Research ArticleNEUROSCIENCE

Tailoring of the axon initial segment shapes the conversion of synaptic inputs into spiking output in OFF-α T retinal ganglion cells

See allHide authors and affiliations

Science Advances  11 Sep 2020:
Vol. 6, no. 37, eabb6642
DOI: 10.1126/sciadv.abb6642
  • Fig. 1 Spiking responses are weaker in ventral versus dorsal OFF-α T RGCs.

    (A) Left: Whole-cell patch clamp recordings were used to record the responses of OFF-α T RGCs to current injections (range, −500 to 1000 pA). Right: Schematic of the whole mount mouse retina depicting the approximate sampling locations for ventral (V) and dorsal (D) cells. (B) Representative current clamp recordings from OFF-α T RGCs in the ventral (red) and dorsal (blue) retina. Pulse duration was 400 ms (gray shading), amplitudes ranged from 100 to 600 pA (ventral) and 100 to 1000 pA (dorsal), respectively. Scale bar, 100 ms/20 mV. Horizontal arrows indicate failure threshold (see main text). (C) Comparison of failure threshold between ventral (red, n = 20) and dorsal (blue, n = 22) cells (495 ± 29 pA versus 718 ± 40 pA, P = 2 × 10−4). (D) Plots of firing rate versus time for all stimulus amplitudes tested; same cells as (B). Transient (T) and sustained (S) response phases correspond to 0 to 100 ms and 100 to 400 ms, respectively. (E) Mean firing rate over time across the population of ventral (red) and dorsal (blue) cells in response to a stimulus amplitude of 300 pA [two-way repeated measures analysis of variance (ANOVA): interaction, P = 0.0002; subgroups, P = 9 × 10−6]. (F) Same analysis as in (E) but for the amplitude that elicited maximum responses in each cell (two-way repeated measures ANOVA: interaction, P = 4 × 10−6; subgroups, P = 3 × 10−8). Insets in (E) and (F) show the ratio of sustained and peak firing rate for ventral (red) and dorsal (blue) cells. (G) The number of elicited spikes is plotted versus injected current amplitude for ventral (red) and dorsal (blue) cells for the whole duration of the injection (left), the initial 100 ms (middle), and the last 300 ms (right) of the stimulus. Insets show the population mean. **P < 0.01; ***P < 0.001.

  • Fig. 2 AIS properties are different in ventral and dorsal OFF-α T RGCs.

    (A) Left: Tracings of representative ventral (red; dendritic field diameter ~ 290 μm) and dorsal (blue; dendritic field diameter ~ 370 μm) cells. Scale bar, 50 μm. Right: Comparison of dendritic field diameter between ventral (red; n = 21) and dorsal (blue; n = 20) cells (280 ± 7 μm versus 347 ± 8 μm, P = 3 × 10−7). (B) Axon diameter was traced along the first 100 μm of the proximal axon in ventral (red; n = 13) and dorsal (blue; n = 17) cells (two-way repeated measures ANOVA: interaction, P = 0.8101; subgroups, P = 0.2336). (C) Confocal images of the soma and AIS from typical ventral (left) and dorsal (right) OFF-α T RGCs. Staining for AnkyrinG (blue) is overlaid with the axon (green); the start and end of the AIS are indicated by white arrowheads. Scale bars, 20 μm. (D) Scatter plot of AIS length (AIS L) versus AIS distance (AIS D) for ventral (red; n = 11) and dorsal (blue; n = 16) cells. Box plots reveal statistically significant differences for both distance (22.3 ± 1.2 μm versus 29.2 ± 1.2 μm, P = 0.0004) and length (16.9 ± 1.4 μm versus 25.5 ± 1.4 μm, P = 0.0003). (E) Immunochemical staining for AnkyrinG (blue) and Nav1.6 (red) along the proximal axon. Solid vertical lines indicate the start and end of the AnkyrinG staining; the dashed vertical line indicates the start of the Nav1.6 portion. Scale bar, 20 μm. ***P < 0.001.

  • Fig. 3 Sustained firing is altered by disruption of AIS function.

    (A) Confocal images of one cell with an intact axon (left) and three axotomized cells in the three rightmost panels. The severed edge of each axon is indicated by an arrowhead. Scale bar, 20 μm. (B) Number of spikes elicited for current injections in ventral (red; n = 4) and dorsal (blue; n = 2) cells that had their axons removed between the soma and the AIS. Three time periods were examined: (i) the full duration of the stimulus (left), (ii) the initial 100 ms (middle), and (iii) the final 300 ms of the stimulus (right). Population means of intact cells are shown by thin lines. (C) Comparison of failure thresholds between ventral (red; n = 4) and dorsal (blue; n = 2) cells that had axotomies between the soma and the AIS (425 ± 25 pA versus 450 ± 50 pA, P = 0.6328). n.s., not significant. (D) Mean firing rate over time across the population of axotomized ventral (red; n = 4), intact ventral (black; data from Fig. 1F), and dorsal (blue; n = 2) cells in response to a stimulus amplitude that elicited maximum responses in each cell. (E) Recordings before (left), during (middle), and after (right) the addition of aTTX (4,9-anhydrotetrodotoxin) (500 nM) to the perfusion bath for a ventral (red; top) and dorsal (blue; bottom) cell. (F) Comparison of peak/transient (left) and sustained (right) components of the response under control (C), during the application of aTTX, and after wash (W) in ventral (red; n = 4) and dorsal (blue; n = 3) OFF-α T RGCs. Population means are shown in black. Error bars represent one SD.

  • Fig. 4 Longer AISs facilitate higher levels of depolarization.

