Using ASAP4.4-Kv for in vivo DRG voltage imaging
For in vivo DRG voltage imaging, we intrathecally injected adeno-associated viruses (AAVs) encoding ASAP4.4-Kv into spinal cord to allow for expression in DRG neurons. At 5–7 weeks after injection, in vivo single photon confocal imaging experiments were performed on the right lumbar (L5) DRG, which innervates parts of right hindpaw, leg, and back of the mouse. Fluorescent signals from ASAP4.4-Kv were acquired by confocal microscopy in frame mode to capture the entire population of L5 DRG neurons. We verified transduction of ASAP4.4-Kv virus into DRG neurons by imaging ASAP4.4 fluorescence in DRG neurons. The basal ASAP4.4-Kv green fluorescence intensity was relatively low under in vivo conditions; however, inflammation in hindpaw caused by complete Freund’s adjuvant (CFA) injection or chronic constriction injury22 of sciatic nerves (SNs) yielded a stronger ASAP4.4-Kv fluorescent signal (Fig. 1a and Supplementary Fig. 1a, b). The results showed that ASAP4.4-Kv can be sparsely but highly expressed in DRG neurons in vivo, and can dynamically respond to voltage in the physiological range (Supplementary Fig. 2). These are essential properties for carrying out the functional analysis at the cellular level in vivo.
We noticed that many DRG neurons showing brightly fluorescent ASAP4.4 were in close proximity to each other, more prominent in CFA and SN-CCI animals (Supplementary Fig. 1c), implying that “cross-excitation23” or “coupled activation4” may arise in proximal parts of the in vivo primary sensory neurons. This phenomenon led us to explore the utility of voltage indicators to reveal neuronal crosstalk within the peripheral nervous system of live animals.
Electrical coupling synchronization between adjacent DRG neurons revealed by ASAP4.4
DRG neurons are covered with satellite glial cells (SGCs), grouping with or without SGCs in between neighboring neurons24. Under normal circumstances, DRG neurons are loosely connected to each other or to SGCs. In contrast, extensive dye transfer and electrical coupling between adjacent neurons are often observed in many pain conditions, including inflammation in mouse hindpaw and sciatic nerve injury25, 26, which is attributed to gap junctions present in both DRG neurons and the surrounding SGCs. As a consequence of electrical coupling by gap junction, DRG neurons exhibit coupled activation following peripheral tissue injury examined by Ca2+ imaging in DRG of live mice4.
We thus attempted to simultaneously record electrical activity between two adjacent DRG neurons using ASAP4.4. We randomly selected pairs of adjacent DRG neurons in different regions of the DRG, and performed a single-line scan at about 1.1 kHz across the membrane regions of the two neuronal cells (Fig. 1a–c, images). We analyzed paired data sets from naïve, CFA, or SN-CCI animals by quantification of fluorescence intensity changes in scanned areas of individual adjacent cells. In naïve mice, very few DRG neurons displayed rhythmic spontaneous subthreshold voltage fluctuations, and no temporal cell-to-cell coherence or synchronization of voltage signals in neuronal membranes was observed (Fig. 1a). Under the context of inflammation or nerve injury, subthreshold voltage fluctuations were readily detectable in vivo (Fig. 1b, c), with approximately 7-fold increase in average area under the curve (AUC) of ASAP4.4-Kv fluorescent signal intensity (Fig. 1d). Strikingly, around 6% of recording neuronal pairs exhibited spontaneous suprathreshold (spiking) activity and strong coincident voltage changes in the range of ten to hundreds of milliseconds, regardless of activity patterns (Fig. 1b, c), whereas gap junction blocker, carbenoxolone (CBX), significantly reduced cell-to-cell electrical synchronization (Fig. 1b). Our results indicate that tissue injury increased cell-to-cell connectivity and network communication between DRG neurons leading to enhanced synchronization in DRG neuronal networks, and eventually to better integration and summation of somatosensory signals. To the best of our knowledge, such electrically synchronous neuronal events between cells in the peripheral sensory system in vivo have not been previously described.
