The response of DRG neurons to protons has been extensively documented in the literature, and in many cases, the suspected ASIC subunit or combinations of them have been deduced from the current kinetics. In the DRG, nociceptors have been traditionally divided into myelinated medium diameter Aδ and unmyelinated small-diameter C types. Here, we will focus on reports making an explicit distinction between PEP and nonpeptidergic small DRG neurons (Table 1). The common neuronal marker used in these studies is the binding of the IB4 lectin. Most of the neurons not binding IB4 are peptidergic (i.e., they express CRGP), and most of those binding IB4 are nonpeptidergic (Figure 1). These neuronal subpopulations show different currents and action potential characteristics (Stucky and Lewin, 1999; Fang et al., 2006). More importantly, as will be discussed further, these two major groups of nociceptors fulfil different sensory roles within the DRG.

When exposed to low pH, some small-diameter rat DRG neurons respond with nondesensitizing, sustained ionic currents, while others respond with desensitizing or mixed currents (Petruska et al., 2000). Consistent with the presence of ASIC subunits, amiloride blocks the transient current and the transient part of mixed currents, whereas sustained currents are insensitive to amiloride. The latter currents are attributed to TRPV1 channels and are observed at lower pH (appearing at pH 6.1); in contrast, the transient currents are attributed to ASIC channels (appearing at pH 6.8; Petruska et al., 2000).

Approximately 90% of all rat DRG small neurons respond to pH 5 with inward currents (Liu et al., 2004). These currents were classified by their desensitization rate into three types: transient, sustained, or mixed. The frequency and waveform of these currents were different between the IB4+ and IB4− subpopulations. The IB4+ population produced most of the sustained (63%) currents, whereas IB4- neurons produced the most mixed currents (69%; Liu et al., 2004). Transient-only currents were not prevalent in any of the two subpopulations (<20% of total observed currents). These transient inward currents begin to activate at pH 7 and are maximal at pH 5. ASICs likely mediate these transient currents since they were sensitive to amiloride. The activation profile of mixed currents results from transient plus sustained currents. Based on these results, it was suggested that small IB4− rather than IB4+ neurons could play an essential role in acid sensing (Liu et al., 2004). Accordingly, ASIC currents (transient and mixed) are exclusive to small IB4- neurons (Drew et al., 2004).

In mouse DRG neurons, specifically unmyelinated nociceptors, it was reported that ASIC currents are also exclusive to the IB4− peptidergic subgroup and largely absent from IB4+ neurons. Transient-only currents were not detected; only (1) mixed and (2) sustained-only currents were detected, and both types were inhibited by amiloride (Dirajlal et al., 2003). DRG neurons from rats and mice generate ASIC currents mostly in IB4- neurons, and this predominance is seen across a wide acidic pH range (pH 6, 5, and 4; Leffler et al., 2006). These reports indicate that within unmyelinated DRG neurons from both rats and mice, ASIC-like currents can be detected almost exclusively within the peptidergic subgroup.

Poirot et al. (2006) provided a more relevant description of ASIC responses in rat DRG small neurons. Only currents that are initially activated within pH values of 6 and higher were investigated. Small sensory neurons produce a slowly inactivating current (Type 1) and two rapidly inactivating currents (Types 2 and 3). Type 1 current is the most prevalent current, the most sensitive to pH (pH₅₀ 6.6) and might be mediated by ASIC1a homomers. Type 2 is the less frequent current and is mediated by heteromers containing ASIC2. Type 3 also shows high proton sensitivity (pH₅₀ 6.5) and may be mediated by ASIC1a/ASIC3 heteromers (Poirot et al., 2006). Independent of the type, 91% of IB4− but only 36% of IB4+ neurons expressed an ASIC current. The distribution of type 1 and type 3 currents is heterogeneous among small DRG neurons. Type 1 current is expressed at higher levels in IB4− neurons than in IB4+ neurons (6-fold). Type 3 current is also more common in IB4− than IB4+ neurons (Poirot et al., 2006). These results suggest that within small, likely unmyelinated neurons, ASIC1, and ASIC3 might play specific roles in the IB4− peptidergic subpopulation, in which transient proton-gated currents are highly expressed. In addition to the detailed biophysical characterization of ASIC currents in the work by Poirot et al. (2006), this study provided two important demonstrations: (1) activation of ASICs in small DRG neurons can induce action potentials, and the probability of this induction increases with lower extracellular pH, in the pH 6.8–6.0 range; and (2) most H⁺-gated currents in small DRG neurons are mediated by ASICs, with the notable exception of a small fraction of neurons (approximately 15%) expressing the type 2 current, in which there is a high current component mediated by TRPV1 channels (i.e., blocked by capsazepine).

