Optogenetic evaluation of the ability of different cutaneous C-fiber afferents to evoke aversive behaviors

Most cutaneous C-fibers, including both peptidergic and non-peptidergic subtypes are presumed to be nociceptors and respond to noxious input in a graded manner. However, mechanically sensitive, non-peptidergic C-fibers also respond to mechanical input in the innocuous range, and so the degree to which they contribute to nociception remains unclear. To address this gap, we investigated the function of non-peptidergic afferents using the MrgprdCre allele. In real time place aversion studies, we found that low frequency optogenetic activation of MrgrpdCre lineage neurons was not aversive in naïve mice, but became aversive after spared nerve injury (SNI). To address the underlying mechanisms of this allodynia, we recorded from lamina I spinoparabrachial (SPB) neurons using the semi-intact ex vivo preparation. Following SNI, innocuous brushing of the skin gave rise to abnormal activity in lamina I SPB neurons, consisting of an increase in the proportion of recorded neurons that responded with excitatory post synaptic potentials or action potentials. This increase was likely due, at least in part, to an increase in the proportion of lamina I (LI) SPB neurons that received input upon optogenetic activation of MrgprdCre lineage neurons. Intriguingly, in SPB neurons there was a significant increase in the EPSC latency from MrgprdCre lineage input following SNI, consistent with the possibility that the greater activation post SNI could be due to the recruitment of a new polysynaptic circuit. Together, our findings suggest MrgprdCre lineage neurons can provide mechanical input to the dorsal horn that is non-noxious before injury but becomes noxious afterwards due the engagement of a previously silent polysynaptic circuit in the dorsal horn.


INTRODUCTION
Traditionally, cutaneous C-fibers have been categorized into two major classes: peptidergic afferents that generally express substance P and/or CGRP, as well as the so-called non-peptidergic afferents that express Mrgprd and/or bind the isolectin IB4 [3; 9]. Because both populations respond vigorously to noxious stimuli, it was generally assumed that both types function mainly as nociceptors. Consistent with this idea, ablation studies have suggested that peptidergic afferents are responsible for thermal pain, whereas non-peptidergic afferents are responsible for mechanical pain [6]. Moreover, because these nociceptive-responsive neurons collectively make up the vast majority of C-fibers, it was not uncommon to generally equate C-fiber activity with nociception and, by extension, pain.
But other findings have challenged the presumption that these populations are exclusively pain-inducing nociceptors. In particular, it remains unclear whether the nonpeptidergic subset of IB4-binding afferents signal nociception, or whether their function is more nuanced. For instance, although non-peptidergic, IB4-binding neurons respond to noxious stimuli, they typically show a graded response over a wide range of mechanical forces, and indeed have the capacity to detect even low threshold input that is clearly non-noxious [21]. In support of this idea, human microneurography studies have shown that the vast majority of mechanically sensitive C-fibers are activated by stimulus intensities that are reported as non-painful [12; 27]. Finally, although transient optogenetic activation of Mrgprd neurons in mice was found to cause withdrawal, prolonged exposure was not sufficient to elicit conditioned place aversion [4]. Thus, the degree to which activity in the non-peptidergic, IB4-binding population is sufficient to drive aversion and/or pain remains ambiguous.
A second major gap in our understanding is the role of non-peptidergic afferents in chronic pain. Injuries that give rise to chronic pain, such as spared nerve injury (SNI), cause central changes that alter the way sensory information is integrated by the nervous system [2; 28]. One of the most common consequences of these injury-induced changes is allodynia: a phenomenon in which innocuous mechanical stimuli, such as light brushing of the skin, are perceived as noxious. It is generally assumed that the afferents responsible for allodynia are those that are most responsive to innocuous mechanical input, i.e. low threshold mechanoreceptors (LTMRs). However, many nonpeptidergic IB4-binding neurons frequently respond vigorously to innocuous stimuli, such as brushing, raising the possibility of their involvement in allodynia [21]. Thus, there remains significant uncertainty about both the afferent subtype(s) and the spinal circuits downstream thereof that give rise to allodynia.
Here, we used the Mrgprd Cre allele to investigate the function of non-peptidergic IB4binding C-fibers in normal sensation and in chronic neuropathic pain. In a real time place aversion assay, we show that activation of Mrgprd Cre lineage neurons is not aversive in naïve mice. However, in the context of SNI-induced neuropathic pain, activation of Mrgprd Cre lineage neurons became aversive. We subsequently show that SNI increases the proportion of lamina I SPB neurons that respond to innocuous brush input, and that this increase is likely to be mediated, at least in part, by the activity of DRG recordings and peripheral stimuli Intracellular recordings from L3 DRG cells in the were made using quartz microelectrodes, (> 100 MΩ filled with 1 mol/L K-acetate). An electric search stimulus was applied at 1.5 Hz through a glass suction electrode applied to the saphenous nerve to locate cells with axons in the saphenous nerve. Cutaneous receptive fields (RFs) were located with a fine paint brush, blunt glass probe, and von Frey hairs. When cells were driven by the nerve but had no mechanical RF, a thermal search was performed by gently applying hot (52 • C) and cold (0 • C) saline to the surface of skin using a syringe with a 20-gauge needle. If a thermal RF was located, the absence of mechanical sensitivity was confirmed by searching the identified RF using a glass probe. In most cases, the response characteristics of the DRG cell were then determined by applying computer-controlled mechanical and thermal stimuli ( Fig. 2A). The mechanical stimulator consisted of a constant-force controller (Aurora Scientific) attached to a 1mm-diameter plastic disc. Computer-controlled 5 s square waves of 5, 10, 25, 50, and 100 mN were applied to the RF of the cell. Following the functional characterization, responsiveness to laser stimulation was determined using an 80-mW, 473-nm wavelength laser and a 200-micron fiber optic cable affixed to a micromanipulator (Laserglow Technologies, Toronto, Canada). The opening of the fiber optic cable was positioned approximately 5 mm from the skin surface.