To determine whether electrically synchronous events corresponded to global neuronal activity, we included in vivo DRG Ca2+ imaging of neuronal populations using Pirt-GCaMP3 mice, in which the GECI GCaMP3 was exclusively expressed in primary sensory neurons under the control of the Pirt promoter27. Using Pirt-GCaMP3 Ca2+ imaging, we could simultaneously monitor neuronal activity of the entire population of DRG neurons4. We imaged the entire DRG at ~6.4 to 7.9 s/frame and found that spontaneous activity was rarely seen in naïve animals (1–3 neurons/DRG), but in the presence of inflammation or nerve injury, increased spontaneous neuronal activity was observed (>10 neurons/DRG) (Supplementary Fig. 3a). This Ca2+ activity could represent either sporadic Ca2+ oscillations or steady-state high Ca2+(Supplementary Fig. 3b). To this point, however, no synchronized spontaneous activity was observed in GCaMP3 signals. In comparing voltage dynamics seen by ASAP4.4-Kv imaging with Ca2+ signals seen by GCaMP3 imaging, we found that ASAP4.4-Kv imaging preserved fast temporal signal information, which GCaMP3 imaging failed to convey. ASAP4.4-Kv detected numerously dynamic membrane voltage signal changes associated with inflammation or nerve injury but GCaMP3 did not (Fig. 1d and Supplementary Fig. 3c). In contrast, GCaMP3 Ca2+ signals reflected an increasing number of spontaneously activated neurons in the entire DRG after inflammation or nerve injury (Supplementary Fig. 1d,e).
Noninvasive optical readout of different afferent subtypes in vivo with ASAP4.4
Primary sensory neurons diverge in function as they express their own unique receptors and ion channels. Classically, DRG neurons are categorized into three subtypes based on somatic action potential shapes and conduction velocity; namely, myelinated Aβ (large diameter and fast conducting) low threshold-mechano receptor (LTMR) and Αδ (medium diameter and medium conducting) afferent nociceptor, along with slowly conducting small diameter unmyelinated C-type nociceptor28, 29. Identification and functional characterization of different neuronal subclasses in vivo have been a challenge, thus current investigations have mostly relied on the invasive in vitro or ex vivo electrophysiologic recordings30, 31. As shown in Fig. 1g, we were able to visualize single action potentials by inspecting small spiking area (1–3 µm) of line scan image. We found that the kinetic properties of action potentials were closely related to the sizes of DRG neurons, consistent with conventional electrophysiologic recordings30, 32. This indicates the feasibility of in vivo voltage imaging for noninvasive optical readout of electrophysiologic features, to the point where afferent subtypes can be inferred and targeted.
ASAP4.4-Kv imaging permits visualization of mechanical stimuli (non-noxious to noxious)-evoked temporal summation of fast voltage signals
To understand how DRG neurons encode painful or non-painful mechanical stimuli, we applied stimulation of different strengths to the hindpaw, and visualized evoked ASAP4.4-Kv signals in DRG neurons. At low stimulation strength (light brush, 0.4 g, or 2 g von Frey; Fig. 2), small and transient subthreshold potential changes could be observed in mechanosensitive neurons (Fig. 2a), and only a few neurons exhibited hindpaw stimulation-evoked transient Ca2+ increases in naïve animals (Fig. 2b). However, peripheral inflammation or nerve injury led to a significant increase in membrane electrical signal summation, including both subthreshold and suprathreshold voltage signals (Fig. 2a, d), but not in Ca2+ responses (Fig. 2b-d). At an intermediate stimulation strength (100 g press), high-frequency voltage dynamics were observed in neurons of naïve mice (Fig. 3a), while inflammation or nerve injury treatment produced exacerbated voltage fluctuations with larger amplitude and longer membrane depolarization (Fig. 3b-d). On the other hand, GCaMP3 Ca2+ imaging revealed increased population level activities in injured mice upon exposure to the same press stimulus (Fig. 3d, e). However, large variations in the magnitude of Ca2+ transients were found within the same DRG and across different treatment groups. Consequently, while the data were grouped, neither average amplitudes nor the mean AUCs of Ca2+ transients differed significantly between naïve or injured animals (Fig. 3d, g), despite the fact that increased activated cell numbers (Fig. 3d) and increased amplitudes of Ca2+ transients (Fig. 3f) were evident in some CFA-injured mice.