In DRG neurons from mice, ASIC3 is the most abundant subunit, whereas ASIC1b is the least abundant subunit (Schuhmacher and Smith, 2016). Regarding its distribution, ASIC subunits, especially ASIC3, do not show heterogeneous expression in immunohistochemistry studies. ASIC3 is more abundant in large-diameter neurons (Alvarez de la Rosa et al., 2002). ASIC3 is expressed mainly in TrkA+ neurons (Molliver et al., 2005), which is the receptor for nerve growth factor (NGF) and a marker of most peptidergic neurons, with medium Aδ and small C fibers. Small sensory neurons that innervate skeletal muscle express more ASIC3 than those that innervate the skin. Moreover, most ASIC3+ muscle afferents (80%) also express CGRP (Molliver et al., 2005). Another study analyzed the expression of ASIC3 in the DRG of eGFP knock-in mice (Lin et al., 2016). One-third of neurons expressed the ASIC3 subunit. ASIC3 localizes to 50% of large-diameter N52+ neurons and 39% of small-diameter peripherin+ neurons. Small peptidergic CGRP+ and IB4+ neurons show lower ASIC3 expression (27 and 23%; Lin et al., 2016). Thus, the expression of the ASIC3 subunit is higher in myelinated Aβ and Aδ fibers.

Analysis of mRNA levels shows that ASIC3 and ASIC1a/b subunits are the most abundant in the complete DRG (Poirot et al., 2006). In a recent work, Papalampropoulou-Tsiridou et al. (2020) analyzed the distribution of ASIC channels in the mouse DRG using RNAscope probes. This tool is a variant of in situ hybridization with higher sensitivity. Neurons were classified by the markers NF200 (marker of medium- to large-diameter myelinated neurons), IB4, and CGRP. None of the five ASIC subunits showed a similar distribution among the three different groups of DRG neurons (Papalampropoulou-Tsiridou et al., 2020). IB4+ neurons showed low expression of ASIC3 (~10% of total neurons) and the highest levels of ASIC2a expression (~70% of total neurons), while ASIC1a and 1b were not detected. This is contrasting to the IB4− subpopulation, in which more than 60% express the ASIC3 subunit and approximately 25% express ASIC1a and ASIC1b. Finally, NF200+ myelinated neurons showed the highest levels of ASIC3 subunit expression.

These differences in mRNA levels agree with the conclusions of the electrophysiological studies previously mentioned. First, they corroborate that ASIC3 is mostly expressed in medium to large NF200+ neurons (Alvarez de la Rosa et al., 2002; Lin et al., 2016). This population corresponds to myelinated Aβ and Aδ fibers. In this population, ASIC1 and ASIC1b are expressed at considerably higher levels (>70%) than in small sensory neurons. In fact, in large-diameter DRG neurons, two slowly and two rapidly inactivating H⁺-gated currents have been detected (Poirot et al., 2006). At least two of these currents have a pH dependence of activation (pH₅₀ 6.6) similar to that of the more prevalent type 1 current in small neurons (Poirot et al., 2006). Second, the lack of ASIC1 and the lowest expression of ASIC3 subunits in IB4+ neurons explain the scarcity of transient ASIC currents in IB4+ neurons (Dirajlal et al., 2003; Liu et al., 2004; Leffler et al., 2006). Finally, the coexpression of three ASIC subunit mRNAs per single neuron was also analyzed. The three neuron subpopulations (NF200+, IB4+, and CGRP+) show different combinations of ASIC subunits, indicating that heterotrimeric ASIC channels are the dominant form present in most primary sensory neurons. The asymmetric distribution of ASIC subunits in DRG neurons suggests a specialized response to acidification from specific DRG neuronal groups (Papalampropoulou-Tsiridou et al., 2020).

Transient receptor potential vanilloid 1 colocalizes with ASIC1a and ASIC3 within single small sensory neurons to a considerable extent (Ugawa et al., 2005). Indeed, several reports of ASIC-like currents in cultured DRG neurons have simultaneously detected H⁺-gated TRPV1-like currents (Neelands et al., 2010; Blanchard and Kellenberger, 2011). In response to protons, mouse DRG neurons produce a sustained inward current that inactivates slowly (>20s; Dirajlal et al., 2003). Small C-type neurons show an unequal distribution of these TRPV1-like H⁺-activated currents. At pH 5, 86% of small IB4− neurons generate sustained and, to a lesser extent, sustained and transient currents. Only 33% of IB4+ neurons responded in a similar way. However, not all neurons generating a sustained TRPV1-like H⁺-gated current are capsaicin-responsive (Dirajlal et al., 2003). In another study, only a quarter of small IB4+ neurons showed sustained currents in response to pH 5.3, and half of IB4− neurons showed either sustained or mixed currents (Drew et al., 2004). As expected, in DRG neurons from double-knockout ASIC2/3 mice, the distribution of these sustained currents is not affected (Drew et al., 2004). This heterogeneous distribution of low pH sustained currents was not observed in cultured DRG neurons from rats (Liu et al., 2004).