Parabrachial injections
Four-to 6-week-old mice were anesthetized with isoflurane and placed in a stereotaxic apparatus. An incision was made to expose the bone and a small hole was made in the skull with a dental drill. A glass pipette was used to inject 100 nL of FAST DiI oil (2.5 mg/mL; Invitrogen, Carlsbad, CA) into the left lateral parabrachial area at the following coordinates: from lambda,1.3 mm lateral; from lambdoid suture, 0.5 mm posterior; from surface, -2.4 mm. The incision was closed with sutures and the mice were allowed to recover and returned to their home cages. These injections were made at least 5 days before electrophysiological recordings.

Whole-cell spinal neuron recordings
Neurons were visualized using a fixed-stage upright Olympus microscope equipped with a 40x water immersion objective, a CCD camera (ORCA-ER; Hamamatsu Photonics, Hamamatsu City, Japan) and monitor. A narrow-beam infrared LED (L850D-06; Marubeni, Tokyo, Japan, emission peak, 850 nm) was positioned outside the bath, as previously described [10]. Projection neurons in lamina I were identified by DiI fluorescence following retrograde labeling in the parabrachial nucleus. Whole-cell patch clamp recordings were made using borosilicate glass microelectrodes pulled using a PC-10 puller (Narishige International, East Meadow, NY). Pipette resistances ranged from 6 to 12 MΩ. Electrodes were filled with a solution containing the following (in mM): 135 K-gluconate, 5 KCl, 0.5 CaCl2, 5 EGTA, 5 HEPES, and 5 MgATP; pH 7.2. Alexa fluor 488 (Invitrogen; 25 mM) was added to confirm recording from the targeted cell. Recordings were acquired using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). The data were low-pass filtered at 2 kHz and digitized at 10 kHz using a Digidata 1322A (Molecular Devices) and stored using Clampex version10, (Molecular Devices).

Cutaneous stimulation
The cell's receptive field was determined using a paint brush and 1-to 4-g von Frey filaments. Once the cell's receptive field was located, subsequent stimuli were applied directly to the receptive field for 1 s to determine the cell's response properties. MiniAnalysis (Synaptosoft, Decatur, GA) was used for detecting excitatory post synaptic currents (EPSCs) and action potentials (APs).

