1.

Introduction

The detection of painful stimuli takes place at the nociceptive peripheral sensory nerve terminals in the skin or the viscera. These miniature structures (0.5 to $5\mu \mathrm{m}$ in diameter, Fig. 1) are pivotal for the detection of noxious stimuli.^1,2 Consequently, the comprehensive understanding of nociception and hence mechanisms of pathological pain can be achieved only by detailed exploration of nociceptive terminal function. However, not much is known about their properties because they are barely accessible by conventional electrophysiological methods. The saphenous skin–nerve preparation and ex vivo somatosensory system models developed by Reeh³ and Koerber and Woodbury,⁴ respectively, as well as other models using extracellular recordings from nerve fibers^5–7 monitor axonal activity following activation of the terminals, in different conditions, but not the activity at the terminals themselves. Electrophysiological recordings and ion imaging from terminal axons provided essential information about ${\mathrm{Na}}^{+}$ currents, which underlie action potential generation^8–10 and terminal ${\mathrm{Ca}}^{2+}$ signaling.^11,12 Nevertheless, spatial constraints of the electrophysiological method make it unsuitable for studying biophysical properties of signal propagation along the cylindrical terminal and distal axon. Conventional imaging assays overcome these spatial limitations; however, they are lacking in sufficient temporal resolution crucial for exploring ion dynamics underlying action potential generation and propagation. Many molecular and biophysical aspects of nociception have been described using nociceptor cell bodies, situated in dorsal root ganglions (DRG) or trigeminal ganglions.^13–16 The functional environment^17–19 and geometry of terminals differ from that of cell bodies, and likely, their passive membrane properties, density, and specific repertoire of transducer and voltage-gated channels. Hence, detailed characterization of ion signal onset, kinetics, and magnitude, along the terminals and terminal axons, have yet to be achieved, albeit being highly valuable for understanding nociceptive physiology. Consequently, the basic questions in nociceptive physiology, such as where action potentials are generated and how they propagate along nonmyelinated tiny axons in normal and pathological conditions, still remain obscure.

Fig. 1

GCaMP6s-based ${\mathrm{Ca}}^{2+}$ imaging of nociceptive-cultured DRG neurite terminals and distal processes. (a) Scheme depicting the experimental protocol (see Sec. 2). The cell bodies of DRG neurons were seeded in the middle compartment of the chamber and neurites were induced to grow to the lateral compartment. Upper inset, schematic representation of the neurite terminals and stimulating pipette. Lower inset, bright field image of a representative neurite terminal and puff pipette location. (b) Puff calibration experiment. Dispersion of a fluorophore (sulfarhodamine 101, $2\mu \mathrm{M}$) following a 500-ms puff (see Sec. 2) is presented in relation to the representative neurite terminal [rendered from (a), lower inset], as a reduction profile of the fluorescence ($F-F_0$, indicated by arbitrary units—au, and color coded, left) with distance from the puff pipette located at coordinates of $x$, $y=0$ (see Sec. 2). Coordinates of the neurite tip are indicated in parenthesis. Note that on the $x$-axis, the fluorescent intensity was reduced by about 50% when reaching a distance of $10\mu \mathrm{m}$ from the tip of the puff pipette. On the $y$-axis, intensity diminished almost completely after $15\mu \mathrm{m}$. (c) Left, fluorescent image of the neurite’s terminal process expressing GCaMP6s. Arrows indicate locations of ROIs from which the recordings shown to the right were performed. Right, representative traces of changes in ${[{\mathrm{Ca}}^{2+}]}_{\text{in}}$ following focal puff application of vehicle and 300-nM capsaicin (far right) measured every $1\mu \mathrm{m}$ along the terminal (“0” indicates the terminal tip), at a sample rate of 1000 fps. Note that the puff of vehicle does not elicit a response. Also note the spatial resolution, allowing analysis of ROIs situated $1\times 1\mu \mathrm{m}$ apart, along the terminal. Shadowed areas indicate time period of puff application of vehicle or capsaicin. Representative of 20 out of 20 ($20/20$) terminals.

