The ability to sense pain is critical to our survival. Normally, pain is provoked by intense heat or cold temperatures, strong force or a chemical stimulus, for example, capsaicin, the pain-provoking substance in chili peppers. However, if nerve fibers in the arms or legs are damaged, pain can occur in response to touch or pressure stimuli that are normally painless. This hypersensitivity is called mechanical allodynia.

A protein called calcitonin gene-related peptide, or CGRP, has been implicated in mechanical allodynia and other chronic pain conditions, such as migraine. CGRP is found in, and released from, the neurons that receive and transmit pain messages from tissues, such as skin and muscles, to the spinal cord. However, only a few distinct groups of CGRP-expressing neurons have been identified and it is unclear if these nerve cells also contribute to mechanical allodynia.

To investigate this, Löken et al. genetically engineered mice so that all nerve cells containing CGRP produced red fluorescent light when illuminated with a laser. This included a previously unexplored group of CGRP-expressing neurons found in a part of the spinal cord that is known to receive information about non-painful stimuli. Using neuroanatomical methods, Löken et al. monitored the activity of these neurons in response to various stimuli, before and after a partial nerve injury. This partial injury was induced via a surgery that cut off a few, but not all, branches of a key leg nerve.

The experiments showed that in their normal state, the CGRP-expressing neurons hardly responded to mechanical stimulation. In fact, it was difficult to establish what they normally respond to. However, after a nerve injury, brushing the mice’s skin evoked significant activity in these cells. Moreover, when these CGRP cells were artificially stimulated, the stimulation induced hypersensitivity to mechanical stimuli, even when the mice had no nerve damage. These results suggest that this group of neurons, which are normally suppressed, can become hyperexcitable and contribute to the development of mechanical allodynia.

In summary, Löken et al. have identified a group of nerve cells in the spinal cord that process mechanical information and contribute to touch-evoked pain. Future studies will identify the nerve circuits that are targeted by CGRP released from these nerve cells. These circuits represent a new therapeutic target for managing chronic pain conditions related to nerve damage, specifically mechanical allodynia, which is the most common complaint of patients with chronic pain.

Calcitonin gene-related peptide (CGRP) is the most prominent molecular marker of the peptidergic subpopulation of primary afferent nociceptors (Basbaum et al., 2009). When released from peripheral terminals of sensory neurons, CGRP acts on endothelial cells that line blood vessels, producing pronounced vasodilation (Brain et al., 1985). Recent efforts to develop novel therapeutics in the management of migraine led to the successful development of antibodies that scavenge CGRP, reducing the vasodilation that triggers migraine (Ho et al., 2010). When released into the superficial dorsal horn from the central branches of sensory neurons, CGRP, along with its co-occurring neuropeptide, substance P, potentiates the glutamatergic excitation of postsynaptic neurons, contributing to injury-provoked central sensitization (Ryu et al., 1988; Woolf and Wiesenfeld-Hallin, 1986). The latter process, in turn, contributes to the ongoing pain and profound hypersensitivity characteristic of both inflammatory and neuropathic pains. Interestingly, a recent study showed that pharmacological inhibition of CGRP receptor signaling in the periphery alleviates incision-induced mechanical and heat hypersensitivity, but not neuropathic pain, suggesting that primary sensory neuron-derived CGRP differentially influences injury-induced persistent pain (Cowie et al., 2018).

Despite much earlier reports, which used colchicine to enhance somatic CGRP levels (Kruger et al., 1988; Tie-Jun et al., 2001) and a more recent report (McCoy et al., 2012) of small CGRP-positive cells in the dorsal horn of a reporter mouse, the prevailing view is that dorsal horn CGRP derives exclusively from afferents. Here, we took advantage of a tamoxifen-inducible Calca^CreER mouse line, which when crossed with a tdTomato reporter mouse, reveals a discrete population of CGRP-expressing interneurons that are concentrated in lamina III and inner lamina II of the spinal cord dorsal horn and trigeminal nucleus caudalis. Unlike dorsal horn vertical cells, which have ventrally directed dendrites and a dorsally directed axon, the CGRP interneurons have mainly dorsally directed dendrites and ventrally directed axons. A comprehensive functional analysis showed that these interneurons are minimally responsive to a host of acute, innocuous or noxious mechanical and chemical stimuli, despite the fact that electrical stimulation of Aβ afferents readily activates the cells. On the other hand, an innocuous mechanical stimulus evoked significant Fos expression in the setting of peripheral nerve injury and chemogenetic activation of the interneurons produced clear mechanical hypersensitivity. Conversely, caspase-mediated ablation of the neurons increased mechanical thresholds. We conclude that these CGRP-expressing interneurons engage deep dorsal horn nociresponsive circuits that contribute either to ascending circuits originating in deep dorsal horn or to the reflex circuits in baseline conditions, but not in the setting of nerve injury.

To map the distribution of CGRP-expressing neurons in the dorsal horn, we first crossed the Calca^CreER mouse line with a ROSA26^fs-tdTomato (Ai14) mouse line, hereafter referred to as CGRP-tdTomato. Adult mice were administered tamoxifen twice (150 mg/kg, at postnatal days 21–23), and as reported previously, this triggered tdTomato expression in primary sensory neurons (Patil et al., 2018). However, we also recorded significant labeling of neurons in the dorsal horn and trigeminal nucleus caudalis (N. Caudalis; Figure 1). Importantly, because the tamoxifen is administered at 3–4 weeks of age, we conclude that the pattern of expression is reflective of that found in the adult.