Cell dissociation and pickup for RT-PCR
DRG containing labeled cells from Mrgprd Cre ; Ai32 mice were removed and dissociated as described previously [1]. Briefly, DRG were treated with papain (30 U) followed by collagenase CLS2 (10 U)/Dispase type II (7 U), centrifuged (1 min at 1000 r/min), triturated in Minimal Essential Medium, plated onto laminin-coated coverslips in 30 mm diameter dishes, and incubated at 37 °C for 45 min. Dishes were removed and flooded with collection buffer (140 mM NaCl, 10 mM Glucose, 10 mM HEPES, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 ). Single, labeled cells were identified using fluorescence microscopy, picked up using glass capillaries (World Precision Instruments) held by a 4axis micromanipulator under bright-field optics, and transferred to tubes containing 3 μL of lysis buffer (Epicentre, MessageBOOSTER kit). Cells were collected within 1 h of removal from the incubator and within 4 h of removal from the animals.

Single-cell amplification and qPCR
The RNA isolated from each cell was reverse transcribed and amplified using T7 linear amplification (Epicentre, Message BOOSTER kit for cell lysate), run through RNA Cleaner & Concentrator-5 columns (Zymo Research), and analyzed using qPCR, as described previously [1], using optimized primers and SsoAdvanced SYBR Green Master Mix (Bio-Rad). Threshold cycle time (Ct) values were determined for each well.

Real time place aversion (RTPA)
Behavioral boxes were constructed from plexiglass that were 12 (l) x 5 (w) x 10 (h) inches with guides for dividing the boxes into three chambers: 2 (5 x 5 inches) and 1 (2 x 5 inches) in the middle. For optical stimulation, we used Blue LED Strip Light, 120/m, 10 mm wide, by the 5m Reel, 12 VDC, 8.9 Watts/meter, 742 mA/meter (centered at 460 nM) and Amber LED Strip Light, 120/m, 10 mm wide, by the 5m Reel, 12 VDC, 8.9 Watts/meter, 742 mA/meter (centered at 595 nM) (Environmental Lights, San Diego, California). Five-inch strips of LEDs were affixed to a 5-X 12-inch metal box (heat sink) covering a 5-x 5-inch area on the ends of the box (one area blue the other amber) leaving a 2-inch area in the middle free of lights. This box could then be placed under the plexiglass box to illuminate the floor (Fig. 3D). The LEDs were controlled using a CED 1401 interface. The LEDs were positioned 10 mm below the surface of the plexiglass floor as we found that at this distance there was no heating of the plexiglass floor over the 15-min period of observation. Mice used in the RTPA were first acclimated to the behavioral boxes for 3 days (15 minutes/day) before testing. On the day of testing the mice were placed in the center compartment and the LEDs turned on and the sliding doors removed so the mice could move freely throughout the box for 15 min. The mice were recorded during this period using a video camera. The videos were then scored off line. All experimenters were blinded to genotype and experimental condition of the mice.

Spared nerve injury
Mice were anesthetized with isoflurane and the posterior right hindlimb was shaved and cleaned using an aseptic solution. An incision was made over the popliteal fossa and the underlying muscle incised to expose the fossa. The tibial and peroneal nerves were isolated. The two nerves were tightly ligated and cut just distal to the ligature. The muscle and skin were sutured and the animal allowed to recover. For sham surgeries the procedures were the same except for the ligation and transection.

Immunohistochemistry
Deeply anesthetized mice were perfused with 4% paraformaldehyde and tissue sections of the L3 spinal cord, DRG, and glabrous skin were embedded in OCT and cut on a cryostat at 20 μm. The sections were stained for IB4, CGRP and eYFP using the following antibodies or probes: anti-GFP (chicken 1:1250; Aves Labs), anti-CGRP (rabbit; 1:1000; Chemicon) and IB4 (1:250; IB4-congugated AlexaFluor 647; Molecular Probes, Eugene, OR). After incubation in primary antiserum for at least 2 h, tissue was washed and incubated in appropriate fluorescently tagged secondary antibodies (1:500; Jackson Immunoresearch) for 1 h followed by Hoescht staining (1:10,000) or 30 min. After washing and mounting in Fluoromount (Sigma), sections were viewed and imaged on an Olympus BX53 fluorescent microscope with UPanSApo 10x or 20x objectives.