Here, we introduce a new approach for high-temporal resolution optical recordings of ion dynamics from a single nociceptive neurite terminal in vitro. This approach allows us to monitor capsaicin-induced calcium (${\mathrm{Ca}}^{2+}$) and sodium (${\mathrm{Na}}^{+}$) dynamics at the terminal neurites and characterize their propagation along the distal neurites. Using this approach, we have demonstrated that the capsaicin-induced ${\mathrm{Ca}}^{2+}$ and ${\mathrm{Na}}^{+}$ signals change as they propagate from the terminal and along the neurite. We showed that capsaicin-induced calcium signals initiated at the terminals were not dependent on voltage-gated ${\mathrm{Na}}^{+}$ channels, whereas signals measured $15\mu \mathrm{m}$ away and onward, toward the cell body, were faster, stronger, and at least partially mediated by voltage-gated ${\mathrm{Na}}^{+}$ channels.

2.

Materials and Methods

3.

Results and Discussion

To study processing of nociceptive information directly at its most relevant location, with sufficient time and space resolution, we introduced an approach for ultrafast optical recording of cultured nociceptive processes. To that end, we used compartmented chambers [Fig. 1(a), see Sec. 2] that have been previously used to study neurites of DRG neurons.^21–24 In these chambers, DRG cell bodies are prompt to grow neurites, and terminal processes can be easily identified and accessed [Figs. 1(a) and 1(c)].

We selectively stimulated these neurite terminals with a calibrated focal puff application [Fig. 1(b)] of capsaicin, an agonist of the noxious heat-sensitive transient receptor potential cation channel subfamily V member 1 (TRPV1) channel, expressed by nociceptive neurons.²⁵ We placed the pipette, containing 300 nM capsaicin, about $10\mu \mathrm{m}$ from the terminal tip [Figs. 1(a) and 1(b)] because at this distance, the application of vehicle did not induce changes in GCaMP6s fluorescence [Fig. 1(c); $n=20$ terminals] or detectable movement of the processes. First, we calibrated the dispersion profile of the puffed substances [see Sec. 2; Fig. 1(b)]. Our data show that the predicted concentration at the terminal tip is $\sim 50\%$ of the initial value [Fig. 1(b)], i.e., in our conditions, the concentration of capsaicin at the terminal is close to the half maximal effective concentration (${\mathrm{EC}}_{50}$) of TRPV1 channels to capsaicin ($\sim 150\mathrm{nM}$²⁶). This is an estimate of the peak concentration during a 500-ms puff, which dissipates in the medium when the puff ends. Additionally, considering that the minimal concentration of capsaicin to activate TRPV1 channels is $\sim 30\mathrm{nM}$,²⁶ the prominent decrease in concentration along the $y$-axis [Fig. 1(b)] renders the concentration mostly ineffective once it reaches other parts of the fiber or adjacent terminals. Indeed, in all the performed recordings, we did not observe ${\mathrm{Ca}}^{2+}$ responses in neighboring terminal branches of the neurite.

We measured capsaicin-induced increase in ${[{\mathrm{Ca}}^{2+}]}_{\text{in}}$ at the neurite’s terminal process using the genetically encoded ${\mathrm{Ca}}^{2+}$ indicator GCaMP6slow [GCaMP6s, Fig. 1(c)]. We chose the slow variant of the GCaMP6 indicator due to its better signal-to-noise ratio and sensitivity (compared with other GCaMP6 variants²⁷), which enabled the detection of weak fluorescent emission signals from fine neurite processes. To study transducer channel- and action potential-mediated changes in terminal ${[{\mathrm{Ca}}^{2+}]}_{\text{in}}$, we optically recorded changes in terminal GCaMP6s fluorescent intensity at a sampling rate of 1000 fps. As it has been shown that the half width of the action potential recorded from the distal neurite of nociceptive neurons is $\sim 2.5\mathrm{ms}$,⁸ this sampling rate should be sufficient to detect ${\mathrm{Ca}}^{2+}$ reflections of single action potentials. We achieved this using a $60\times$ oil 1.4NA Plan Apo objective, in combination with a fast acquisition back-illuminated $80\times 80\text{pixel}$ CCD camera (see Sec. 2). Puff application of capsaicin, but not vehicle [Fig. 1(c)], led to prominent increase in GCaMP6s fluorescence, detectable at a spatial resolution of $1\times 1\mu \mathrm{m}$ along the terminal tip [Fig. 1(c)].

Next, utilizing the high functional spatial resolution of our approach, which allows precise tracing of signal propagation along the terminal, we measured the amplitude, time of onset, and rise rate of capsaicin-induced ${\mathrm{Ca}}^{2+}$ signals along the terminal and terminal neurite [Figs. 2(b), 2(d)–2(f), black]. It is noteworthy that due to short acquisition time (see Sec. 2), we could not explore the mechanisms underlying the decay phase of the capsaicin-induced response at the neurite’s processes and, therefore, we focused our efforts on the rising phase of the response.

