/− compared with RhoB^+/+ mice in the CA1 pyramidal neurons. Representative EPSP traces at 20 ms show the facilitation of the second EPSP compared with the initial response in both RhoB^+/+ and RhoB^−/− animals. C, High-frequency stimulation (100 Hz, 1 s) of the synaptic response in area CA1 (arrow) produced a significant and long-lasting potentiation of the synaptic response in RhoB^+/+ animals. PTP of the response was significantly reduced in RhoB^−/− animals, as was the eLTP. A later phase LTP (60 min post-tetanus) however, was unaffected in RhoB^−/− animals compared with their RhoB^+/+ counterparts. Representative EPSP traces from RhoB^+/+ and RhoB^−/− animals at t = −5 (1), 2 (2), 15 (3), and 60 min (4) show that the early deficit in synaptic potentiation is recovered over time. Data are expressed as mean ± SEM [n = 9–19, *p < 0.05, **p < 0.01: two-way ANOVA followed by Tukey's post hoc analysis at t = 2 min (PTP), t = 15 min (eLTP), t = 60 min (lLTP) compared with t = −5 min (baseline)].

Figure 3.

Localization of RhoB in corticohippocampal cultured neurons. A, B, Cultured corticohippocampal neurons were stained with anti-RhoB (red) and either anti-synaptophysin (A) or anti-PSD-95 (B) (both green). Images show immunoreactive RhoB is in close apposition, but not colocalized, with immunoreactivity for the presynaptic marker synaptophysin (A, merged image and inset), while there is a clear postsynaptic colocalization of immunoreactive RhoB with the immunoreactivity for the postsynaptic marker PSD-95 (B, merged image and inset), strongly suggesting a postsynaptic localization of RhoB. Scale bars, 10 μm in merged images and 2 μm in insets.

Figure 4.

Western blot analysis of cofilin and LIMK phosphorylation in the CA1 hippocampal region from RhoB^+/+ and RhoB^−/− mice, following tetanus-induced LTP. Ai, Western blot images illustrating levels of pCofilin in isolated CA1 regions of hippocampal slices previously subjected to HFS and 15 min synaptic potentiation. Bands from RhoB^+/+ (+/+) animals show an increase in intensity following LTP induction. No change in band intensity is seen in tissue from RhoB^−/− (−/−) slices after LTP induction. Levels of total cofilin remained constant throughout the time course of the experiment. Aii, In acute RhoB^+/+ hippocampal slices, levels of phosphorylated cofilin were significantly increased 15 min following the delivery of a single high-frequency stimulation to the Schaffer collaterals of CA1 pyramidal cells. In RhoB^−/− animals, no increase in cofilin activation was observed following induction of LTP. Data are expressed as mean ± SEM. Bi, Western blot images illustrating levels of pLIMK in isolated CA1 regions of hippocampal slices treated as in A. Bii, Levels of phosphorylated LIMK showed a tendency to increase in RhoB^+/+ tissue following LTP induction, while pLIMK levels were significantly reduced in tissue from RhoB^−/− mice. n = 7–8; *p < 0.05 vs RhoB^+/+ control: two-way ANOVA followed by Tukeys post hoc analysis. Numbers (Ai, Bi) represent size markers (kilodaltons).

Figure 5.

Morphological analysis of Golgi-stained neurons from cortical layer II/III and the CA1 region of the hippocampus. A, B, Sholl analysis of cortical neurons highlighted significant increases in branch points in cortical cells of RhoB^−/− (−/−) mice (open circles) when compared with RhoB^+/+ (+/+) cells (black squares) in areas proximal (40, 60, and 80 μm) to the cell body layer. *p < 0.01 versus RhoB^+/+ group: by four-way ANOVA followed by Tukey's post hoc analysis. No significant differences in branch pattern were observed in dendrites located further from the cell body. C, Sholl analysis of CA1 pyramidal cells in RhoB^−/− and RhoB^+/+ mice following Golgi–Cox staining showed no significant differences in branching patterns throughout the dendritic tree. Data in B and C are expressed as a mean ± SEM (n = 3 animals, 20–71 cells/genotype). D, E, Analysis of dendrite number (D) and summed dendrite length (E) separated into primary apical, secondary apical, primary basal, and secondary basal dendrites from cortical pyramidal neurones. *p < 0.05 vs RhoB^+/+ group, t test.

Figure 6.

Dendritic spine analysis of labeled RhoB^−/− (−/−) and RhoB^+/+ (+/+) CA1 pyramidal cells. A, Rhodamine red-filled dendrites from a CA1 pyramidal cell. Left, Confocal analysis generated high-resolution images of dendrites and dendritic spines from CA1 pyramidal cells. Right, Further manipulation of the image allowed increased resolution to measure the length and head and neck diameters of individual spines. M, Mushroom spine; S, stubby spine; T, thin spine. B, Spines that demonstrated a large head (H)-to-neck (N) ratio (>1.4) were defined as mushroom spines. Those spines whose length (L)-to-neck ratio was >1.4 were defined as thin spines. Stubby spines were labeled as such if their head-to-neck and length-to-neck ratio was ≤1. C, Spine counts on secondary and tertiary dendritic branches in RhoB^−/− CA1 pyramidal cells show a dramatic reduction in the number of spines in both secondary and tertiary branches compared with RhoB^+/+ mice. No significant change in spine number on quaternary branches was observed. D, Comparison of spine subtypes between RhoB^−/− and RhoB^+/+ mice highlights a significantly increased percentage of stubby spines and a corresponding decrease in thin spines in dendritic branches located in stratum radiatum when expressed as a percentage of total spine number. Data are expressed as mean ± SEM (n = 4 animals, 8 dendrites/genotype, 399 RhoB^+/+ spines, 459 RhoB^−/− spines, *p < 0.05, **p < 0.01: two-way ANOVA followed by Tukey's post hoc analysis).

Figure 7.

Morphological analysis of fluorescently labeled dendritic spines of RhoB^−/− (−/−) and RhoB^+/+ (+/+) CA1 pyramidal neurons. A, Significant increases in overall spine length, head width, and neck widths were observed in dendritic spines from RhoB^−/− pyramidal cells compared with those from RhoB^+/+ neurons. B, Subclassification of spines into either mushroom-, thin-, or stubby-type spines demonstrates that both mushroom- and thin-type spines are significantly longer in RhoB^−/− than in RhoB^+/+ animals. However, no change in the length of stubby spines was observed. C, Only mushroom-type spines demonstrate a significant increase in head size. D, No significant effect of genotype was observed on neck widths of individual subtypes. Data are expressed as mean ± SEM (n = 4 animals/ genotype, 8 dendrites/genotype, 399 spines (RhoB^+/+), 459 spines (RhoB^−/−). *p < 0.05, **p < 0.01: Kolmogorov–Smirnoff test). E, Example of deconvolved stack images of pyramidal neuron tertiary dendrites showing spines from RhoB^+/+ and RhoB^−/− mice; scale bar, 1 μm.

Figure 8.

Roles of the Rho GTPase family in the regulation of neuronal morphology. While several Rho GTPases are involved in the complex and dynamic regulation of neuronal morphology, the data suggest that activity-dependent control over dendritic arborization and spine morphology involves RhoB via the pathway illustrated.