Neuromodulation of Axon Terminals

Abstract

Understanding which cellular compartments are influenced during neuromodulation underpins any rational effort to explain and optimize outcomes. Axon terminals have long been speculated to be sensitive to polarization, but experimentally informed models for CNS stimulation are lacking. We conducted simultaneous intracellular recording from the neuron soma and axon terminal (blebs) during extracellular stimulation with weak sustained (DC) uniform electric fields in mouse cortical slices. Use of weak direct current stimulation (DCS) allowed isolation and quantification of changes in axon terminal biophysics, relevant to both suprathreshold (e.g., deep brain stimulation, spinal cord stimulation, and transcranial magnetic stimulation) and subthreshold (e.g., transcranial DCS and transcranial alternating current stimulation) neuromodulation approaches. Axon terminals polarized with sensitivity (mV of membrane polarization per V/m electric field) 4 times than somas. Even weak polarization (<2 mV) of axon terminals significantly changes action potential dynamics (including amplitude, duration, conduction velocity) in response to an intracellular pulse. Regarding a cellular theory of neuromodulation, we explain how suprathreshold CNS stimulation activates the action potential at terminals while subthreshold approaches modulate synaptic efficacy through axon terminal polarization. We demonstrate that by virtue of axon polarization and resulting changes in action potential dynamics, neuromodulation can influence analog-digital information processing.

Figure 1.

Recording set-up and passive property alterations in experimental and modeling results. Custom made experimental set-up with Ag-AgCl₂ electrodes producing uniform electric fields across the slice. Soma electrode stimulated and recorded the response, while the bleb electrode only recorded the respective response. The reference microelectrode used to measure the bath polarization (a). For parallel field stimulation, the electrodes were placed across the slice such that the electric field was parallel to the dendro-axonic axis of the layer-V pyramidal cells, whereas for the orthogonal orientation the electric field is perpendicular to the dendro-axonic axis (b, left). The axonal alignment, θ, relative to the electric field was determined for each cell (b, right). Sample image of a layer-V pyramidal cell, filled with Alexa-488 presented here (c). (red arrow: axon hillock, blue arrow: axon initial segment, yellow arrow: axon bleb). Cellular orientation in the slice is illustrated in the schematic (d). The axon bleb is patched at the surface of the slice whereas the dendrite remains intact deep inside the slice. Average polarization length of P14–19 (N = 12, n = 250) is significantly lower than in young adult (2 months) (N = 5, n = 41) (e). Relative bleb–soma polarization as a function of the axonal length (f). In the parallel orientation, the relative polarity-specific (dep and hyper) polarization was evident started only in the axons longer than 100 μm (N = 19, n = 19). In the orthogonal orientation, the relative polarization was not affected significantly (N = 15, n = 15). Compartmentalization of a neuron for the polarization modeling (g), where, AIS1 represents the axon hillock, AIS2 is the axon initial segment and AIS3 denotes the axon. Models of relative polarization predicted similar polarization as experimental data, but only when persistent sodium current (Na_p) and persistent potassium current (K_p) were considered in addition to the passive membrane properties (h). (Depolarized passive: yellow, hyperpolarized passive: green, depolarized active: red, hyperpolarized active: blue).

Figure 2.

AP amplitude and midwidth ratio alterations to Φ change of current under parallel or orthogonal electric field. For parallel electric field orientation, the axonal AP amp ratio to Φ change of current is linearly correlated to axonal length for both depolarizing and hyperpolarizing polarities (a) (red, dep field, R² = 0.53, P < 0.001; blue, hyp field, R² = 0.61, P < 0.0001) while the soma is not affected (b) (red, dep field, R² = 0.0008, P = 0.906; blue, hyp field, R² = 0.0414, P = 0.389). For orthogonal electric field orientation axonal AP amplitude ratio to Φ change of current is linearly correlated to axonal length for hyperpolarizing polarity but not in depolarizing polarity (c) (red, dep field, R² = 0.24, P = 0.062; blue, hyp field, R² = 0.34, P = 0.02) with no effect at the soma (d) (red, dep field, R² = 0.079, P = 0.3089; blue, hyp field, R² = 0.0034, P = 0.8359). With parallel field orientation, AP mid-width ratio to Φ change of current also linearly correlates with the axonal length in axon (e) (red, dep field, R² = 0.6, P < 0.0001; blue, hyp field, R² = 0.36, P < 0.0057) but not in soma (f) (red, dep field, R² = 0.0004, P = 0.9298; blue, hyp field, R² = 0.07097, P = 0.2562). Whereas in the orthogonal field the ratio is not linearly correlated, neither in axon (g) (red, dep field, R² = 0.04, P = 0.45; blue, hyp field, R² = 0.04, P = 0.5) nor in soma (h) (red, dep field, R² = 0.0001, P = 0.968; blue, hyp field, R² = 0.2263, P = 0.0731). In parallel orientation AP amp ratio and mid-width ratio to Φ change of current are linearly correlated to terminal polarization, (i) (R² = 0.3985, P < 0.0001) and (j) (R² = 0.3003, P < 0.0001), respectively. (Parallel, N = 19, n = 19; orthogonal, N = 15, n = 15).

Figure 3.

Direct electric field alters the threshold latency and conduction velocity. Schematic of threshold latency measurement (a). If the distance from the soma to AIS is ‘L’ and the axon length is ‘2L’, the AP generated in AIS takes a similar time to reach the soma and axon bleb (upper figure in a). However, when the axon is lengthier, the AP takes a longer time to arrive at the bleb than the soma (lower figure in a), this delay is the threshold latency. Threshold latency is linearly correlated to the axonal length in both parallel (b) (red, dep, R² = 0.9787; black, no, R² = 0.9836; blue, hyp, R² = 0.984) and orthogonal (c) (red, dep, R² = 0.9915; black, no, R² = 0.9935; blue, hyp, R² = 0.9915) field (parallel, N = 19, n = 19; orthogonal, N = 15, n = 15). The slope of the curve in figure (b) and (c) is AP conduction velocity, indexed for axons longer than 100 μm, in (d), values are mean ± SEM.