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.2021 Sep 1;126(3):888-905.
doi: 10.1152/jn.00237.2021. Epub 2021 Aug 4.

Analog transmission of action potential fine structure in spiral ganglion axons

Affiliations

Analog transmission of action potential fine structure in spiral ganglion axons

Wenke Liu et al. J Neurophysiol..

Abstract

Action potential waveforms generated at the axon initial segment (AIS) are specialized between and within neuronal classes. But is the fine structure of each electrical event retained when transmitted along myelinated axons or is it rapidly and uniformly transmitted to be modified again at the axon terminal? To address this issue, action potential axonal transmission was evaluated in a class of primary sensory afferents that possess numerous types of voltage-gated ion channels underlying a complex repertoire of endogenous firing patterns. In addition to their signature intrinsic electrophysiological heterogeneity, spiral ganglion neurons are uniquely designed. The bipolar, myelinated somata of type I neurons are located within the conduction pathway, requiring that action potentials generated at the first heminode must be conducted through their electrically excitable membrane. We used this unusual axonal-like morphology to serve as a window into action potential transmission to compare locally evoked action potential profiles to those generated peripherally at their glutamatergic synaptic connections with hair cell receptors. These comparisons showed that the distinctively shaped somatic action potentials were highly correlated with the nodally generated, invading ones for each neuron. This result indicates that the fine structure of the action potential waveform is maintained axonally, thus supporting the concept that analog signaling is incorporated into each digitally transmitted action potential in the specialized primary auditory afferents.NEW & NOTEWORTHY Diverse action potential shapes and kinetics resulting from dynamic heterogeneity in spiral ganglion neurons are axonally transmitted as multiplexed signals that retain the fine structure of each distinctive waveform within a digital code.

Keywords: action potential waveform; analog transmission; endogenous heterogeneity; hybrid signaling; spiral ganglion neurons.

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Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