Statistical analyses
Data are expressed as the mean ± SEM. For electrophysiological experiments, dots represent data points from individual neurons combined from all animals. The following tests were used for statistical analysis: unpaired Student's t for continuous normal data (comparison of 2 groups), Chi-squared or Fisher's exact test for categorical data, ordinary one-way ANOVA with Tukey's multiple comparisons post hoc test if indicated by the main effect (comparison of >2 groups), or two-way repeated-measures ANOVA with Tukey's multiple comparisons post hoc test if indicated by the main effect (time course with comparison of 2 or more groups). All tests were two-tailed (unless otherwise indicated) and a value of P < 0.05 was considered statistically significant in all cases. All statistical analyses were performed using GraphPad Prism 8.4 software.

Histological and molecular characterization of Mrgprd Cre lineage neurons.
To investigate the functional role IB4-binding neurons, we targeted these neurons genetically using the Mrgprd Cre knockin allele that was originally developed by David Anderson [21]. Because Mrgprd shows some developmental expression [16], we carefully characterized the neurons that are captured by this genetic tool, as assessed using Ai32, a Cre-dependent reporter embedded in the Rosa locus that enables expression of a ChR2-eYPF fusion protein [17]. Analysis of cell bodies in lumbar DRG suggested that the Mrgprd Cre allele captured ~80% of IB4-binding neurons, and similarly that ~80% of IB4-binding neurons expressed ChR2-eYFP (Figs. 1A-C). In contrast, the majority of the CGRP-expressing afferents did not express ChR2-eYPF (Figs. 1D-E). The analysis of the innervation pattern in the lumbar spinal cord showed a similar pattern: Mrgprd Cre lineage neurons showed a very high degree of colocalization with IB4 in a band that corresponds approximately to inner lamina II, but limited colocalization with CGRP, which primarily targets lamina I and outer lamina II (Figs. 1F-J). In the glabrous skin, Mrgprd Cre -targeted neurons colocalized with the pan-neuronal marker PGP9.5, and innervated the most superficial aspect of the epidermis (Figs. 1K-M).
A number of recent single-cell sequencing studies have suggested that non-peptidergic afferents can be further subdivided into more refined subtypes characterized by expression of Mrgprd, Mrgpra3, or Sst/Nppb [20; 23; 26]. To assess whether the Mrgprd Cre lineage captures these populations, we performed quantitative real-time PCR of individual eYFP-labeled DRG neurons that were freshly dissociated from adult Mrgprd Cre ; Ai32 mice. These experiments revealed that all genetically-marked neurons fell into one of three apparent cell types: those with high Mrgprd (and lacking Mrgpra3, Sst, Calca and Tac1); those with high levels of Mrgpra3 (as well as Calca, but little or no Mrgprd, Sst and Tac1); and those with high levels of Sst (and low/moderate levels of Mrgpra3 and Tac1, but lacking Mrgprd, Calca) (Fig. 1N). Importantly, this single-cell analysis of eYPF-labeled cells indicated that Mrgprd is no longer detected in the Mrgpra3 or Sst populations in the adult, suggestive of developmental expression (Fig.  1O). Thus, the Mrgprd Cre lineage faithfully captures the majority of afferents that have traditionally been termed 'non-peptidergic' nociceptors, which includes most IB4-binding neurons, but not peptidergic nociceptors, C-LTMRs, or Trpm8-expressing cold/cooldetectors. For purposes of comparison, we also visualized the recombination mediated by the Trpv1 Cre allele. Consistent with previous reports [5], we observed that Trpv1 Cre lineage neurons include the vast majority of cutaneous C-fibers, though not C-LTMRs or Trpm8-expressing afferents (summarized in Fig. 1P).