Rat DRG neurons are more responsive to capsaicin than mouse DRG neurons and have a higher prevalence and magnitude of H⁺-activated currents. When DRG neurons from TRPV1(−/−) mice were analyzed, there was a high reduction in proton responses. In comparison to WT neurons, TRPV1(−/−) neurons show significantly smaller currents at pH 5 and 4 (Leffler et al., 2006). As expected, transient ASIC-like currents remained unaltered in TRPV1(−/−) neurons. Moreover, in a skin nerve preparation in vitro, the response of myelinated Aδ fibers to pH 5 and 4 was unaffected in TRPV1(−/−) neurons in comparison to WT neurons. This suggests that the observed TRPV1 responses to low pH are predominantly generated by small unmyelinated neurons (Leffler et al., 2006).

Cavanaugh et al. (2011) used two lines of knock-in mice to study the distribution of TRPV1 in the DRG. During development, a wide variety of DRG neurons, both peptidergic and nonpeptidergic, express TRPV1 (Cavanaugh et al., 2011). In adult mice, however, TRPV1 expression is more restricted to the peptidergic population. Of the total TRPV1-expressing neurons, 64% were peptidergic, and only 11% were nonpeptidergic IB4+ neurons. Of all SP+ neurons in the DRG, 82% are TRPV1+, and of all CGRP+ neurons, 49% are TRPV1+ (Cavanaugh et al., 2011). Substance P (SP) is a marker of unmyelinated peptidergic neurons, while CGRP marks all peptidergic neurons (Aδ and C fibers). Previous studies reported a predominant expression of TRPV1 in the peptidergic subpopulation (Tominaga et al., 1998; Zwick et al., 2002). Since the detection of reporter genes is a more sensitive tool than immunostaining, the study of Cavanaugh et al. (2011) overcame the underestimation of TRPV1 expression in previous studies (Breese et al., 2005; Hjerling-Leffler et al., 2007). Interestingly, 20% of all TRPV1+ neurons are neither IB4+ nor CGRP+ (Cavanaugh et al., 2011).

Patil et al. (2018) classified DRG neurons based on their electrophysiological properties. The authors assessed DRG neurons in culture from several transgenic mouse lines carrying reporter genes for Nav1.8, TRPV1, and CGRP. They compared these against those of transgenic mice expressing well-established neuronal markers. Four of the resulting groups of neurons expressed TRPV1. The four TRPV1+ groups are small-diameter neurons. Three of those subpopulations are indeed peptidergic (CGRP+), and only one is nonpeptidergic (CGRP-; Patil et al., 2018).

Dorsal root ganglia neurons reveal a gradient of expression of TRPV1. Small neuronal bodies and a range of lightly stained neurons of various sizes are densely stained (Goswami et al., 2014). In the rat DRG, colocalization of TRPV1 with the NF200 marker was reported to occur in 11% of the total TRPV1+ neurons (Mitchell et al., 2014). In the mouse, however, TRPV1-induced reporter genes marked approximately 5% of myelinated NF200+ neurons. Thus, within the peptidergic population, there is one small group of myelinated TRPV1+ neurons (Cavanaugh et al., 2011).

Transient receptor potential Ankiryn subtype 1 is expressed by small-DRG neurons. Neurons expressing TRPA1 almost always express TRPV1 channels, and approximately half of the neurons that respond to capsaicin also respond to mustard oil or some other TRPA1 agonist (Bautista et al., 2005). Additionally, TRPA1 has a high degree of colocalization with CRGP, SP, and IB4 (Bautista et al., 2005; Kim et al., 2010). The expression of TRPA1 in medium- or large-diameter DRG neurons was almost absent (less than 6% of large neurons). TRPA1 was found to be functionally expressed by a significant proportion of nonpeptidergic neurons (NP; 80% of TRPA1-responsive neurons to MO bind IB4) but not in a great proportion of CRGP+ neurons (only 20% of CRGP+ neurons responded to MO; Barabas et al., 2012). This last is supported by transcriptomic studies in DRGs, where higher TRPA1 expression was detected in nonpeptidergic neurons than in peptidergic nociceptors (Usoskin et al., 2015; Zeisel et al., 2018).

