H-Ras induces exuberant de novo dendritic protrusion growth in mature neurons regardless of cell type

Abstract

Dendritic protrusions, mainly spines and filopodia, correlate with excitatory synapses in pyramidal neurons (PyNs), but this relationship may not apply universally. We found that ectopic H-Ras expression increased protrusions across various cortical cell types, including layer 2/3 PyNs, parvalbumin (PV)-, and vasoactive intestinal peptide (VIP)-positive interneurons (INs) in the primary motor cortex. The probability of detecting protrusions correlated with local H-Ras activity, indicating its role in protrusion formation. H-Ras overexpression led to high turnover rates by adding protrusions. Two-photon photolysis of glutamate induced de novo spine formation in mature H-Ras expressing neurons, suggesting H-Ras's effect is not limited to early development. In PyNs and PV-INs, but not VIP-INs, spine neck lengths shifted to filopodia-like phenotypes. H-Ras primarily induced filopodia in PyNs and spines in PV- and VIP-INs. Increased protrusions in H-Ras-transfected PyNs lacked key excitatory synaptic proteins and did not affect miniature excitatory postsynaptic currents (mEPSCs), suggesting multifaceted roles beyond excitatory synapses.

Keywords: Cell biology; Molecular biology; Neuroscience.

Graphical abstract

Figure 1

H-Ras increases protrusion number in cortical pyramidal neurons (A) Left: schematic depiction of H-Ras sensor constructs and its controls (H-Ras_ONLY, RBD_RAF, ΔH-Ras, and Control). Right: schematic of the mode of action of ddFP-based H-Ras sensor. (B) Virus injection scheme and experimental timeline. (C) Two-photon microscopy images showing representative dendrites (apical distal, apical proximal, and basal) of pyramidal neurons expressing Flex-tdTomato in the primary motor cortex of a typical acute brain slice made from C57Bl6 mice at ∼ P60 injected 4-week prior with CaMKII-Cre, Flex-tdTomato, and either Flex-pCAG-B3-RafRBD-2A-GA-HRas (H-Ras), Flex-pCAG-B3-2A-GA-HRas (H-Ras_ONLY), Flex-pCAG-B3-RafRBD-2A-GA (RBD_RAF) or Flex-pCAG-B3-2A-GA (ΔH-Ras). Scale bar, 2 μm. (D) A summary graph showing the protrusion density. Dots represent average protrusion number from each neuron (dots) and bars indicate mean ± SEM, respectively for each condition (control: 0.7259 ± 0.05162, n = 17; H-Ras:1.0316 ± 0.0743, n = 15; H-Ras_ONLY: 1.4443 ± 0.0657, n = 11; ΔH-Ras: 0.73881 ± 0.10413, n = 10; and RBD_RAF: 0.6554 ± 0.07848, n = 7). The dotted line represents the average protrusion density of the tdTomato only control. ∗p < 0.05 (one-way ANOVA, post-hoc: Tukey test). (E–G) Light shaded box shows graphs related to dendritic spines (E) and dark shaded box shows data related to filopodia (F and G). Separation of protrusions into spines (E) and filopodia (F) showing their density. Dots represent average numbers from each neuron and bars indicate mean ± SEM, respectively, for each condition; (E) control: 0.6096 ± 0.739, n = 14; H-Ras: 0.8479 ± n = 13; H-Ras_ONLY: 1.338 ± 0.0577, n = 11; ΔH-Ras: 0.6507 ± 0.0764, n = 13; RBD_RAF: 0.6398 ± 0.0902, n = 9; (F) Control: 0.0068 ± 0.0027, n = 14; H-Ras: 0.0605 ± 0.0094, n = 13; H-Ras_ONLY: 0.0427 ± 0.006, n = 11; ΔH-Ras: 0.0106 ± 0.003, n = 13; RBD_RAF: 0.0115 ± 0.0031, n = 9. The H-Ras effect seems to be mostly due to an increase in filopodia number, however, H-Ras_ONLY showed an increase in both filopodia and spine number. ∗p < 0.05, ∗∗∗p < 0.001 (one-way ANOVA, post-hoc: Tukey test). (G) Percentage of filopodia of individual neurons (dots) and the mean ± SEM filopodia percentage (control: 0.6059 ± 0.1377, n = 12; H-Ras: 6.9017 ± 1.0834, n = 13; H-Ras_ONLY: 3.067 ± 0.3917, n = 11; ΔH-Ras: 1.4622 ± 0.3541, n = 14, and RBD_RAF: 1.2259 ± 0.3908, n = 9). Pyramidal neurons expressing ectopic H-Ras have higher percentages of filopodia. ∗∗∗p < 0.001 (one-way ANOVA, post-hoc: Tukey test). (H) Separation of protrusion density analysis into the three dendritic locations: apical distal, apical proximal and basal. Apical distal: control (0.931 ± 0.0776), H-Ras (1.06549 ± 0.09451), H-Ras_ONLY: (1.58348 ± 0.1445), ΔH-Ras (0.79241 ± 0.14282), and RBD_RAF (0.77855 ± 0.09053); apical proximal: control (0.37316 ± 0.0448), H-Ras (0.84397 ± 0.0987), H-Ras_ONLY: (1.39342 ± 0.13986), ΔH-Ras (0.30431 ± 0.04335), and RBD_RAF (0.35444 ± 0.06879); basal: control (0.5743 ± 0.05578), H-Ras (0.95856 ± 0.06823), H-Ras_ONLY: (1.3534 ± 0.08495), ΔH-Ras (0.58031 ± 0.09471), and RBD_RAF (0.5017 ± 0.107). control: n = 17; H-Ras: n = 15; H-Ras_ONLY: n = 11, ΔH-Ras: n = 10; RBD_RAF: n = 7. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (two-way ANOVA, Post-hoc: Tukey test). See also Figures S1, S2, and S3.