Optogenetic manipulation of Mrgprd Cre lineage neurons
We next characterized the responses of Mrgprd Cre lineage neurons to optogenetic stimulation of the skin with Mrgprd Cre ; Ai32 mice and the ex vivo preparation, performing intracellular recordings of L3 DRG neurons ( Fig. 2A). Afferents were categorized as cutaneous C-fibers if they responded to electrical stimulation of the saphenous nerve with a conduction velocity of 1.2 m/s or less. Next, the receptive field of a given cutaneous C-fiber was identified manually through application of mechanical and/or thermal stimuli. Thereafter, quantitative phenotyping to natural and optogenetic stimuli was performed using a Peltier thermode, a mechanical stimulator, and a 473-nm laser applied to the skin within the identified receptive field. Typically, Mrgprd Cre lineage neurons responded weakly to cooling and vigorously to noxious heat; they also responded to both low threshold (10 mN) and high threshold (50 mN) mechanical input in a graded manner, consistent with previous characterizations of non-peptidergic nociceptors [14; 21]. Both optogenetic and natural stimulation of the skin elicited action potentials (Fig. 2D). We found that a 5-ms pulse of light was sufficient to drive a single action potential, whereas a 300-ms pulse of light was required to observe a doublet. Finally, Mrgprd Cre lineage neurons could follow optogenetic stimulation of at least 5 Hz (Fig. 2C). Overall, of 21 cutaneous C-fibers that were recorded from Mrgprd Cre ; Ai32 mice, 15 showed responses to optogenetic stimulation (~70%), consistent with the idea that Mrgprd Cre lineage neurons represent the majority of cutaneous C-fibers. For comparison, we found that 11 of 12 (~90%) cutaneous C-fibers from Trpv1 Cre ; Ai32 mice could be opto-tagged in this fashion (Fig. 1D), as expected given the broad recombination mediated by this allele [5]. Importantly, though the Trpv1 Cre allele captured slightly more C-fibers than the Mrgprd Cre allele, optogenetic stimulation of individual C-fibers from mice of either genotype gave rise to similar numbers of action potentials, thereby enabling direct comparison of the two alleles in behavioral studies (Fig. 1E).

Behavioral responses to optogenetic manipulation of Mrgprd Cre lineage neurons
Withdrawal in animals has traditionally been interpreted to represent a pain behavior, but the percept associated with this response is difficult to infer. Previous work has shown that selective optogenetic activation of Mrgprd neurons elicits withdrawal, but not flinching, licking, or guarding [4]. We anticipated that optogenetic stimulation of Mrgprd Cre lineage neurons would be at least as aversive, if not more so, because the genetic tool we used captures a significantly larger number of C-fibers than that used by Beaudry et al. [4]. Although both alleles target the same genetic locus, the degree of recombination using the Mrgprd Cre allele is more extensive than the Mrgprd CreER allele for two reasons: the constitutive Cre allele captures more than the adult Mrgprd population due to developmental expression (i.e., Sst and Mrgpra3 populations), whereas the Mrgprd CreER allele likely captures less than the adult Mrgprd population due to incomplete recombination. Contrary to our expectations, however, we found that the responses of Mrgprd Cre mice to optogenetic stimulation of the hindpaws appeared quite modest, and not qualitatively different than that described for the Mrgprd CreER mice [4]. Resting mice generally withdrew their hindpaw for a brief moment, as if startled, and then replaced it on the floor, and alert mice rarely responded at all. In sharp contrast, activation of Trpv1 Cre lineage neurons invariably caused rapid, robust withdrawal that was frequently accompanied by flinching and/or licking (Fig. 3A), just as previously described [4]. These observations raised the possibility that low-frequency optogenetic activation of non-peptidergic 'nociceptors' might not actually be aversive.
Because reflexive behaviors can be hard to interpret and do not readily assess aversion, we turned to non-reflexive behaviors for quantitative analysis. When a stimulus is quite noxious, such as intraplantar formalin, mice will develop conditioned place aversion to the side of the chamber that is associated with the aversive experience. Intriguingly, Beaudry et al. showed that optogenetic activation of Trpv1 Cre afferents was sufficient for conditioned place aversion, whereas activation of Mrgprd CreER afferents was not [4]. However, conditioned place aversion can be difficult to achieve unless the paired stimulus is fairly injurious. We therefore developed a real time place aversion (RTPA) assay to enable the detection of aversive stimuli with higher sensitivity.
Our previous optogenetic characterization had been done by laser, a coherent light source that is ideal for focal stimulation. However, for the RTPA studies we were planning to use LEDs, an incoherent light source which is more suitable for widespread activation. To compare the efficacy of optogenetic stimulation by laser and LED, we performed ex vivo skin nerve recordings (Fig. 3B). Importantly, stimulation of the skin with either light source gave rise to a similar number of action potentials in ChR2expressing Mrgprd Cre lineage neurons, indicating that optogenetic stimulation by laser or LED was equally effective (Fig. 3C). With this knowledge, we built a custom RTPA box comprising a stimulation side in which the floor was lined an array of blue LED lights and a control side in which the floor was lined an array of amber LED lights, which were separated by a small, unlit middle chamber. These lights were set to run in either the sustained mode, in which the lights were constantly on, or the 'wind-up' mode, in which the lights would flash at 2 Hz for 30 ms per flash. Mice were placed in the RTPA box for 15 minutes, during which time they were allowed to move freely from one chamber to another (Fig. 3D).
Next, we performed RTPA assays to quantify the aversiveness of optogenetic stimulation. Just as expected, we found that Trpv1 Cre ; Ai32 mice showed significant avoidance of the blue-light side, consistent with the idea that optogenetic activation of nociceptors is unpleasant. In sharp contrast, the time that Mrgprd Cre ; Ai32 mice spent on the blue-light side was not different than control littermates harboring Ai32 alone (Fig.  3E). Similar findings were observed when the LED lights were flashing at 2 Hz to simulate wind-up (Fig. 3F). These striking observations suggest that optogenetic activation of Mrgprd Cre ;Ai32 fibers was not aversive, at least not in naïve mice.