We analyzed the difference in time of onset between two adjacent segments, consecutively for all adjacent segments (see Sec. 2), which reflects the time it took the signal to travel along a segment. We found that the signal traveled along $m_1$ for $58.5\pm 10\mathrm{ms}$, $m_2$ for $62.2\pm 10\mathrm{ms}$, and $m_3$ for $15.6\pm 5\mathrm{ms}$ ($n=4$). After segment $m_3$, time of onset difference between two ROIs was $3.2\pm 2\mathrm{ms}$ [segments $m_3$ to $m_8$; Fig. 2(d), black, $n=4$]. Using these data, we calculated the fluorescent propagation velocity of capsaicin-induced ${\mathrm{Ca}}^{2+}$ signals between segments (see Sec. 2) and demonstrated that the signal propagated along $m_1$ with a velocity of $97.2\pm 23\mathrm{nm}/\mathrm{ms}$, $m_2$ with $85.2\pm 11\mathrm{nm}/\mathrm{ms}$, and $m_3$ with $382\pm 95\mathrm{nm}/\mathrm{ms}$ ($n=4$). Signals propagated between segments $m_1$ to $m_3$ did not vary in their response peak amplitude or maximum rise rate [$\max (\mathrm{d}F/\mathrm{d}t)$; Figs. 2(b), 2(e), 2(f) black]. After $\sim 15\mu \mathrm{m}$ (segment $m_3$), the fluorescence propagation velocity increased significantly and reached a peak of $870\pm 300\mathrm{nm}/\mathrm{ms}$ at $m_4$ ($\sim 20\mu \mathrm{m}$), and the speed of propagation between $5\mu \mathrm{m}$ segments remained similar along the rest of the measured terminal ($p=0.47$, one-way ANOVA, $n=4$). It is noteworthy that the values for propagation velocity that we have measured represent the propagation of fluorescent signals along the process and not the electrical conductance. The propagation of GCaMP6s fluorescence is dependent on electrical conductance; however, there are many additional factors that are involved, such as the ${\mathrm{Ca}}^{2+}$ channels’ and indicator kinetics (see also Ref. 28).

Fig. 2

Propagation of capsaicin-induced ${\mathrm{Ca}}^{2+}$ transients along the neurite terminal. (a) Fluorescence image of representative nociceptive terminals expressing GCaMP6s with illustration of pipette location and with the arrows indicating the locations of ROIs from which the measurements shown in (b) and (c) were performed. (b, c) Representative traces of capsaicin-induced changes in ${[{\mathrm{Ca}}^{2+}]}_{\text{in}}$ measured every $5\mu \mathrm{m}$ at distances shown in left, from the terminal tip before (b) and 10 min after (c) application of $10\text{-}\mu \mathrm{M}$ TTX together with $10\text{-}\mu M$ A803467. Changes in fluorescents were measured at 1000 fps. Note the substantial increase in signal amplitude after $15\mu \mathrm{m}$, which was abolished by applying TTX and A803467. Insets: Expanded timescale of the traces presented in (b) and (c) emphasizing the signal onset. Arrows indicate calculated time of signal onset (see Sec. 2). Note that the timescale of the inset for (b) is further expanded to better illustrate the differences in time of signal onset along the neurite in the control conditions. (d) Time of onset of capsaicin-induced ${\mathrm{Ca}}^{2+}$ signals measured from terminal tip (point “0”) in control conditions (SES, black squares) and after bath application of TTX together with A803467 (red circles), plotted versus distance from the terminal tip. Note much smaller changes in time of onset after $15\mu \mathrm{m}$ in control conditions. Application of TTX together with A803467 significantly increased the difference in time of onset after $15\mu \mathrm{m}$. ns, not significant; *$p<0.05$, **$p<0.01$, ***$p<0.001$; black letters and symbols, comparison between values measured at each location in control condition, $n=4$, one-way ANOVA with posthoc Bonferroni; orange letters and symbols, comparison between “SES” and “TTX + A803467;” $n=2$; two-way ANOVA, with posthoc Bonferroni. (e) Peak amplitudes of capsaicin-induced changes in fluorescence, measured every $5\mu \mathrm{m}$ and plotted as a function of distance from terminal tip, in control conditions (SES, black) and after (red) application of TTX together with A803467. Note that in control conditions, signal amplitude significantly increased after $15\mu \mathrm{m}$. TTX together with A803467 did not affect the signal measured at the first $15\mu \mathrm{m}$, but significantly reduced the signal amplitude thereafter. Symbols and statistics as in (d). (f) Maximum rate of rise of ${\mathrm{Ca}}^{2+}$ transients, $\max (\mathrm{d}F/\mathrm{d}t)$, at the different locations along the neurite terminal in control condition (SES, black), and TTX together with A803467 (red) plotted as a function of distance from the terminal tip. Note that in control conditions, the rise rate significantly increased at $\sim 15\mu \mathrm{m}$ and plateaued afterwards. Application of TTX together with A803467 prevented the increase in rise rate. Statistics as in (d).

