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, 594 (1), 39-57

Stimulation-induced Ca(2+) Influx at Nodes of Ranvier in Mouse Peripheral Motor Axons

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Stimulation-induced Ca(2+) Influx at Nodes of Ranvier in Mouse Peripheral Motor Axons

Zhongsheng Zhang et al. J Physiol.

Abstract

In peripheral myelinated axons of mammalian spinal motor neurons, Ca(2+) influx was thought to occur only in pathological conditions such as ischaemia. Using Ca(2+) imaging in mouse large motor axons, we find that physiological stimulation with trains of action potentials transiently elevates axoplasmic [C(2+)] around nodes of Ranvier. These stimulation-induced [Ca(2+)] elevations require Ca(2+) influx, and are partially reduced by blocking T-type Ca(2+) channels (e.g. mibefradil) and by blocking the Na(+)/Ca(2+) exchanger (NCX), suggesting an important contribution of Ca(2+) influx via reverse-mode NCX activity. Acute disruption of paranodal myelin dramatically increases stimulation-induced [Ca(2+)] elevations around nodes by allowing activation of sub-myelin L-type (nimodipine-sensitive) Ca(2+) channels. The Ca(2+) that enters myelinated motor axons during normal activity is likely to contribute to several signalling pathways; the larger Ca(2+) influx that occurs following demyelination may contribute to the axonal degeneration that occurs in peripheral demyelinating diseases. Activity-dependent Ca(2+) signalling is well established for somata and terminals of mammalian spinal motor neurons, but not for their axons. Imaging of an intra-axonally injected fluorescent [Ca(2+)] indicator revealed that during repetitive action potential stimulation, [Ca(2+)] elevations localized to nodal regions occurred in mouse motor axons from ventral roots, phrenic nerve and intramuscular branches. These [Ca(2+)] elevations (∼ 0.1 μm with stimulation at 50 Hz, 10 s) were blocked by removal of Ca(2+) from the extracellular solution. Effects of pharmacological blockers indicated contributions from both T-type Ca(2+) channels and reverse mode Na(+)/Ca(2+) exchange (NCX). Acute disruption of paranodal myelin (by stretch or lysophosphatidylcholine) increased the stimulation-induced [Ca(2+)] elevations, which now included a prominent contribution from L-type Ca(2+) channels. These results suggest that the peri-nodal axolemma of motor axons includes multiple pathways for stimulation-induced Ca(2+) influx, some active in normally-myelinated axons (T-type channels, NCX), others active only when exposed by myelin disruption (L-type channels). The modest axoplasmic peri-nodal [Ca(2+)] elevations measured in intact motor axons might mediate local responses to axonal activation. The larger [Ca(2+) ] elevations measured after myelin disruption might, over time, contribute to the axonal degeneration observed in peripheral demyelinating neuropathies.

