Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jul 18;7(1):4744.
doi: 10.1038/s41598-017-03541-y.

Optical Read-out of Neural Activity in Mammalian Peripheral Axons: Calcium Signaling at Nodes of Ranvier

Affiliations
Free PMC article

Optical Read-out of Neural Activity in Mammalian Peripheral Axons: Calcium Signaling at Nodes of Ranvier

Arjun K Fontaine et al. Sci Rep. .
Free PMC article

Abstract

Current neural interface technologies have serious limitations for advanced prosthetic and therapeutic applications due primarily to their lack of specificity in neural communication. An optogenetic approach has the potential to provide single cell/axon resolution in a minimally invasive manner by optical interrogation of light-sensitive reporters and actuators. Given the aim of reading neural activity in the peripheral nervous system, this work has investigated an activity-dependent signaling mechanism in the peripheral nerve. We demonstrate action potential evoked calcium signals in mammalian tibial nerve axons using an in vitro mouse model with a dextran-conjugated fluorescent calcium indicator. Spatial and temporal dynamics of the signal are presented, including characterization of frequency-modulated amplitude. Pharmacological experiments implicate T-type CaV channels and sodium-calcium exchanger (NCX) as predominant mechanisms of calcium influx. This work shows the potential of using calcium-associated optical signals for neural activity read-out in peripheral nerve axons.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Single sweep action potential elicited calcium-responses at nodes of Ranvier (a) Response signal to 50 action potentials (100 Hz). Top panel images show an individual node of Ranvier before the action potential stimulus (i), during (ii), and after (iii) (arrows indicate the node location). Bottom panel is the quantitative optical signal produced by nodal image pixels (ROI is approximately 3 × 2 µm). Thick black bar indicates action potential stimulus. (b) Field of tibial nerve axons with at least six nodes of Ranvier yielding a calcium-coupled fluorescence change in response to a 1 s train of action potentials (100 Hz). Signal amplitudes among the six nodes range from 11–24%. Inset scale bars: 1 s and 5% signal change. (c) Longitudinal calcium signal propagation in an axon segment with node of Ranvier) (d) Activity dependent calcium fluorescence in response to a 50 action potential burst (100 Hz) recorded at incremental distances away from the node epicenter (signal trace colors correspond to ROIs in panel c). Plotted signals illustrate the latency in signal onset as well as reduced signal amplitude with distance from the node. (e) Peak normalized signal amplitude versus distance from node from five axon nodes. Yellow trace corresponds to the nodal data of panels c and d.
Figure 2
Figure 2
Calcium response to a single action potential and short bursts of action potentials. (a) Fluorescence response to a single action potential at a node of Ranvier, showing from left to right: five raw recordings, largest amplitude response trace, mean signal, and mean signal decay with exponential fit. (b) Mean fluorescence amplitude change with increasing number of action potentials in a stimulus train: a 100 Hz pulse train was applied for increasing durations to include more action potentials (1–80 APs). The signal amplitude begins to plateau as it reaches a relative steady state. Panel (left) shows mean amplitude measurements for three nodes acquired as the number of action potentials is ramped ‘up’ as well as ‘down’, showing no hysteresis. Panel (right): mean amplitude measurements are well fit by a double exponential function (R2 0.997).
Figure 3
Figure 3
Activity-dependent calcium signal amplitude is modulated by action potential frequency and train duration. (a) Calcium signals in response to a 2 s stimulus, and (b) to a 0.5 s stimulus at a range of action potential frequencies (c) Calcium-fluorescence signals modulated by action potential train duration. Constant frequency (125 Hz) action potential trains are applied at 0.5, 1, 1.5 and 2 s (colored bars, bottom) and the calcium response duration closely follows the stimulus while preserving steady amplitude. The average decay constant for the four signals is 205 ± 15 ms (d) Frequency-modulated calcium fluorescence traces with bars indicating mean steady-state amplitude. (e) Stimuli/recordings were performed while stepping action potential frequency up and down showing no significant hysteresis effect. (f) Signal amplitude versus frequency of nodes from three independent nerve samples as recorded in panel d, and their mean (g) illustrates linear dependence with a slope of 0.07% fluorescence/Hz. Data shown are from single sweep recordings with the exception of panel f in which each trace is the mean of the up and down sweeps, and panel g which contains the sample mean.
Figure 4
Figure 4
Comparison of frequency modulation slopes to nodal size (a) Frequency modulation slopes from nodes of six independent nerve sample. The nodal dF/Fo vs. frequency traces are the ramp up and down (two sweep) means with the exception of Node4 and Node6 which are single sweep traces. (b) Images of each axon node (scale bar 5 μm). (c) Slopes versus nodal diameter, juxtaparanodal diameter, and juxtaparanodal/nodal ratio do not reveal correlation between nodal size and slope.
Figure 5
Figure 5
Extracellular calcium removal causes abolishment of axonal calcium-fluorescence transients. (a) Nodal calcium response to 20 action potential bursts (100 Hz) in normal calcium, zero-calcium, and replete calcium. Single sweep recordings are shown. Scale bar: 1s & 3% fluorescence change. (b) Results are summarized for three independent experiments indicating unequal means between normal calcium and zero calcium states (p = 0.014) and between zero calcium and replete calcium states (p = 0.017) (ANOVA with Tukey-Kramer test for multiple comparisons, F = 11.04, df = 6). (c) Application of the ER Ca2+-ATPase blocker thapsigargin (6 μM) and the mitochondrial Na/Ca exchanger blocker CGP37157 (50 μM) did not significantly reduce activity-dependent nodal calcium amplitude.
Figure 6
Figure 6
Nodes in close proximity show differential response to blockers. (a) Signals of two adjacent nodes in the same mibefradil-treated nerve sample show the blockage of signal in node (1) and the persistence of signal in node (2). (b) Two adjacent nodal signals in a KBR7943-treated nerve similarly shows a differential drug effect, with nodal signal (1) blocking and nodal signal (2) remaining unblocked. Single sweep recordings are shown. Inset scale bars are 1s, and 3% dF/F o.
Figure 7
Figure 7
CaV channel and NCX blockers show differential inhibition of activity-dependent calcium response. (a) T-type CaV channel blocker mibefradil (25 μM) nearly abolishes the calcium signal in some nodes while other nodes are unblocked. (b) NCX inhibitors ORM10103 (15 μM) and (c) KBR7943 (15 μM) also show differential blockage: a set of nodal signals are significantly diminished while others show little or no blockage. (d) Co-application of mibefradil and KBR7943 shows similar results as seen for single drug application. (e) Activity-dependent calcium signals were not blocked or diminished by 10 μM nifedipine (L-type inhibitor). (Recordings from each data panel came from 2–4 mouse nerves).