    (A) Responses to current injection consisted of a series of action potentials “riding” on top of a change in the baseline membrane voltage (green); the change in V (from resting) is referred to as ΔV. Scale bar, 100 ms/10 mV. (B) Plots of the number of elicited spikes as a function of ΔV in ventral (red; n = 17) and dorsal (blue; n = 20) cells elicited during the full extent of the stimulus (left), during the initial response phase (middle), and during the sustained response phase (right). Gray shading indicates ΔV > 10 mV. (C) Left: ΔVfail was defined as the change in V at failure threshold. Scale bar, 100 ms/10 mV. Right: ΔVfail was significantly lower in ventral (red; n = 17) versus dorsal (blue; n = 20) cells (15.5 ± 0.8 mV versus 23.8 ± 1.6 mV, P = 4 × 10−5). One ventral cell was considered an outlier and excluded from analysis (gray arrow). (D) Each circle is a plot of ΔVfail versus AIS length for an individual ventral (red; n = 8) or dorsal (blue; n = 12) RGC. The gray line is the best-fit linear regression (P = 0.0001). ***P < 0.001.

  • Fig. 5 Spike dynamics are altered by removal of the AIS.

    (A) Overlay of spontaneous spikes from three axotomized cells (different distances from the soma for each). The inset shows a comparison of the spike with the longest axon segment remaining with a spike from an intact cell (green); an arbitrary horizontal offset was used to enhance visibility of the two traces. (B) Overlay of phase plots for the same spikes on the left. The arrow indicates the initial segment-somatodendritic break in the cell with the longest axonal segment. The inset shows a comparison of the same spike with a spike from an intact cell (green); an arbitrary horizontal offset was again used to enhance visibility of the two traces. (C) Two parameters were extracted from phase plots: (i) threshold depolarization (green) and (ii) onset rapidness (black) (see main text). Scale bar, 10 mV/20 V/s. (D) Threshold depolarization (green) and onset rapidness (black) are plotted versus the location of the axotomy (n = 16). Solid lines are best-fit Hill functions, and dashed lines indicate potential other transitions of onset rapidness and threshold depolarization. Average values from cells with intact axons are shown at right. Schematic on top and shaded box indicate the approximate location of the AIS (brown); points to the left of the shading indicate axotomies between the soma and the AIS, whereas points right to the shading indicate axotomies beyond the AIS. (E) Scatter plot of AIS length versus onset rapidness (P = 0.2454) and comparison of onset rapidness between ventral (red; n = 18) and dorsal (blue; n = 18) cells (26.4 ± 1.1 ms−1 versus 28.1 ± 1.4 ms−1, P = 0.3981). (F) Scatter plot of AIS length versus threshold depolarization (P = 0.0484) and comparison of threshold depolarization between ventral (red; n = 18) and dorsal (blue; n = 18) cells (6.5 ± 0.4 mV versus 5.2 ± 0.4 mV, P = 0.0119).

  • Fig. 6 Simple models of AIS-specific spiking properties.

    (A) A cylindrical single-compartment model was equipped with either Nav1.1 or Nav1.6 channels. (B) Firing rate in response to a current injection of 10 pA was higher when Nav1.6 channels were inserted into the model. Gray arrows indicate spontaneous spiking. Scale bar, 50 ms/20 mV. (C) Phase plots derived from (B) reveal a lower threshold for the Nav1.6 model than for the Nav1.1 model. Scale bar, 20 mV/200 V/s. (D) Left: A 1-mm axon model was created with 201 compartments. All compartments were uniform except each had only Nav1.6 channels or only Nav1.1 channels; the percentage of compartments with Nav1.6 channels increased from 0 (top) to 100% (bottom) across different simulation trials; Nav1.6 channels were always centered around the middle compartment. Right: Spiking response to a 10-pA current injection into the center compartment for the four configurations on the left. Scale bar, 200 ms/20 mV. (E) Firing rate (top), threshold depolarization (middle), and spontaneous firing rate (bottom) at the center compartment as a function of the Nav1.6 portion within the axon.

  • Fig. 7 Realistic models of AIS-specific spiking properties.

    (A) Overlay of simulated spikes from the realistic model cell with short (10 μm; red) and long (35 μm; blue) AISs. Dashed horizontal lines indicate thresholds for spike initiation. (B) The influence of AIS L (unfilled circles) and AIS D (filled circles) on sustained firing rate in response to a somatic current injection of 300 pA. (C) Scatter plots of AIS length versus transient (left; P = 0.0036) and sustained firing rates (right; P = 0. 0347) from physiological experiments. The stimulus amplitude used was the one that resulted in the strongest responses in each cell. Solid lines in indicate best-fit linear regressions. *P < 0.05; **P < 0.01. (D) Firing rate in response to a 300-pA stimulus is plotted as a function of threshold depolarization for recorded data (red and blue correspond to ventral and dorsal cells, respectively) and simulated spike trains [black; data from (B)]. (E) Inset shows the input currents elicited during light stimulation in a typical ventral (dashed) and dorsal (solid) cell as measured by (9). Scale bar, 250 ms/500 pA. These currents were injected into the somas of model RGCs and the membrane voltage over time was plotted in ventral (red; n = 6) and dorsal (blue; n = 6) cells (main) with all voltage sensitive ion channels blocked. Red and blue horizontal shadings indicate failure threshold ± one SD as measured in ventral and dorsal RGCs, respectively. Gray shading indicates the 400-ms time window that was used in the current clamp experiments of Fig. 1.

Supplementary Materials

  • Supplementary Materials

    Tailoring of the axon initial segment shapes the conversion of synaptic inputs into spiking output in OFF-α T retinal ganglion cells

    Paul Werginz, Vineeth Raghuram, Shelley I. Fried

    Download Supplement

    This PDF file includes:

    • Figs. S1 to S8
    • Table S1

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article