Figure 2

H-Ras increases protrusion number in cortical parvalbumin interneurons (A) Virus injection scheme and experimental timeline. (B) Representative two-photon microscopy images of parvalbumin-positive interneurons (PV-INs). Scale bar: 10 μm. (C) Two-photon microscopy images showing representative dendrites (proximal and distal) of PV-INs expressing Flex-tdTomato and either Flex-pCAG-B3-RafRBD-2A-GA-HRas (H-Ras), Flex-pCAG-B3-RafRBD-2A-GA (RBD_RAF), or Flex-pCAG-B3-2A-GA (ΔH-Ras). Scale bar, 2 μm. (D) A superimposed bar and dot graph showing the protrusion, spine, and filopodia density of each neuron (dot) and mean ± SEM (top and middle) or median ± IQR (bottom), respectively, for each condition (control, H-Ras, RBD_RAF, and ΔH-Ras) in PV INs. The dotted line represents the median or average density of the tdTomato only control. (Top) Protrusion density averaged from all imaged dendritic branches; control (0.14191 ± 0.00433); H-Ras (0.61928 ± 0.06211); ΔH-Ras (0.18324 ± 0.01037); RBD_RAF (0.20159 ± 0.01719). control: n = 13; H-Ras: n = 22; ΔH-Ras: n = 14; RBD_RAF: n = 13. ∗∗∗p < 0.001 (one-way ANOVA, post-hoc: Tukey test); (Middle) Spine density averaged from all imaged dendritic branches; control (0.11723 ± 0.01079); H-Ras (0.50923 ± 0.05125); ΔH-Ras (0.2538 ± 0.01699); RBD_RAF (0.26233 ± 0.02447). Control: n = 15; H-Ras: n = 23; ΔH-Ras: n = 15; RBD_RAF: n = 14. ∗∗∗p < 0.001 (one-way ANOVA, post-hoc: Tukey test); (Bottom) filopodia density averaged from all imaged dendritic branches; control (0 ± 0.00132); H-Ras (0.00153 ± 0.01984); ΔH-Ras (0 ± 0.00137); RBD_RAF (0 ± 0.00099). Control: n = 16; H-Ras: n = 22; ΔH-Ras: n = 14; RBD_RAF: n = 13. ∗∗∗p < 0.001 (Kruskal-Wallis one-way ANOVA, post-hoc: Kolmogorov-Smirnov test). (E) Representative two-photon images of dendritic protrusions and their morphology. Scale bar = 2 μm. (F) Graph displaying percentage of filopodia at each expression condition from individual neurons (dots) and the median ± IQR (bar). PV-INs expressing ectopic H-Ras has higher percentages of filopodia. (G) Separation of protrusion density analysis into proximal and distal. (Left) Protrusion density of proximal dendrites; control (0.14573 ± 0.01189); H-Ras (0.42605 ± 0.03174); ΔH-Ras (0.16542 ± 0.01075); RBD_RAF (0.18838 ± 0.01431). Control: n = 18; H-Ras: n = 21; ΔH-Ras: n = 15; RBD_RAF: n = 14; (Bottom) protrusion density of distal dendrites; control (0.16693 ± 0.01614); H-Ras (0.71365 ± 0.07629); ΔH-Ras (0.19031 ± 0.01679); RBD_RAF (0.25007 ±. 0.03138). Control: n = 15; H-Ras: n = 24; ΔH-Ras: n = 13; RBD_RAF: n = 13. The dotted line represents the average protrusion density of dendrites expressing tdTomato only (control). Neurons expressing H-Ras showed a higher rate of dendritic protrusions throughout all dendritic regions. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (two-way ANOVA, post-hoc: Tukey test). Control (green), H-Ras (magenta), ΔH-Ras (light magenta), and RBDRAF (pink). Light shaded box shows graphs related to dendritic spines (D “middle”) and dark shaded box shows data related to filopodia (D “bottom” and F). See also Figures S4 and S5.