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Graphical abstract
Figure 1.
Figure 1.
Innervation of inner hair cells is retained in acute organ of Corti-spiral ganglion neuron (OC-SGN) preparations. Low (A) and high (B andC) magnification images of an acute OC-SGN preparation of aThy1-YFP cochlea acquired immediately after dissection without fixation. Both the cell somata (SGN) and peripheral terminals (asterisks) remain intact.C: fluorescent image inB merged with a bright field view. Low (D) and high (E) magnification images of a recording electrode patched on a P2 mid-turn neuron in the acute OC-SGN preparation.F–H: intracellular labeling of the same neuron shown inD andE using biocytin filling and streptavidin-mediated horseradish peroxide (HRP) reaction. Arrows indicate the cell soma; arrowheads indicate the synaptic terminal region.I–K: example images of a recorded neuron from the apical region of a P7 OC-SGN preparation. Fluorescent staining of the intracellular marker biocytin revealed the trajectory of peripheral process (I andJ) and the bipolar morphology (K).J: pseudo-colored image of the biocytin labeling inI (red) merged with a bright field view.K: high magnification image of the cell soma region of the neuron shown inI–J.L: afferent terminal of the neuron shown inI–J bifurcated at the base of its target inner hair cell. Red, biocytin, green, Myosin-7a. Arrows indicate the cell soma; arrowheads indicate the synaptic terminal region.M–P: colabeling of biocytin and Myosin-7a in four different neuronal terminals. Scale bar inK applies toK–P. IHCs, inner hair cells; OHC, outer hair cells.
Figure 2.
Figure 2.
Somatic recording of local and invading action potential activity in the organ of Corti-spiral ganglion neuron (OC-SGN) preparation.A: the relation between maximum number of spikes (APmax) and the temporal difference between the first and second spike (interspike interval, ISI) for 65 recordings obtained from the mid-cochlea ofP0–8 animals, in which spontaneous activity was observed. Recordings with both −80 mV and −60 mV holding potentials were included.B: exemplar traces for cells in the UA (top), RA (middle), and SA (bottom) categories. Subthreshold (red), suprathreshold (black) and APmax traces (gray) for both −60 mV and −80 mV holding potentials are shown. The same recordings (blue traces) also exhibited ongoing activity of invading action potentials (arrowhead) and EPSPs (arrows). Double arrowhead indicates an action potential with an abrupt inflection, below threshold, and before the onset of the constant current stimulus (vertical arrowhead).C: successive current clamp traces (left toright) from aP6 spiral ganglion neuron somatic recording.D: an exampleP6 recording showing bursting activity. Note that the action potential amplitude decay occurs when action potentials fire in rapid succession, similar to that observed in responses evoked locally with constant current injections.Insets show shaded portions of the traces at higher resolution.E: voltage clamp traces from aP0 spiral ganglion neuron showed complex waveforms of synaptic and action currents. EPSPs, excitatory postsynaptic potentials; RA, rapidly adapting; SA, slowly adapting; UA, unitary adapting.
Figure 3.
Figure 3.
Putative synaptic currents are 6,7-dinitroquinoxaline-2,3-dione (DNQX) sensitive.A: low (left) and high (right) magnification images of the recording pipette patched on a neuron in the basal turn of a P1 acute organ of Corti-spiral ganglion neuron (OC-SGN) preparation.B: current clamp recordings from a holding potential of −80 mV. Subthreshold (black), suprathreshold (dark gray), and AP max (light gray) traces are shown. An excitatory postsynaptic potential (EPSP) was observed on the baseline of one subthreshold trace (arrow).C: with square pulse depolarization from −80 mV holding potential to −70 mV ∼50 mV, inward and outward whole cell currents were revealed in voltage-clamp mode. Traces were leak subtracted with a hyperpolarization trace.D: examples of putative EPSCs and action currents recorded in voltage-clamp mode with −80 mV (black), −70 mV (dark gray) and −60 mV (light gray) holding potentials. Ongoing action currents (left trace) were observed from −70 mV holding potential.E: putative EPSCs and ongoing action currents were blocked by application of DNQX (final concentration ∼10 μM) in the bath.
Figure 4.
Figure 4.
Invading action potentials were blocked by tetrodotoxin (TTX).A: locally evoked action potentials, invading action potentials, and putative EPSPs were observed in the cell soma of a P5 spiral ganglion neuron in the acute organ of Corti-spiral ganglion neuron (OC-SGN) preparation recorded at a holding potential of −80 mV.B: after bath application of TTX (final concentration ∼10 µM), both the locally evoked action potentials and the invading action potentials were abolished.C: frequency histograms of putative EPSP amplitudes before and after TTX application. EPSP, excitatory postsynaptic potential.
Figure 5.
Figure 5.
Somatically observed excitatory postsynaptic potential (EPSPs) show intracellular and intercellular heterogeneity.A: monophasic (arrowhead) and multiphasic (double arrowhead) EPSPs observed in aP4.5 neuron. Action potentials were blocked by ∼10 µM TTX in the bath.B: three exemplar recordings showing different distributions of EPSP properties.Top: raw traces;middle: fitted traces normalized to the same amplitude;bottom: scatter plot of EPSP τ rise and EPSP amplitude with marginal histogram and Gaussian fits. The subgroup Gaussian mean values for both tau rise and amplitude are shown as dashed lines in the scatter plot. The three recordings shown here exhibited one (cyan), two (red), and three (blue) Gaussian components in the histogram of EPSP τ rise, as could be observed in the normalized fitted traces. EPSP amplitude of the same potentials was plotted against the tau rise, and the amplitude histogram and Gaussian fits are displayed on the y axis.C: comparison of somatic EPSP (C1 andC3) and subthreshold (C2 andC4) responses between two different cells (blue and red).C1: raw and average traces of EPSPs.C2: raw and average responses to just subthreshold level of current injection.C3: average traces normalized by amplitude.C4: subthreshold responses depolarized to comparable voltage levels.D: relation between averaged onset τ of evoked subthreshold traces and average EPSP τ rise and EPSP amplitude in eight different cells. The examples shown inC were color coded accordingly in the graph as blue and red symbols.