Effect of injury: behavioral responses to optogenetic manipulation of Mrgprd Cre lineage neurons in the context of neuropathic pain
Although our data suggested that Mrgprd Cre lineage neurons can be activated without causing acute pain, a key remaining question was whether they contribute to chronic pain. Allodynia is one of the hallmarks of chronic pain, particularly neuropathic pain [13]. For people that suffer from allodynia, innocuous everyday experiences, such as fabric moving across the skin, can give rise to shooting pain. Since many non-peptidergic afferents can respond robustly to dynamic low threshold stimulation [21], we hypothesized that Mrgprd Cre lineage neurons might contribute to this allodynia. To address this question, we selected to use the spared nerve injury (SNI) model [8] of chronic neuropathic pain (Fig. 4A). Although the saphenous nerve remains intact in this model, there is nevertheless the potential for the function of afferents to be affected by the injury. We therefore measured the responses of Mrgprd Cre lineage afferents to optogenetic stimulation of the skin, comparing naïve and SNI mice (Fig. 4B). Importantly, the number of action potentials that were elicited was not affected by SNI (Fig. 4C). Thus, neuropathic injury did not cause sensitization of Mrgprd Cre lineage afferents to optogenetic stimulation.
To address whether SNI altered how the input from Mrgprd Cre lineage neurons was integrated in the central nervous system, we performed RTPA assays. Once again, at baseline, the amount of time that naïve Mrgprd Cre ; Ai32 mice spent on the blue-light side was not different than that of control mice, confirming that activity in Mrgprd Cre lineage neurons is not aversive in the absence of injury. In contrast, however, optogenetic activation of Mrgprd Cre lineage neurons in mice with SNI caused significantly more avoidance than activation of these neurons in naïve mice (Fig. 4D). This finding -in essence, optogenetic allodynia -suggested that activity in non-peptidergic afferents becomes aversive following nerve injury. Moreover, the lack of sensitization post-injury in Mrgprd Cre lineage afferents to optogenetic stimulation implied that central mechanisms must mediate optogenetic allodynia due to SNI.

Modulation of spinoparabrachial neurons by Mrgprd Cre lineage afferent input
To investigate the spinal circuitry underlying this phenomenon, we performed whole-cell patch-clamp recordings of lamina I neurons using the ex vivo somatosensory preparation (Fig. 5A). Optogenetic stimulation of either the peripheral or central terminals gave rise to EPSCs onto recorded neurons in lamina I, albeit with different latencies (Fig. 5B). We also noted that the EPSC magnitude was larger when the optogenetic stimulation occurred at the skin rather than at the dorsal horn (Fig. 5C). However, there were several instances in which optogenetic stimulation at the dorsal horn was efficacious, whereas stimulation at the skin was not, likely reflecting the fact that not all recorded neurons had receptive fields in the skin of the dorsal hindpaw. For this reason, we selected central stimulation for subsequent experiments.
We found that optogenetic activation of the central terminals of Mrgprd Cre lineage neurons gave rise to EPSCs in only 6 of 27 random lamina I neurons (Figs. 5D-E). In contrast, optogenetic activation of the central terminals of Trpv1 Cre lineage neurons gave rise to EPSCs in 6 of 8 random lamina I neurons, which represented a significantly higher proportion relative to Mrgprd Cre (Fisher's exact test; p < 0.05). Moreover, optogenetic activation of afferents in Trpv1 Cre ; Ai32 mice was sufficient to generate action potentials in 5 of 6 neurons that exhibited EPSCs, whereas the optogenetic activation in Mrgprd Cre ; Ai32 mice was insufficient to generate action potentials in lamina I cells (Figs. 5F-G), which again represented a significantly lower proportion than that observed upon activation of Trpv1 Cre lineage neurons (Fisher's exact test; p < 0.05). Since randomly targeted neurons are predominantly interneurons, these observations suggest that Mrgprd Cre lineage neurons do not provide strong excitatory input, either directly or indirectly, onto lamina I interneurons.