Figure 3

H-Ras increases protrusion number in cortical vasoactive intestinal peptide interneurons (A) Virus injection scheme and experimental timeline. (B) Representative two-photon images of vasoactive intestinal peptide-expressing interneurons (VIP-INs). Scale bar: 10 μm. (C) Two-photon microscopy images showing representative dendrites (proximal and distal) of VIP INs expressing Flex-tdTomato in the primary motor cortex of a typical acute brain slice made from VIP-Cre mice at ∼ P60 injected 4-week prior with Flex-tdTomato and either H-Ras, or ΔH-Ras. Scale bar, 2 μm. (D) A superimposed bar and dot graph showing the protrusion density of each neuron (dots) and mean ± SEM (bar), respectively, for each condition (H-Ras, and ΔH-Ras) in VIP INs; H-Ras (0.43456 ± 0.04397); ΔH-Ras (0.13683 ± 0.13375) H-Ras: n = 20; ΔH-Ras: n = 12; ∗∗∗p < 0.001 (two sample t test). (E) Separation of protrusion density analysis into spines and filopodia. (Left) Spine density averaged from all imaged dendritic branches; H-Ras (0.42397 ± 0.0354); ΔH-Ras (0.14144 ± 0.0175); H-Ras: n = 22; ΔH-Ras: n = 14. ∗∗∗p < 0.001 (two-sample Kolmogorov-Smirnov test). (Right) Filopodia density averaged from all imaged dendritic branches; H-Ras (0.00298 ± 0.00585); ΔH-Ras (0.00082 ± 0.00291). H-Ras: n = 19; ΔH-Ras: n = 12. ∗∗∗p < 0.001 (two-sample Kolmogorov-Smirnov test). (F) Graph displaying percentage of filopodia at each expression condition from individual neurons (dots) and the median ± IQR (bar). (G) Separation of protrusion density analysis into proximal and distal. (Left) Protrusion density of proximal dendrites; H-Ras (0.42397 ± 0.0354); ΔH-Ras (0.14144 ± 0.0175); (Right) protrusion density of distal dendrites; H-Ras (0.43697 ± 0.04737); ΔH-Ras (0.13057 ± 0.01376). The dotted line represents the average protrusion density of dendrites expressing ΔH-Ras. Neurons expressing H-Ras showed a higher protrusion density throughout all dendritic regions. H-Ras: n = 20; ΔH-Ras: n = 12; ∗∗∗p < 0.001 (two sample t test, post-hoc: Sidak Holm’s test).