Figures

Figure 1
Figure 1. Stimulation‐induced elevations of [Ca2+] originate at nodes of Ranvier
All records are from the phrenic nerve axon illustrated in A–C, which was iontophoretically injected with the Ca2+ indicator OG‐1 and stimulated at 50 Hz for 5 s. The low magnification field in Fig. 1 B shows a fluorescence image of the injected axon (green) superimposed on a phase image (grey) of the phrenic nerve trunk. C shows 3 fluorescence micrographs (coded as F/F rest) of this axon before, during and after stimulation. In the pseudocoloured images of C and D, blue indicates low resting [Ca2+] and warmer colours indicate elevated [Ca2+]. The stimulated image in C shows two areas of elevated [Ca2+], both of which were identified as nodes in the phase (greyscale) and fluorescence (green) images shown in A and B. Distance calibration bar in B also applies to C. Records in D–G came from node no. 1; each plot is shown adjacent to a greyscale fluorescence image defining nodal and paranodal regions. D, pseudocoloured image shows the time course of [Ca2+] changes (measured as F/F rest) before, during and after the stimulus train (duration indicated by dashed lines) along the length of the axon (vertical axis indicates location, horizontal axis indicates time). E, superimposed time courses of stimulation‐induced F/F rest changes at different distances from the node. Coloured bars in the fluorescence image indicate the location at which each F/F rest trace was measured. F and G, spatial plots of F/F rest for the indicated times (0.2–5 s) after the onset (F) and termination (G) of stimulation. Recovery curves in G began at the peak of the stimulation response shown in F.
Figure 2
Figure 2. Responses to stimulation averaged for 11 phrenic nerve axons
A, average change in F/F rest and Δ[Ca2+] as a function of distance, calculated for the 1st and 10th second of stimulation trains at 50 Hz. Phase (greyscale) and coloured fluorescence images indicate the nodal (N) and internodal (IN) locations corresponding to the plotted distances. B, averaged time course of changes in F/F rest and Δ[Ca2+] for nodes and locations 10 and 20 μm distant from nodes. Shading indicates duration of stimulation. Bars indicate ± SEM. Equations used to convert F/F rest measurements into estimates of Δ[Ca2+] are given in Methods.
Figure 3
Figure 3. Frequency dependence of responses before and after stimulation in a phrenic axon
Each stimulus train consisted of 500 stimuli administered at the indicated frequency (12.5 Hz for 40 s, 25 Hz for 20 s, 50 Hz for 10 s and 100 Hz for 5 s). A, time course of F/F rest changes for 100 Hz and 25 Hz stimulation measured at the node and flanking regions of the axon illustrated in the fluorescence image in B. B, superimposed spatial profiles of F/F rest changes measured during (upper) and after (lower) stimulation at the indicated frequencies. Traces in A and B are averages of 3–4 repetitions at each frequency (repeated cycles of 12.5, 25, 50, 100 Hz). C, mean (± SEM) change in nodal F/F rest during (filled circles) and after (open circles) stimulation at the indicated frequencies (SEM was usually smaller than point diameter). In B, each trace labelled ‘during stimulation’ was generated by averaging spatial profiles (acquired each second) at times when nodal F/F rest stabilized at a plateau level. These times are the last 38 s of the 12.5 Hz train; last 18 s of 25 Hz train; last 8 s of 50 Hz train; last 3 s of 100 Hz train. Each trace labelled ‘after end of stimulation’ was generated by averaging spatial profiles over a period during which nodal F/F rest was relatively stable, 5–20 s after the end of stimulation, for all trains.
Figure 4
Figure 4. Stimulation‐induced elevations of nodal [Ca2+] in a ventral root axon (A) and in a motor axon coursing through the levator auris muscle (B)
Both A and B show fluorescence images of an OG‐1‐filled axon (a), an elevation of nodal [Ca2+] during stimulation at 50 Hz, coded as F/F rest (b), the spatial extent of the Δ[Ca2+] response (c), and the time course of the response at nodal and flanking internodal regions (d). The intramuscular axon had 2 nodes, each at a branch point, and a heminode (HN) at the junction between the myelinated axon and the motor nerve terminal (MNT). Bb–d, also shows the much larger, prolonged [Ca2+] elevation averaged over the motor nerve terminal. For analysis the intramuscular axon (dotted lines in Bb inset) was digitally straightened to yield the image shown in Bc, as described in Methods. The spatial distributions in Ab and c and Bb and c were taken at the 5th and 10th second after beginning of stimulation at 50 Hz, respectively.
Figure 5
Figure 5. Stimulation‐induced elevations in nodal [Ca2+] are reversibly abolished by removal of bath [Ca2+], and are inhibited by blockers of T‐type Ca2+ channels and NCX
A–E, each panel illustrates a control response to stimulation (50 Hz, 10 s), and a response to the indicated treatment/drug or drug combination. All recordings from phrenic nerve, with multiple traces superimposed in each record. Drug targets are indicated in F. A, reversible inhibition of [Ca2+] response after removal of extracellular Ca2+ (replaced by equimolar Mg2+). B, lack of effect of 30 and 60 min exposure to CPA. C, lack of effect of nimodipine and of nimodipine + agatoxin. D, partial inhibition with mibefradil. E, partial inhibition with KB‐R7943, and nearly complete inhibition with KB‐R7943 + mibefradil combination. F, summary of the mean (± SEM) results of similar experiments. Each drug/treatment response (ΔF/F rest) was normalized to control ΔF/F rest; 3–13 axons per group. *Significant difference from control at P < 0.001, after correction for multiple comparisons to control. Concentrations: CPA (10 μm), nimodipine (4 μm), ω‐agatoxin IVA (AgTx, 1 μm), ω‐conotoxin GVIA (CgTx, 3 μm), mibefradil (2 μm), ML218 (6 μm), SN‐6 (10 μm), KB‐R7943 (10 μm).