ASIC1 and ASIC3 expression is abundant in two clusters of DRG neurons: NF and PEP. The highest expression of ASIC1 channels is reported in large myelinated Aβ neurons (NF4/5). ASIC1 is also expressed in PEP1 and TRPM8+ cells but is absent from NP neurons. Large myelinated Aβ neurons (NF4/5) also express high levels of ASIC3, which has been linked to proprioceptive mechanotransduction in this population (Lin et al., 2016). However, the highest expression of ASIC3 was found in Aδ nociceptors (PEP2), and similar to ASIC1, ASIC3 was also absent from NP neurons (Figure 3). This largely coincides with the results of mRNA detection showing that neither of the two ASIC1 isoforms, ASIC1a or ASIC1b, can be detected in IB4+ neurons and that ASIC3 expression is lowest in this group (Papalampropoulou-Tsiridou et al., 2020). Contrary to the ASIC1 and ASIC3 subunits, the expression of the least sensitive ASIC2 subunit is almost homogenous across the DRG. Thus, ASIC1 and ASIC3 are not expressed by nonpeptidergic nociceptors, which agrees with electrophysiological, immunohistochemical, and in situ hybridization studies.

Transient receptor potential vanilloid 1 expression is a feature of all PEP neurons, including C-type nociceptors, Aδ nociceptors, and TRPM8+ neurons. Additionally, TRPV1 was also present in the two groups of NP nociceptors. This pattern of TRPV1 largely coincides with the expected results of electrophysiological (Patil et al., 2018) and reporter gene studies (Cavanaugh et al., 2011). The expression of TRPA1 is very restricted: it is found in specific subpopulations of PEP but mostly in NP neurons. The expression of both TRP channels is negligible in Aβ myelinated neurons. Thus, TRPV1 is characteristic of all peptidergic populations, whereas TRPA1 expression is more restricted (Figure 3).

Only three subpopulations of PEP neurons combine acid-sensitive ASIC and TRP channel expression: PSPEP1 (Aδ nociceptors), PSEP2, and PSEP3 (both mechano-heat noxious neurons; Figure 3). This observation agrees with the findings of the distribution of acid-induced currents, we reviewed in the previous section, in which we concluded that there is a prevalence of acid-induced currents in the peptidergic C-nociceptor population. Thus, based on these two lines of evidence, we hypothesize that peptidergic nociceptors are better suited to transduce H⁺ stimuli than nonpeptidergic nociceptors.

For clarity, in all sections ahead, we will exclude the PSNP6–8 TRPM8+ cluster of cold-related neurons, which are unrelated to acid nociception, and the PSNP1 TH+ cluster of C-LTMRs, as that population does not sense noxious stimuli.

PEP2 (PSEP1) neurons are lightly myelinated Aδ nociceptors. This cluster of Aδ fibers is different from the Aδ-LTMRs innervating longitudinal lanceolate endings (Zimmerman et al., 2014), which are likely represented by the PSNF1 cluster (Figure 3). In addition to the myelin marker NF200, they also express CGRP and the TrkA receptor (Fang, 2005), the latter of which do not stain Aβ myelinated neurons. Aδ nociceptors have some degree of overlap with Aβ fibers with respect to diameter and conduction velocities, and they constitute less than 4% of total DRG neurons (Djouhri and Lawson, 2004; Lawson et al., 2019). Additionally, Aδ nociceptors are considered to be sensors of the “first pain” to noxious heat (which humans perceive as a cold sensation; Treede et al., 1998). Cutaneous Aδ nociceptors sensitize to mechanical stimuli after inflammation, similar to their C-fiber counterparts (Andrew and Greenspan, 1999; Potenzieri et al., 2008). Thus, Aδ nociceptors have a mechano-heat-noxious role as PEP1 C fibers do but with faster conduction velocities.