Figure 4

Local H-Ras activity facilitates de novo growth of protrusions (A) Schematic of organotypic slice preparation and timeline. (B) Representative dendrite images expressing tdTomato (top) and H-Ras (tdTomato + pCAG-B3-RafRBD-2A-GA-HRas) (bottom) collected via a two-photon microscope at P12. DNA was biolistically transfected to organotypic cortical slices at P1. Scale bar: 2 μm. (C) A summary graph showing the protrusion density of each analyzed dendritic section (∼30 μm) (dots) and the mean protrusion density ±SEM (bar) of Control (0.49719 ± 0.02549) and H-Ras neurons (0.6445 ± 0.02608). The dotted line represents the average protrusion density of control dendrites. Control: n = 53, H-Ras: n = 52; ∗∗∗p < 0.001 (two sample t test). (D) tdTomato and H-Ras biosensor expression in a representative organotypic pyramidal neuron: red = tdTomato, green = H-Ras biosensor. (E) Magnification of dendritic branch shown in D (white box) (top) tdTomato, (middle) H-Ras biosensor GFP, and (bottom) merge. Colored arrow heads indicate examples of high H-Ras activity (orange) or low H-Ras activity (light blue). Scale bar: 2 μm. (F) Scatterplot showing that higher H-Ras activity (relative green/red ratio) correlates with a higher percentage of detecting a spine in close proximity (1 μm). The sigmoidal curve fitting to the data shows an R value of 0.87415. (G) Correlation matrix comparing H-Ras activity values with the existence of protrusions in a 1 μm radius; (left) experimental data, (right) shuffled data control. H-Ras activity correlates to a higher extent with the existence of dendritic protrusions in the experimental data in comparison to shuffled data. (H) Virus injection scheme and experimental timeline. (I) Example images of high-frequency glutamate uncaging (HFU) experiments (circles, 40 pulses at 10Hz) on adult pyramidal neurons (∼P60): (top left) ΔH-Ras + MNI glutamate, (top right) H-Ras + MNI glutamate, (bottom left) ΔH-Ras without MNI-glutamate, and (bottom right) H-Ras without MNI-glutamate. All neurons expressed tdTomato as cell markers. Arrowheads indicate de novo spine formation after HFU. Scale bar, 2 μm. (J) Success rate of de novo spine formation by HFU at P60 in pyramidal neurons expressing tdTomato and either ΔH-Ras (19.2%) or H-Ras (52%). In the absence of MNI-glutamate (mock) neither ΔH-Ras nor H-Ras exhibited any de novo spine formation. ΔH-Ras: n = 24 trials, 16 cells; H-Ras: n = 26 trials, 15 cells; ΔH-Ras mock: n = 4 trials, 4 cells; H-Ras mock: n = 8 trials, 7 cells. ∗∗∗p < 0.001 (Chi-square test). See also Figure S5.

Figure 5

Increased protrusion number does not represent features of functional excitatory synapses (A) Virus injection scheme and experimental timeline. (B) Light microscopy images of layer 2/3 pyramidal neuron recorded in whole-cell voltage-clamp-mode in acute slices: whole motor cortical brain region (left), magnified image: light microscopy and fluorescent (right bottom), schematic of pyramidal neurons being fluorescent positive (+) or fluorescent negative (−) (right top). (C) Examples traces of mEPSCs in ΔH-Ras⁺ (light magenta), H-Ras⁺ (magenta), ΔH-Ras^- (gray), and H-Ras^- neurons (black) (1.25 Hz low-pass fft filter). (D–G) mEPSC frequencies (D), amplitudes (E), rise times (F), and decay time constants (τ) (G) in ΔH-Ras⁺ (light magenta), H-Ras⁺ (magenta), ΔH-Ras^- (gray), and H-Ras^- neurons (black). Each dot represents an average value from one neuron, and the dotted line represents mean ± SEM. (H) Example confocal images of ΔH-Ras and H-Ras of pyramidal neurons (left) and magnified dendrites (right). From top to bottom: image of pyramidal neuron expressing tdTomato, postsynaptic protein Homer1 stained with Alexa Fluor 633 (green), presynaptic protein bassoon stained with Alexa Fluor 405 (blue), merge of all channels. Scale bars: 10 μm (pyramidal neuron), 3 μm (dendrite). (I) Magnification image of (H) visualizing single protrusions of ΔH-Ras (left) and H-Ras (right). From top to bottom: postsynaptic protein Homer1 (green) stained with Alexa Fluor 633 with and without dendrite visualization, presynaptic protein bassoon with Alexa Fluor 405 (blue) stained with Alexa Fluor 405 with and without dendrite visualization, merge of channels with and without dendrite visualization. Scale bar: 0.75 μm. (J) Imaris reconstructed image of dendrites and dendritic protrusions in conjunction with bassoon reconstruction (blue) or Homer1 reconstruction (green) of ΔH-Ras (left) and H-Ras neurons (right). Scale bar: 0.75 μm. (K) Imaris reconstruction of dendrites/protrusions together with Homer1 (green, top) or bassoon (blue, bottom) of ΔH-Ras (left) and H-Ras neurons (right). (L) A summary graph showing the protrusion density of PyNs with confocal imaging. ΔH-Ras: n = 12, H-Ras: n = 9; ∗p < 0.05 (two-sample t test). (M and N) Analysis of fraction of protrusions having close contact (0.1 μm) to bassoon puncta (M) or encompassing Homer1 puncta (N) in either ΔH-Ras or H-Ras neurons shows that neurons expressing ectopic H-Ras have reduced number of protrusions expressing Homer1 as well as reduced bassoon contacts. ΔH-Ras: n = 12, H-Ras: n = 9; ∗∗p < 0.01, ∗∗∗p < 0.001 (two-sample Kolmogorov-Smirnov test). See also Figures S7 and S8.