Remarkably, at $15\mu \mathrm{m}$ (segment $m_3$), a two-phase response appeared [Fig. 2(b)]. While the first phase was similar to the one observed at the terminal tip ($p=0.35$ for rise rate; $p=0.58$ for the amplitude, one-way ANOVA with posthoc Bonferroni), the second phase possessed a significantly larger peak amplitude and maximum rise rate [$p<0.05$, one-way ANOVA, $n=6$ terminals; Figs. 2(e), 2(f) black], reaching a plateau of about 3.5 times larger response peak amplitude and 7 times larger rise rate at the maximum. This large and fast component was completely abolished following application of a combination of TTX and A803467 [Figs. 2(c), 2(d)–2(f) red] to block TTX-s- and Na(v)1.8-mediated TTX-r ${\mathrm{Na}}^{+}$ channels,²⁹ which are expressed by nociceptive terminals.⁸ Moreover, blocking Na(v) channels delayed the time of onset [Fig. 2(d), red], reduced the fluorescence propagation velocity to $57\pm 11\mathrm{nm}/\mathrm{ms}$, and decreased the peak amplitude and maximal rise rate [Figs. 2(e) and 2(f), red] of capsaicin-evoked responses recorded along the neurite after $\sim 15\mu \mathrm{m}$, such that the values become similar to those recorded at the first $15\mu \mathrm{m}$ [Figs. 2(e) and 2(f)]. These data suggest that the increase in ${[{\mathrm{Ca}}^{2+}]}_{\text{in}}$ beyond the $\sim 15\mu \mathrm{m}$ is partially dependent on activation of voltage-gated ${\mathrm{Na}}^{+}$ channels.

We further supported the latter notion by directly measuring capsaicin-induced ${\mathrm{Na}}^{+}$ fluxes in the neurite’s terminal process using the ${\mathrm{Na}}^{+}$ indicator SBFI-AM. Imaging of ${\mathrm{Na}}^{+}$ fluxes at different locations along the terminal revealed that application of capsaicin induced a small transient increase in ${[{\mathrm{Na}}^{+}]}_{\text{in}}$, in the neurite’s terminal tip and a larger step-like ${\mathrm{Na}}^{+}$ signals at a distance from the tip [Fig. 3(b), $n=3$].

Fig. 3

Capsaicin-induced ${\mathrm{Na}}^{+}$ transients vary along the neurite terminal. (a) Representative image of a nociceptive terminal process loaded with SBFI-AM. Note at the lower right corner, the illustration of pipette location. Arrows indicating locations of ROIs from which the recordings shown in (b) were performed. (b) Representative traces of changes in ${[{\mathrm{Na}}^{+}]}_{\text{in}}$ measured at the terminal tip (gray) and $20\mu \mathrm{m}$ from the terminal tip (blue), with a sampling rate of 125 fps. Note transient increase in ${[{\mathrm{Na}}^{+}]}_{\text{in}}$ at the tip and step-like continuous response at $20\mu \mathrm{m}$. Dotted lines indicate baseline fluorescence. Traces indicating decrease in florescence emission after excitation with 380 nm. Shadowed area indicates time period of puff application of capsaicin.