Contribution of Mrgprd Cre lineage afferents to aversion following spared nerve injury
Our behavioral experiments suggested that Mrgprd Cre lineage input only became aversive following SNI. To investigate mechanisms that may contribute to this injuryinduced change, we performed whole-cell recordings from lamina I SPB neurons in naïve or SNI animals (Fig. 6A). In naïve mice, less than half of lamina I SPB neurons displayed EPSCs in response to brushing of the skin with a paint brush, and this input led to action potentials in only 2 of 12 cells (Fig. 6B). Following nerve injury, however, 100% (13 of 13) lamina I SPB neurons showed EPSCs in response to the same dynamic, low threshold input. Moreover, brushing of the skin from SNI mice resulted in action potentials in ~70% of LI SPB neurons in SNI mice, representing a 4-fold increase compared to naïve mice. These results raise the possibility that allodynia in SNI mice may be caused by increased LI spinal output to low threshold stimuli, such as brush.
Next, we began investigating the contribution of Mrgprd Cre lineage neurons to this abnormal spinal output. In naïve mice, 7 of 12 lamina I SPB neurons showed optogenetically-induced EPCSs upon stimulation of Mrgprd Cre lineage afferents at 2 Hz. Following SNI, however, this proportion increased significantly, with 12 of 13 lamina I SPB neurons now responding to optogenetic stimulation (Fig. 6C). It should be noted, however, that this emergent Mrgprd Cre lineage neuron input was still rarely sufficient, in itself, to elicit an action potential in the recorded output neuron. Thus, although Mrgprd Cre lineage neurons are unlikely to be the only afferents involved in allodynia, they may nevertheless provide an important contribution. To gain insight into the underlying mechanism, we analyzed the EPCSs in more detail. Although a higher fraction of lamina I neurons showed light-induced EPSCs following SNI, the responses in individual neurons to optogenetic stimulation were similar and we saw no evidence of synaptic sensitization. Instead, we found that the average response latency to optogenetic stimulation was significantly increased following SNI (Fig. 6D). This elevated latency implies that the enhanced number of responders to Mrgprd Cre lineage stimulation occurs through an emergent polysynaptic pathway that is normally silent in naïve mice (Fig. 6E).