Similar articles

See all similar articles

Cited by 4 articles

References

    1. Belter JT, Segil JL, Dollar AM, Weir RF. Mechanical design and performance specifications of anthropomorphic prosthetic hands: a review. J. Rehabil. Res. Dev. 2013;50:599–618. doi: 10.1682/JRRD.2011.10.0188. - DOI - PubMed
    1. Saikia A, et al. Recent advancements in prosthetic hand technology. J. Med. Eng. Technol. 2016;1902:1–10.
    1. Schiefer MA, Polasek KH, Triolo RJ, Pinault GCJ, Tyler DJ. Selective stimulation of the human femoral nerve with a flat interface nerve electrode. J. Neural Eng. 2010;7:26006. doi: 10.1088/1741-2560/7/2/026006. - DOI - PMC - PubMed
    1. Schiefer MA, et al. Selective activation of the human tibial and common peroneal nerves with a flat interface nerve electrode. J. Neural Eng. 2013;10:56006. doi: 10.1088/1741-2560/10/5/056006. - DOI - PMC - PubMed
    1. Sharma A, et al. Long term in vitro functional stability and recording longevity of fully integrated wireless neural interfaces based on the Utah Slant Electrode Array. J. Neural Eng. 2011;8:45004. doi: 10.1088/1741-2560/8/4/045004. - DOI - PMC - PubMed

Publication types

Substances

Feedback