PEP1 (PSPEP2–5) neurons are unmyelinated nociceptors. As a group, they are defined by the expression of the marker Tac1. This nociceptor group is formed by classical mechano-heat noxious neurons (Emery and Ernfors, 2020). Notably, the subpopulations PSPEP2 and PSPEP3 combine TRPV1 and TRPA1 expression and could represent the main thermal nociceptors (i.e., C-type heat nociceptors) given that the triple deletion of TRPV1+TRPA1+TRMP3 abolishes noxious heat perception (Vandewauw et al., 2018). In addition, it is possible that the so-called “silent nociceptors” could correspond to some subpopulation of the PEP1 cluster. These unresponsive neurons are likely represented by mechanical-insensitive and heat-insensitive type C-neurons (cMiHis) and are present in both PEP and NP nociceptors (Lawson et al., 2019). Silent nociceptors are a group of peptidergic (CGRP+, IB4−, and TrkA+) C neurons expressing CHRNA3 and are sensitized to mechanical stimuli upon inflammation (Prato et al., 2017). Interestingly, the inflammation mediator PGE2 sensitizes C-type mechanically insensitive nociceptors (CMi). Whether thermal and/or silent nociceptors are indeed represented by discrete subpopulations of the PEP1 cluster remains to be demonstrated.

Another possibility is that PEP1 subpopulations could represent different transcriptional states of the same neuronal group, as was considered by Usoskin et al. (2015), to reconcile the evidence for polymodality with the evidence for neuronal diversity. A recent study described the stimulus responsiveness (i.e., mechanical, heat, mechanical-heat, etc.) of DRG neurons innervating cutaneous tissue, in which neurons were clustered based on single-cell transcriptional profiling of 28 selected genes (Adelman et al., 2019). Cutaneous PEP neurons include two main types of nociceptors: C-type heat nociceptors (CHs) and polymodal C-type mechanical and heat nociceptors (CMHs), whereas NP neurons mainly include C-type mechanical nociceptors (CMs) and polymodal CMHs (Adelman et al., 2019).

Both groups of cutaneous nociceptors, PEP and NP, include CMHs, which are the most common type of C-type polymodal nociceptors (cPMNs) in the DRG (Dubin and Patapoutian, 2010; Lawson et al., 2019). Since the majority of CMHs and cPMNs in general are nonpeptidergic IB4+ nociceptors (Rau et al., 2009; Adelman et al., 2019), the role of PEP1 neurons as polymodal nociceptors seems less likely. Previous reports in cutaneous peptidergic neurons (SP+) of mice showed that CMHs also responded to the application of acid (pH 5) via ASIC3 (Price et al., 2001).

There are important differences between peptidergic nociceptors innervating muscle and cutaneous tissue. Unlike cutaneous DRG neurons, neurons innervating muscle appear to be clustered in different subpopulations; in particular, the PEP cluster of nociceptors displays more transcriptional variation, suggesting different subtypes in comparison to skin-innervating peptidergic neurons (Adelman et al., 2019). Importantly, more than 50% of Aδ or C muscle afferents (groups III and IV) are chemosensitive, with a very low percentage being polymodal and only 18% of the muscle afferents being silent nociceptors (Jankowski et al., 2013). Chemosensitive neurons innervating muscle are classified into two groups: (1) metaboreceptors, which sense a slight decrease in acidity (pH 7.0), low levels of lactic acid and ATP (Light et al., 2008), and (2) metabonociceptors, which respond to higher levels of lactic acid and ATP and higher acidity (pH 6.6). Both Aδ and C muscle-innervating metabonociceptors were found to be TRPV1+ and ASIC3+, whereas nonnociceptive metaboreceptors were TRPV1− and ASIC3− (Jankowski et al., 2013). This suggests that acid-sensitive Aδ and C muscle nociceptors are likely to be PEP neurons, the only cluster containing subpopulations expressing TRPV1 and ASIC3 together.

Thus, the assignment of distinct functional roles among PEP subpopulations is further complicated by the different innervated target tissues. It is more frequent for peptidergic (CGRP+) fibers to terminate in deeper tissues than nonpeptidergic (IB4+ or MrgprD+) fibers (Zylka et al., 2005). Accordingly, the majority of visceral afferents are peptidergic (CGRP+; Robinson and Gebhart, 2008), and the majority of silent nociceptors also innervate deep tissues (Prato et al., 2017). In fact, it has been argued that the PSPEP2 subpopulation might innervate cutaneous tissue, and the PSPEP3 subpopulation innervates deeper tissues (Emery and Ernfors, 2020).

ASIC3 and TRPV1 were coexpressed in only three peptidergic subpopulations of DRG neurons. PSEP1 neurons have a defined role as myelinated nociceptors and are susceptible to sensitization by inflammation. PSEP2 and PSPEP3 neurons likely represent heat nociceptors but, since they are part of the PEP1 cluster, may include mechanical-heat polymodal fibers, especially in the case of cutaneous afferents. In muscle afferents, it is possible that the PSEP2 subpopulation senses noxious acidosis.