The spread of Ras activity triggered by activation of a single dendritic spine

Abstract

In neurons, individual dendritic spines isolate N-methyl-d-aspartate (NMDA) receptor-mediated calcium ion (Ca2+) accumulations from the dendrite and other spines. However, the extent to which spines compartmentalize signaling events downstream of Ca2+ influx is not known. We combined two-photon fluorescence lifetime imaging with two-photon glutamate uncaging to image the activity of the small guanosine triphosphatase Ras after NMDA receptor activation at individual spines. Induction of long-term potentiation (LTP) triggered robust Ca2+-dependent Ras activation in single spines that decayed in approximately 5 minutes. Ras activity spread over approximately 10 micrometers of dendrite and invaded neighboring spines by diffusion. The spread of Ras-dependent signaling was necessary for the local regulation of the threshold for LTP induction. Thus, Ca2+-dependent synaptic signals can spread to couple multiple synapses on short stretches of dendrite.

Figure 1. Ras activation in individual dendritic spines during plasticity induction

(A) Experimental geometry. (B) Schematic of fluorescence decay curves following pulsed excitation. Slow and fast components correspond to free donor and donor bound to acceptor, respectively. FRET decreases fluorescence lifetime. (C) FLIM images of Ras activity. At time = 0, 30 uncaging pulses (0.5 Hz) were applied to the spine marked by the arrowhead in low (nominally 0 mM) extracellular Mg²⁺. Warmer colors indicate shorter lifetimes and higher levels of Ras activation. (D) Changes in spine volume. Colors correspond to the circles in (C). Arrow, time of stimulus. (E) Spine volumes for the stimulated and nearby (< 4 μm) spines (−5 to 20 minutes: 91 spines; > 20 minutes: 9 spines, mean ± sem). ΔVol_sustained is the volume difference between 15.5-19.5 minutes and the baseline volume. (F) Ras activation. Colors correspond to the circles in (C). (G) Ras activation in the stimulated and nearby spines (82 spines, mean ± sem). (H) Pathways to Ras activation. Ras activation was the average binding fraction at 1-3 minutes minus baseline, normalized to the control condition. Numbers of spines: 82 Ctrl, 11 Low [Ca²⁺]_ex (200 μM), 12 CPP (10 μM), 27 KN62 (10 μM), 20 LY294002 (20 μM), and 23 Gö6976 (1 μM). Error bars indicate mean ± sem. Asterisks indicate P < 0.05 versus control.

Figure 2. Spatial spread of Ras activity

(A) Fluorescence lifetime images of Ras activity. At time = 0 LTP was induced at the spine marked by an arrowhead. (B) The spatial spread of Ras activation at different time points. Black circles indicate distances along the dendrite relative to the stimulated spine (gray circle; n = 11, mean ± sem). The solid line shows the fitted profile of Ras activation derived from a 1-D diffusion-reaction model (22). (C) The spatial spread of Ras activation (Ras activation, normalized to peak Ras activation in the stimulated spine, two minutes post-stimulus) at various expression levels. (D) Normalized Ras activation 3-5 μm from the stimulated spine. r = 0.36, P > 0.3, n = 8.

Figure 3. Ras mobility and the time constant of Ras inactivation

(A) Left: Spine before and after photoactivation (green, paGFP-Ras; red, mCherry). Right: Time course of activated paGFP-Ras in the spine (black) and parent dendrite (blue). (B) Decay time constants of paGFP fluorescence in the photoactivated spine. Horizontal bars, mean. Numbers of spines: 17 cytosolic, 46 membrane, 21 Ras, 84 Ras G12V, 52 Ras G12V (33° C). Asterisks indicate P < 0.05 versus paGFP-Ras G12V (23° C). (C) Decay time constants of paGFP-Ras G12V fluorescence at varying expression levels. r = −0.05, P > 0.8, n = 13. (D) Fraction of paGFP fluorescence remaining in the spine 30 seconds after photoactivation. Horizontal bars, mean. Number of spines: 46 membrane, 84 Ras G12V. P > 0.7. (E) Fraction of paGFP-Ras G12V fluorescence remaining in the spine at 30 seconds at varying expression levels. r = 0.25, P > 0.4, n = 13. (F) Fluorescence lifetime images before and after trains of back-propagating action potentials (40 APs at 83 Hz, repeated 4 times every 5 seconds). (G) Time course of Ras activation for the experiment shown in (F). Arrows, action potential stimuli. (H) τ_inactivation at 23° C and 33° C. Ras inactivation was measured at both temperatures in individual cells. n = 5, P < 0.001. (I) Ras inactivation at varying expression levels. r = 0.8, P < 0.01, slope = 0.6 minutes per fold over-expression, intercept = 2.9 minutes. n = 12.

Figure 4. Spread of Ras signaling and synaptic crosstalk

(A) Time-lapse images of a GFP-expressing pyramidal neuron in an acute hippocampal brain slice. At time = 0, 30 uncaging pulses (0.5 Hz, 4 ms pulse duration, LTP protocol) were applied to the spine marked by a white arrowhead (LTP spine) in low extracellular Mg²⁺. At time = 3 minutes, vehicle (0.1% DMSO) was pressure applied locally from a glass pipette until time = 17 minutes. At time = 5.5 minutes, the subthreshold protocol (30 uncaging pulses, 0.5 Hz, 1 ms pulse duration) was applied to a nearby spine (sub spine, red arrowhead). (B) Spine volume changes in the vehicle condition (11 spines, mean ± sem). (C) Spine volume changes in the 20 μM U0126 condition (11 spines, mean ± sem). (D) Sustained changes in spine volume. Error bars indicate mean ± sem. Asterisk indicates P < 0.05. (E) Ras activation in the LTP and sub spines during the crosstalk paradigm in cultured hippocampal slices. At time = 0, the LTP protocol was applied to the LTP spine, and 3.5 minutes later the subthreshold protocol was applied to the sub spine (13 spines, mean ± sem). (F) Relationship between Ras activation and the sustained spine enlargement in the sub spine during crosstalk. ΔBinding fraction was measured before the subthreshold protocol (1.5 to 3.5 minutes after the LTP protocol). r = 0.54, P = 0.05.