Article Figures & Data
Figures
- Figure 1.
AP-induced axonal swelling and subsequent migration of microglial processes to the periaxonal area. A, A low-magnification image of a cortical slice obtained from an Iba1-GFP mice in which microglia express GFP (green). Note the largely ramified morphology suggesting a “resting state,” except for some surface microglia. A recording pipette and a pyramidal neuron, from which a recording was obtained, are loaded with Alexa Fluor 594 (red). Scale bar, 50 µm. B, Time-lapse images of a layer 2/3 somatosensory cortical neuron axon filled with Alexa Fluor 594 (red) and surrounding microglial processes (expressing eGFP, green), acquired at different times before and after a 3 min current stimulation protocol applied to the the soma of the neuron to evoke repetitive APs (at 10 Hz). Scale bar, 3 µm. C, Higher-magnification images of axonal fluorescence signals from the areas indicated by the rectangular boxes at −12 and 6 min in B. Note the increase in FI. Scale bar, 1 µm. D, G, Mean change in relative axonal and microglial FIs; before, during, and after 6 min of 10 Hz soma current stimulation applied from t = 0 (n = 16). Plots of the relative neuronal (red) FI (D) and perineuronal (green) microglial FI (G) for axons (including no stimulation controls) and dendritic compartments are superimposed (apical dendrite, black; basal dendrite, gray). E, H, Poststimulus change in relative axonal (H) and microglial (H) FIs for different durations of stimuli. F, Pooled data showing the correlation between the relative stimulus-induced changes in axonal FI and microglial FI obtained from a range of 10 Hz stimulus train durations, as indicated by each symbol.
- Figure 2.
Investigating roles of VAACs in AP-induced axonal swelling and subsequent migration of microglial processes to the periaxonal area. A–E, Plots of the prestimulus and poststimulus data points for axonal swelling (red) and periaxonal microglial (green) FI, for the control condition (A) and for each of the applied drugs (B–E). Individual, paired data, and mean values (±SEM) are shown. FI was averaged over the prestimulus and poststimulus time points, and these were compared using the paired t test: *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 3.
Microglial wrapping of an axon and rescuing the depolarized somatic membrane potential. A, Time-lapse images of an axon (red) and surrounding microglial processes (green) before (Ai) and at various times as indicated (Aii–Avi) after a 6 min period of supramaximal AP stimulation. Scale bar, 5 µm. Arrows at Aiii and Av indicate extensive microglial accumulation around the axon. B, Vm (top) and changes in relative axonal (red) and periaxonal microglia (green) FI (bottom panels) obtained from the experiment shown in A. Bi–Bvi correspond to times shown in A. Note that the Vm and FI are shown on the same timescale to illustrate the temporal associations among Vm depolarization, axonal swellings, and the extensive accumulation of microglial processes around the axons that precedes repolarization and reduced swelling. C, D, The temporal relationships among changes in axonal volume, membrane potential, and the fluorescence intensity of periaxonal microglial processes. The graphs plot data for each neuron of C; the relative times of Vm depolarization and how this relates to increased axonal swelling and microglial FIs; and how the recovery of Vm relates to the recovery of axonal swelling (top) and microglia FI (bottom; D). In C and D, each colored line (with associated points) was obtained from a single experiment (n = 7). The criterion for FI increase was >5% from the prestimulus intensity, and for depolarization was >20 mV from resting Vm. Vm recovery (time = 0 in D) was defined as the return to within 10 mV of the initial resting Vm, and microglial FI recovery (process retraction) was defined as the return to within 5% of the initial value.
- Figure 4.
Repolarization of membrane potential is associated with a decreased membrane conductance. A, Sample trace of Vm before, during, and after the application of a 6 min strong depolarizing stimulus. The regular positive and negative deflections reflect voltage responses to steps of current used to measure membrane conductance. Ai–Aiii indicate periods before, during, and after the marked spontaneous, transient depolarization. B, Current pulses (top) and corresponding membrane potential responses (bottom) as used to assess passive membrane properties. Current steps ranged from −200 to +120 pA in 20 pA increments. C, Representative membrane potential responses evoked by current pulses before the spontaneous depolarization (Ci), during the depolarization (Cii), and after the depolarization (Ciii). Red dashed lines indicate the slope of the current–passive voltage relationship used to derive the membrane conductance. Note the action potentials in Ci evoked at more depolarized potentials. D, Relative membrane conductance before, during, and after the sustained depolarization derived from fitting a linear regression to the relationship between applied current and subsequent voltage responses (as shown in C) to quantify the slope conductance (ΔI/ΔVm). Circles indicate the mean values. Individual values are shown alongside means and SEM. Means were compared using a paired t test. *p < 0.05
- Figure 5.
Inhibition of microglial migration to swollen axons by block of VAACs. A, Time-lapse representative images of an axon (red) and microglial processes (green) at different times before and after a strong (6 min) depolarizing stimulus, all in the presence of VAAC block. Scale bar, 5 µm. B, Representative traces of Vm and axonal and microglial FIs in the presence of NPPB, from a different neuron as shown in A. A sudden, large depolarization followed the stimulation-induced increase in axonal FI (red), but no increase in microglial FI (green) was seen adjacent to the axon, and no recovery of the depolarized Vm was seen (Vm continued to further depolarize to 0 mV). C, Pooled data showing the mean change of Vm following 6 min of AP stimulation, in control conditions (n = 16, solid line) and in the presence of NPPB (n = 8, dashed line). ***p < 0.005. D, Kaplan–Meier survival curves of neurons treated with NPPB (n = 8, dashed line) and in control (n = 16). *p < 0.05. Neuronal “death” was defined as a neuron whose Vm depolarized to close to 0 mV for more than a few minutes (typically followed by apparent loss of the Giga seal).
Movies
- Movie 1.
Time-lapse video showing an example of an excessively stimulated axon with a substantial volume increase, which was followed by intense contacts with microglial processes. Note the swollen axon being repeatedly wrapped by microglial processes, after which the substantial membrane potential depolarizations were restored to the resting levels (for details, see Fig. 4). x–y-projection images were acquired every 3 min for 78 min.
- Movie 2.
Time-lapse video showing microglial processes migrating to an axon but not to the dendrite. The axon (bottom) was wrapped by microglial processes shortly after a substantial membrane potential depolarization was recorded. The neighboring basal dendrite (top) was not wrapped by microglia during or after this depolarization. x–y-projection images were acquired every 3 min for 60 min.
In this issue