It is noteworthy to mention that TRPV1 channels are also permeable to ${\mathrm{Na}}^{+}$.³⁰ Thus, the signal at the terminal tip may reflect ${\mathrm{Na}}^{+}$ influx via active TRPV1 channels. The fast and step-like nature of the signals recorded away from the neurite may underlie burst-like firing of the action potentials.³¹ From a methodological point of view, this in vitro approach, which is based on a distinctive compartment containing only neurite terminals, is currently the only approach allowing direct study of terminal ${\mathrm{Na}}^{+}$ dynamics. It is advantageous over an in vivo assay as the usage of AM-based dyes in vivo will likely produce a strong background noise due to AM dye loading of not only the terminals but also nonneuronal cells in the imaged area. The lack of genetically encoded ${\mathrm{Na}}^{+}$ indicators for specific measurements of ${\mathrm{Na}}^{+}$ flux in distinct cell types render measurements from small structures in vivo or ex vitro extremely challenging. Importantly, low initial fluorescence intensity of SBFI-AM in the neurite’s terminal, due to multiple factors (e.g., relatively low passage of SBFI excitation wave length by the objective and intraterminal loading) allowed us to sample capsaicin-induced intraterminal changes of ${\mathrm{Na}}^{+}$, only at 125 fps.

Altogether our data suggest that the nociceptive neurite’s terminals and processes, in terms of ${\mathrm{Ca}}^{2+}$ dynamics and its dependence on ${\mathrm{Na}}^{+}$ channel blockers, are composed of two functionally separated zones. Our results imply that signal generation and propagation in the first $15\mu \mathrm{m}$ is not mediated by voltage-gated ${\mathrm{Na}}^{+}$ channel-induced depolarization. This ${\mathrm{Ca}}^{2+}$ signaling could be mediated by ${\mathrm{Ca}}^{2+}$-permeable TRPV1 channels, low threshold voltage-gated ${\mathrm{Ca}}^{2+}$ channels, ${\mathrm{Ca}}^{2+}$-induced ${\mathrm{Ca}}^{2+}$ release, and capsaicin-mediated ${\mathrm{Ca}}^{2+}$ release from TRPV1-expressing internal stores.³² Given the focality of our application and the susceptibility of the late response to ${\mathrm{Na}}^{+}$ channel blockers, TRPV1-mediated ${\mathrm{Ca}}^{2+}$ release from internal stores has a lesser contribution to the capsaicin-induced ${\mathrm{Ca}}^{2+}$ response, which we observed after $15\mu \mathrm{m}$. Hence, as suggested by our data, signal propagation beyond the first $15\mu \mathrm{m}$ is largely driven by voltage-gated ${\mathrm{Na}}^{+}$ channels leading to ${\mathrm{Ca}}^{2+}$ elevation probably via voltage-gated ${\mathrm{Ca}}^{2+}$ channels and ${\mathrm{Ca}}^{2+}$-induced ${\mathrm{Ca}}^{2+}$ release. Interestingly, the maximal propagation velocity, we have measured from terminal neurites of nociceptive processes, was substantially smaller than those measured using single fiber recordings from mice sciatic-tibial nerve in vivo.⁶ This discrepancy could result from the differences between electrical conductance and fluorescent propagation, as stated above. Indeed, the velocities of fluorescence propagation that we and others²⁸ have measured in cultured neurites are slower than that measured for propagation of electrical signals.⁶

In this study, we have analyzed the activity of individual terminal neurites, which are easily identified and traced. It is difficult, however, to trace and record individual neurite activity along its full length because proximal neurites in culture create dense bundles of neurites.^20,24 Utilization of our approach on individually labeled neurons, via micropipette injection of a spectrally different dye, may overcome this difficulty and provide essential information about the calcium dynamics along different compartments of peripheral processes.

In conclusion, we have introduced an approach for studying nociceptive terminal processes by bringing together and utilizing fast and sensitive optical recordings of ion dynamics from easily accessible cultured neurite terminals. Altogether, the data shown here demonstrate that our methodology gains in sensitivity and temporal acquisition capabilities, compared with standard imaging techniques, allowing fast optical recordings of weak signals from minute nociceptive terminal processes. Moreover, our approach gains in functional spatial resolution, compared with single electrode electrophysiological approaches, permitting characterization of signal propagations along the terminal and terminal neurites by sampling the signals in action potential relevant time resolution. Thus, the ultrafast and high-resolution optical recording technique described here provides the only tool for a detailed study of a nociceptive terminal functional molecular network, which underlies noxious stimuli detection and transmission in normal and pathological conditions. This could advance the very much needed knowledge for understanding pain physiology and pathophysiology. Moreover, this platform can be utilized to enable studying the terminals of other neuronal types, such as other primary sensory neuron terminals.