Figure 6.
Figure 6.
Diverse profiles of locally evoked and invading action potentials are highly correlated across different developmental stages. All recordings were performed from a holding potential of −60 mV and filtered at 1 kHz.A1–A5: features of locally evoked action potentials.A1: an exemplar recording showing a slowly adapting neuron with invading action potentials (arrows) when the cell was below threshold level (dotted line).A2: features of suprathreshold-evoked action potentials were consistent among traces from the same neuron. *Onset of stimulus.A3: traces inA2 normalized to the same amplitude and aligned by the time point at which they reached 10% dV/dtmax.A4, dV/dt plots aligned by 10% dV/dtmax.Inset: dV/dt plots on the original time scale ofA2, with the stimulus onset labeled by an asterisk.A5: phase plots of the action potentials as inA2.B1: examples of invading action potentials at −60 mV holding potential without net current injection.B2–B5: invading action potentials observed in the same cells as inA2–A5.B2: invading action potentials temporally aligned to 10% dV/dtmax.B3: traces inB2 normalized to the same amplitude and aligned by the time point they reached 10% dV/dtmax.B4–B5, dV/dt time courses and phase plots of the invading action potentials inB2. AP, action potential; HP, holding potential.
Figure 7.
Figure 7.
Features of locally evoked and invading action potentials are highly correlated.A–D: relation between averaged duration, amplitude, dv/dtmax, and dv/dtmin for locally evoked (x-axis) and invading action potentials (y-axis) from neurons shown in Fig. 6 at different developmental stages ranging fromP0 toP6 (n = 5 neuron recordings; holding potential = −60 mV).E–H: relation between the same parameters in P5–7 neurons shown in Fig. 8 with heterogeneous intrinsic kinetics (n = 8 neuron recordings; holding potential = −80 mV).n represents different neuron recordings made from multiple tissue isolates.
Figure 8.
Figure 8.
Two broad categories of action potential profiles inP5–7 spiral ganglion neurons. Locally evoked action potentials and invading action potentials were recorded from eight P5-7 neuronal somata at −80mV holding potential with 5 kHz filtering frequency. Recordings made from each cell were color-coded accordingly.A1: exemplar traces of evoked action potentials at the onset of the constant current pulse and concurrent invading action potentials (arrows). The afterhyperpolarization and afterdepolarization of the invading action potentials are indicated with double and single asterisks, respectively.A2: evoked action potentials plotted against time from the onset of current injection.A3: action potentials shown inA2 were aligned to the peak and averaged for each neuron. The averaged traces were then normalized and realigned to the time point of 10% dV/dt.Inset: a high magnification view of the action potential inflection.A4–A5: phase plots and dV/dt time courses of evoked action potentials showed two broad categories, indicated as fast and slow inA5.B1: exemplar traces of invading action potentials and EPSPs at −80 mV holding potential.B2; invading action potentials recorded from the same cells as inA, with the peaks of action potentials aligned in time.Inset: average traces of the peak-aligned action potentials in each neuron.B3: amplitude-normalized and peak-aligned average traces of the action potentials inB2.Inset: high magnification view of the action potential onset.B4–B5: phase plots and dV/dt time courses of the invading action potentials. Note, additional components in the fast (arrows) and slow groups (arrowheads) in the rising phase and falling phases, respectively.
Figure 9.
Figure 9.
Latency differences between neurons was amplified between locally evoked action potentials, whereas other aspects of the fine structure remained almost identical to invading action potential waveforms.A: averaged latency values ordered from fast to slow were plotted for each of the eight neuron recordings and fitted with an exponential function (dashed line,R2 = 0.927; error bars are SE between trials).B: averaged duration values ordered as inA, by contrast, were not graded. Locally evoked (filled diamonds) and invading (filled triangles) data were fitted with a two-point running average (dashed lines). Error bars are SE between trials.C: fast, fast-mid, and slow latency invading action potential dV/dt plots (purple, green, and red; Fig. 8A5) were normalized to maximum somatic action potential dV/dt for each of three example neurons (lt. purple, lt. green, orange; Fig. 8B5). The time course, indicated by the calibration bar, applies to all dV/dt plots. Arrows indicate the biphasic depolarization phase of the invading action potentials; arrowheads indicate the biphasic hyperpolarization phase of both action potential classes.
Figure 10.
Figure 10.
Action potentials conducted along axonal-like spiral ganglion neurons retain analog waveform kinetics and are shaped by activity-dependent and subthreshold modulation.A1 andA2: analog signals are a consistent component of both invading and locally evoked action potentials, respectively. Action potential amplitudes were normalized to expedite comparisons between kinetic components; gray shading highlights the onset/latency differences.B1 andB2: action potentials when fired in close succession display amplitude decay (gray shading) for both invading and locally evoked action potentials, respectively.C1: analog synaptic activity can be detected hundreds of microns from the source.C2 whether synaptic activity reaches threshold depends upon endogenous membrane properties of each neuron in addition to the level of activity. Double arrow indicates the difference between the peak of the excitatory postsynaptic potentials (EPSPs) and threshold of the recorded neuron.Inset: action potential amplitude is altered by subthreshold voltage. Unless otherwise indicated, calibration bars are 20 mV by 50 ms. ISC, initial segment central ISP, initial segment peripheral.
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