At the strongest mechanical stimulus (300 g), long-lasting membrane potential fluctuations with sustained membrane depolarization were observed in DRG neurons of naïve mice (Fig, 4a), and voltage fluctuations in neuronal membranes were further aggravated by inflammation or nerve injury treatment (Fig. 4b, c and Supplementary movie 1). Similar to previous results, average Ca2+ transients differed only marginally in groups affected by inflammation or nerve injury (Fig. 4d-f), and increased numbers of activated cells were not evident (Fig. 4d and Supplementary movie 7). In addition, simultaneous in vivo dual color imaging of ASAP4.4-Kv (green) and mCyRFP333, a cyan-excitable red fluorescent protein that can be used as a non-perturbing voltage-independent fluorescent marker as a control signal for ASAP4.4-Kv voltage imaging, demonstrated that the pattern of evoked electrical activity was distinguishable from rhythmic physiological motions arising from respiration or heartbeat (Supplementary Fig. 4).
ASAP4.4-Kv imaging reports thermal (heat or cold)-evoked voltage signals with high temporal fidelity
It has been reported that primary sensory neurons employ different strategies to encode heat vs. cold34, 35. To discern how heat or cold is represented in vivo, we examined the ASAP4.4-Kv voltage signals from heat or cold-sensing neurons. In naïve mice, the membrane voltage dynamics during noxious heat (50℃) stimulation displayed a slowly depolarizing voltage ramp that returned to baseline within 300 ms (Fig. 5a). Noxious cold (0℃) stimulation, however, led to two distinctive forms of voltage activity: bursting or non-bursting (Fig. 6a). Bursting neurons displayed burst-frequency firing behaviors, whereas non-bursting neurons generated only single action potentials followed by small membrane fluctuations (Fig. 6a). Inflammation or nerve injury, in turn, resulted in augmentation of membrane voltage fluctuation and electrical amplitude in both heat- and cold-sensing neurons (Fig. 5b,c and Supplementary movie 2; Fig. 6b,c and Supplementary movie 3). Notably, stimulation by heat or cold was represented by distinct populational signals under various pain conditions.
After CFA-induced inflammation, numerous DRG neurons were activated upon noxious heat stimulation (50℃) but numbers were similar between naïve and CFA groups (Fig. 5d, e and Supplementary movie 8), while fewer neurons displayed Ca2+ activity to noxious cold (0℃) compared to naïve animals (Fig. 6d, e). As with the previous mechanical stimuli, heat-induced increases in Ca2+ transients were observed in some DRGs of individual CFA-treated mice (Fig. 5f), but not in grouped DRGs (Fig. 5d, g). In contrast, cold-sensitive neurons displayed reduced Ca2+ transients after peripheral inflammation, both individually (Fig. 6f) and as a group (Fig. 6d, g). These results are consistent with previous reports that cold-mediated Ca2+ activity was lost in specific types of cold-sensing neurons following peripheral injury35. The discrepancy between voltage and Ca2+ signals in cold-sensing neurons suggests that, following peripheral inflammation, an individual sensory neuron still retains the ability to encode cold-specific sensory input; however, summation of the neuronal response to painful cold is suppressed by network activity in DRG.
ASAP4.4-Kv imaging reveals high potassium or capsaicin-evoked strong membrane voltage fluctuations
Finally, we used the ASAP4.4-Kv voltage sensor to examine how DRG neurons encode noxious chemical nociception. In naïve mice, direct topical application of high potassium (50 mM KCl) or capsaicin (10 µM), a TRPV1 agonist which can initiate activity in nociceptive neurons, onto L5 DRG, resulted in >4-fold increase in voltage fluctuations over baseline (Fig. 7a, d). Both CFA and SN-CCI treatments significantly increased neuronal responses to KCl or capsaicin, with substantial increases in frequency and magnitude of dynamic membrane voltage fluctuations (Fig. 7b–d and Supplementary movie 5, 6). When the same chemical treatments were performed on Pirt-GCaMP3 mice, we observed robust activation of a large population of DRG neurons within the DRG sensory ganglia (Fig. 7f, g). Topical application of capsaicin resulted in DRG neuronal activation primarily in the small and medium diameter neurons within all populations of DRG neurons imaged (Fig. 7g). Small and medium diameter neurons are nociceptors that typically express TRPV1 receptors. On average, the Ca2+ transients in activated neurons from injured mice were significantly higher than those from naïve animals (Fig. 7e). Compared to physical stimulation, direct chemical administration onto DRG neurons produced near-maximal Ca2+ transients and responses in most DRG neurons in vivo. These findings lead us to conclude that neuronal hypersensitivity is a common consequence of peripheral injury.