DISCUSSION
Our study provides evidence that activation of Mrgprd Cre lineage afferents in a naïve state is not aversive, but becomes aversive in the context of chronic neuropathic pain caused by SNI. At the circuit level, we found that lamina I SPB neurons show significantly more brush-induced activity following nerve injury, which is an attractive mechanism to account for the phenomenon of mechanical allodynia. Moreover, Mrgprd Cre lineage afferents may contribute to this abnormal spinal output after SNI because more lamina I SPB neurons receive Mrgprd Cre lineage afferent input following optogenetic activation relative to naïve controls. Finally, this increased input appears to be due to an emergent polysynaptic pathway, as evidenced by an increase in the average response latency to optogenetic stimulation of Mrgprd Cre lineage afferents. Altogether, our study suggests that SNI gives rise to changes in spinal circuitry that enables increases brush-evoked activity in lamina I SPB neurons, and that this effect is likely due, at least in part, to increased input from Mrgprd Cre lineage afferents through a polysynaptic neural pathway that is normally silent in naïve mice.
Previous studies have identified many mechanisms of injury-induced plasticity in the dorsal spinal cord [2; 28]. Our data suggest that one of these mechanisms involves the engagement of a polysynaptic circuit that emerges following SNI, as evidenced by the increased average latency of input from non-peptidergic afferents. These findings are consistent with the idea that allodynia following nerve injury is caused by disinhibition, which results in low threshold stimuli abnormally reaching nociceptive pathways in the superficial dorsal horn [25].
The results presented here show that low frequency activation of cutaneous Mrgprd Cre lineage afferents does not induce aversive behaviors. This lineage represents the majority of C-fibers innervating the skin. Thus, an important question is, what information is being conveyed during low frequency activation of these fibers? One potential answer to this question is found in a recent study looking at parallel ascending SPB pathways projecting to distinct subsets of lateral parabrachial sub-nuclei [7]. They found that activation of one of these SPB pathways (GPR83, which is associated with cutaneous mechanosensation) could have either a positive or negative valence depending on the intensity of the optogenetic stimulation. Therefore, low intensity activation of these fibers could possibly evoke a sensation of innocuous touch.
One of the limitations of this type of study is that optogenetic stimulation does not perfectly mimic natural stimulation. In this case, we found that light-mediated activation gave rise to action potentials in Mrgprd Cre lineage afferents that was similar in frequency to that observed upon stimulation with low threshold stimuli (e.g., 10 mN), but we did not achieve the instantaneous firing frequencies that are typically observed upon stimulation with high threshold stimuli (e.g., 50 mN). For this reason, we cannot exclude the possibility that increasing the frequency of action potentials generated by optogenetic stimulation would alter its valence. Nevertheless, activation of this population at low frequency (2 Hz) is clearly not aversive in naïve mice, whereas activation of afferents that includes the cutaneous peptidergic C-fibers is. This important distinction raises the possibility that the bona fide nociceptors-those whose main function is to warn the organism of tissue damage-are primarily represented by peptidergic C-fibers, and that the function of non-peptidergic C-fibers is likely to be much more nuanced and contextdependent.
An unexpected finding of our work was that control mice lacking ChR2 expression nevertheless showed a modest degree of avoidance for the blue-light side of the RTPA apparatus that emerged after repeated exposure. Since both sides of the chamber produce equally low levels of thermal radiation, it is likely that this avoidance was in response to visual, rather than cutaneous, input. Moreover, mice avoided blue over amber light despite similar lux (illuminance), suggesting that it is the wavelength of light that contributes to the avoidance. We found that the avoidance observed in control mice was most pronounced when light was on continuously. In contrast, the optogeneticallyinduced aversion in Trpv1 Cre ; Ai32 mice was most pronounced when the light was on transiently, flashing at 2 Hz. Because optogenetic stimulation in the wind-up mode gave rise to the highest degree of aversion and thus seemed to be the most effective, we selected this stimulation paradigm for the behavioral experiments involving SNI. However, we note that it will be important to investigate a wide range of stimulation frequencies, including ones that we were not able to achieve with the current tools, to get a more complete picture of the relationship between stimulation frequency and perceptual percept.
Despite the caveat that mice are able to see cutaneous optogenetic stimulation through the floor in RTPA studies, we feel that this approach is still preferable to the implantation of fibers or LEDs through a surgical manipulation because the activation of cutaneous afferents with light does not involve tissue damage, which is likely to be confounding in pain studies. In this regard, it is noteworthy that sham-operated mice that expressed ChR2 in Mrgprd Cre lineage afferents also showed a trend toward avoidance of the blueside of the RTPA chamber that was greater than that observed in naive mice, though not as pronounced as that observed in SNI mice. This intermediate phenotype of sham treatment suggests that the surgical procedure involving the cutting of skin and muscle is in itself an injury model that may be sufficient for ongoing changes in spinal circuitry.
Just as reported previously [16], we found that Mrgpra3-expressing afferents and Sstexpressing afferents are derived from the Mrgprd lineage. Intriguingly, all three of these afferent subtypes show predominant (though not exclusive) cutaneous targeting, and all three have been implicated in itch [11]} [15; 19; 24]. We did not observe itch behaviors -scratching or biting -upon optogenetic activation of Mrgprd Cre lineage afferents in our study. Curiously, significant itch behavior has only been reported upon chemogenetic (and not optogenetic) activation of these populations [11; 19; 22]. We speculate that it is not simply the type of afferent input, but also the pattern and the frequency of activation that is interpreted by the nervous system to differentially represent itch, pain, and some aspects of touch. Understanding this integration is of fundamental importance to our basic understanding of somatosensation and may one day lead to the development of improved strategies for the treatment of pain.

CONFLICT OF INTEREST STATEMENT
The authors have no conflicts of interest to declare.