Activity-dependent relocation of the axon initial segment fine-tunes neuronal excitability

Abstract

In neurons, the axon initial segment (AIS) is a specialized region near the start of the axon that is the site of action potential initiation. The precise location of the AIS varies across and within different neuronal types, and has been linked to cells' information-processing capabilities; however, the factors determining AIS position in individual neurons remain unknown. Here we show that changes in electrical activity can alter the location of the AIS. In dissociated hippocampal cultures, chronic depolarization with high extracellular potassium moves multiple components of the AIS, including voltage-gated sodium channels, up to 17 mum away from the soma of excitatory neurons. This movement reverses when neurons are returned to non-depolarized conditions, and depends on the activation of T- and/or L-type voltage-gated calcium channels. The AIS also moved distally when we combined long-term LED (light-emitting diode) photostimulation with sparse neuronal expression of the light-activated cation channel channelrhodopsin-2; here, burst patterning of activity was successful where regular stimulation at the same frequency failed. Furthermore, changes in AIS position correlate with alterations in current thresholds for action potential spiking. Our results show that neurons can regulate the position of an entire subcellular structure according to their ongoing levels and patterns of electrical activity. This novel form of activity-dependent plasticity may fine-tune neuronal excitability during development.

Figure 1. Activity-dependent changes in AIS position

a, Ankyrin G label in control and 15 mM K⁺ conditions. Right, fluorescence intensity along the axon. Dotted lines indicate soma. b, Ankyrin G positions and length (885 cells, 36 coverslips). c, βIV-spectrin and PanNa_v label. d, Positions for βIV-spectrin (1065 cells, 44 coverslips), PanNa_v (95 cells, 4 coverslips), Pan-neurofascin (NF; 96 cells, 4 coverslips), and FGF-14 (89 cells, 4 coverslips). e, Ankyrin G label after recovery from (K⁺-control), or continued (K⁺-K⁺) depolarization (194 cells, 8 coverslips). f, βIV spectrin label in GAD65+positive neurons (200 cells, 8 coverslips). All scale bars indicate 20 μm; *P < 0.05; **, P < 0.01; ***, P < 0.001. All plots show mean ± s.e.m.

Figure 2. T- and/or L-type calcium channels mediate activity-dependent changes in AIS position

a, Ankyrin G label after 12-14 DIV, 15 mM K⁺ -treatment with different voltage-gated ion channel antagonists. Scale bar, 20 μm; dotted line, soma. b, Mean ankyrin G positions (TTX, 392 cells, 16 coverslips; Ni²⁺, 193 cells, 8 coverslips; SNX, 247 cells, 10 coverslips; mibefradil, 246 cells, 10 coverslips; ω-agatoxin-TK, 199 cells, 8 coverslips; ω-conotoxin-GVIA, 199 cells, 8 coverslips; nifedipine, 200 cells, 8 coverslips). *, P < 0.05; ***, P < 0.001; error bars, s.e.m.

Figure 3. Changes in AIS position with patterned ChR2 photostimulation

a, LED photostimulation of ChR2-positive neurons. b, Ankyrin G label in ChR2-positive neurons after different photostimulation conditions. Scale bar, 20 μm. c, Mean ankyrin G positions for ChR2-positive (green) and ChR2-negative (black) neurons after different photostimulation conditions (0.2 Hz, 133 cells, 4 coverslips; 1 Hz Sparse, 274 cells, 8 coverslips; 1 Hz Bursts, 310 cells, 8 coverslips; 1 Hz Bursts + TTX, 154 cells, 4 coverslips; 1 Hz Bursts + mibefradil, 134 cells, 4 coverslips). *, P < 0.05; **, P < 0.01; error bars, s.e.m.

Figure 4. Differences in AIS position are associated with differences in neuronal excitability

a, Current-clamp traces in control and 15 mM K⁺ conditions Numbers indicate threshold current (pA) and threshold current density (pA pF⁻¹). b, Threshold current, threshold current density and R_m (41 cells, 3 cultures). c,d, YFP-Na_vII-III label and current-clamp traces e, AIS end position versus current threshold (control, 24 cells, 3 cultures; AIS end mean 33.51 ± 2.63 μm; 15 mM K⁺, 26 cells, 1 culture; AIS end mean 37.02 ± 2.40 μm). Lines indicate linear regression. f, g, Left: biocytin and ankyrin G label. Middle and right panels show current-clamp traces for 10 ms and 500 ms current injections. . Numbers indicate input pA pF⁻¹. h, AIS start position versus threshold current density for 10ms current injections (53 cells, 2 cultures). i, Current density versus spike number for 500ms current injections, sample split by AIS start position. j, As for i, but sample split by R_m. All scalebars indicate 20 μm; *, P < 0.05; **, P < 0.01; ***, P < 0.001. All plots show mean ± s.e.m.