Figure 6
Figure 6. Treatments that elevate intra‐axonal [Na+] increase the stimulation‐induced nodal [Ca2+] elevations in phrenic axons
A, diagrams illustrate forward operation of NCX at the resting potential (left), and reverse‐mode NCX operation during action potential depolarization combined with 1 of the 4 listed strategies for elevating intra‐axonal [Na+] (right). B (left to right), control response to stimulation, abolition of response in veratridine (10 μm), enhanced response during veratridine washout, and comparison of control and veratridine wash responses. C, control response to 10 s train (no. 1), response during prolonged conditioning train (no. 2), response to repeated 10 s train (no. 3), and comparison of responses to train no. 1 and no. 3. In the graph below, each line connects the response to train no. 1 and no. 3 measured in each of 4 separate axons; points labelled with upright triangles (∆) correspond to the axon depicted in C. D, effects of blocking Na+/K+‐ATPase activity with ouabain (0.2 mm, upper traces) or by removing and later restoring extracellular [K+] (lower traces). Each record plots sequential responses to 50 Hz stimulation trains repeated at 10 min intervals. In B–D fluorescence (F) is plotted in arbitrary fluorescence units (afu) to indicate changes in both resting and stimulated fluorescence.
Figure 7
Figure 7. BAYK‐8644, which enhances activation of and influx through L‐type Ca2+ channels, increases stimulation‐induced [Ca2+] elevations in phrenic axons
A, superimposed time courses of F/F rest responses to 50 Hz stimulus train, recorded in node (centre) and flanking internodal regions in control solution, after addition of BAYK‐8644 (BAY‐K, 2 μm), and after addition of nimodipine to the BAYK‐8644 solution. B, spatial extent of F/F rest elevation in control, BAY‐K and Bay‐K + nimodipine. Records in A and B came from the axon illustrated in B; records in A plot mean ± SEM of 4 responses. C, ΔF/F rest (mean ± SEM) of peak nodal responses to stimulation in the indicated solutions (n = 5 axons, BAY‐K response significantly different from control at P < 0.01). Spatial profile curves in B were generated by averaging spatial profiles (acquired each second) at times when nodal F/F rest stabilized at a plateau level, between the 5th and 10th second of stimulation at 50 Hz.
Figure 8
Figure 8. Paranodal demyelination increases stimulation‐induced [Ca2+] responses by enabling activation of L‐type Ca2+ channels
A, effects of brief exposure to LPC. Traces plot time courses and spatial extents of F/F rest elevations recorded around the illustrated node before and after exposure to LPC. Bar graph summarizes results of nodal responses in 5 LPC‐treated axons. B, similar recordings in axons subjected to brief mechanical stretch. C and D, time courses and spatial extents of the F/F rest responses recorded in an LPC‐treated axon (C) or a stretched axon (D) before and following application of nimodipine. Other aspects of the format are similar to that in A. P < 0.01 in A, P < 0.05 in B, P < 0.05 in C, P < 0.01 in D. Spatial profile curves were generated by averaging spatial profiles (acquired each second) at times when nodal F/F rest stabilized at a plateau level, between the 5th and 10th second of stimulation at 50 Hz.
Figure 9
Figure 9. Diagrams summarizing the dominant pathways used for stimulation‐induced Ca2+ entry into motor neuron soma, motor axon and motor terminal (A) and the appearance of Ca2+ entry via L‐type channels in axons following acute paranodal demyelination (B)
Diagrams in B show nodes at rest (left) and during repetitive stimulation (right), with a normal node at the top (intact myelin) and a disrupted node at the bottom (paranodal demyelination). In all cases T‐type channels in nodal/paranodal axolemma are activated during stimulation, and the direction of NCX‐mediated Ca2+ transport shifts from extrusion in the resting axon to entry during stimulation. With intact myelin the depolarization of the axolemma during action potentials (shades of red) is largely restricted to the node; the much smaller depolarization in peri‐nodal regions (under myelin) is sufficient to activate LVA T‐type channels but not HVA L‐type channels. Following acute paranodal demyelination, action potentials produce greater depolarization of peri‐nodal axolemma, sufficient to activate L‐type channels. For simplicity, peri‐nodal channels were drawn only on the right side of each node. Information in A concerning soma and initial segment comes from published studies (see Discussion). The exact distribution of T‐type channels and NCX between nodal and peri‐nodal axolemma is not known, and possible contributions of reverse‐mode NCX‐mediated transport to Ca2+ entry into soma and terminal have (to our knowledge) not been determined.

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