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We first confirmed the approach by ensuring that the tdTomato-expressing primary sensory neurons of the dorsal root ganglia (DRG) double-label with an antibody to CGRP. Figure 1 illustrates that 80% of tdTomato-positive neurons in trigeminal ganglia (TG) and DRG immunostained for CGRP and that 78% of the CGRP immunoreactive neurons were tdTomato-positive (Figure 1a–d).

Consistent with the central projection of CGRP-expressing sensory neurons, we also observed very dense tdTomato-positive terminals in the superficial laminae of the dorsal horn and nucleus caudalis. We also recorded many tdTomato-labeled neurons in regions of the central nervous system known to contain significant populations of CGRP-immunoreactive neurons or terminals, including motoneurons in the ventral horn of the spinal cord (Figure 1—figure supplement 1), the parabrachial nucleus (Figure 1—figure supplement 2), subparafascicularis of the thalamus (Figure 1—figure supplement 3; Yasui et al., 1991), and central nucleus of the amygdala (Figure 1—figure supplement 3) and in cranial motor nuclei (Figure 1—figure supplement 4). We conclude that the pattern of CGRP-expression observed in the Calca^CreER mouse provides a reliable marker of CGRP-expressing neurons in the adult.

Unexpectedly, we also found large numbers of small tdTomato-positive neurons in the superficial dorsal horn and nucleus caudalis (notably in lamina III) and occasionally in more superficial layers (Figure 1e–f: Figure 8—figure supplement 1c). Consistent with previous literature, we did not detect CGRP-immunoreactivity in dorsal horn neurons using well-validated antibodies. However, by in situ hybridization we confirmed that Calca mRNA is present in neurons in the same regions of the spinal cord dorsal horn and nucleus caudalis (Figure 1g–h), which is consistent with the single cell PCR reports of Calca message in subpopulations of dorsal horn neurons (Häring et al., 2018; Sathyamurthy et al., 2018). We speculate that the lack of CGRP immunostaining reflects rapid transport of the peptide from the cell body to its axon, which undoubtedly underlies the requirement for colchicine to demonstrate these neurons by immunocytochemistry (Kruger et al., 1988; Tie-Jun et al., 2001). We found the CGRP-positive interneurons to be particularly abundant at the most caudal levels of the nucleus caudalis, markedly decreasing rostrally as the hypoglossal nucleus appears (Figure 1—figure supplement 4).

CGRP dorsal horn neurons are excitatory interneurons

We next asked whether these CGRP-expressing neurons include both projection and interneurons. First, we injected the retrograde tracer Fluorogold (1%) into several brain areas that receive projections from the spinal cord dorsal horn. Despite an extensive analysis, which included injections into the ventrobasal and nucleus submedius (Yoshida et al., 1991) of the thalamus, lateral parabrachial nucleus (see Figure 5—figure supplement 2b–c for injection site in the parabrachial nucleus), and dorsal column nuclei, which are targeted by postsynaptic dorsal column neurons located in the region of lamina IV of the dorsal horn, we found no evidence of CGRP-expressing projection neurons. This finding was confirmed with an anterograde-tracing approach in which we injected an AAV1-flex-GCaMP6s virus unilaterally into the nucleus caudalis of Calca^CreER/tdTomato mice (Figure 5—figure supplement 2a). After 4 weeks, we examined the brainstem, thalamus, and hypothalamus for GFP-labeled fibers, but found no evidence of long-distance axonal projections deriving from the lamina III CGRP cells.

By immunolabeling the CGRP-tdTomato neurons, we next determined that these cells are excitatory and define a unique subset of interneurons. First, the CGRP-tdTomato cells co-express Lmx1b (98%; 92/94 tdTomato cells), but not Pax2 (Figure 2—figure supplement 1), which are excitatory and inhibitory markers, respectively. Some of the CGRP-tdTomato cells populate inner lamina II, and here approximately 16% co-expressed PKCγ (31/187 tdTomato cells), a marker of a large population of excitatory interneurons (Malmberg et al., 1997). Sixty-three (97/158 tdTomato cells) and 9% (9/97 tdTomato cells) of the CGRP interneurons co-expressed calbindin and calretinin, respectively, calcium binding proteins that mark subpopulations of excitatory dorsal horn interneurons (Figure 2). The incomplete immunohistochemical overlap with major neurochemical classes of dorsal horn interneurons indicates that the CGRP interneurons are heterogeneous consistent with previously described populations of dorsal horn neurons. However, as there is a limited number of quality antibodies that can be used for comprehensive neurochemical profiling we turned to in situ hybridization (Figure 3).

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Figure 3

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Consistent with the concentration of tdTomato-CGRP interneurons in lamina III, particularly notable is that 56% of the Calca mRNA-expressing (CGRP) cells double-labeled for Rora message (639/1134 Calca mRNA-expressing cells), a marker of excitatory interneurons in lamina III (Bourane et al., 2015b). Interestingly, however, only 4% co-expressed Cck (27/595 Calca mRNA-expressing cells), which marks a significant subset of the RORα population (Liu et al., 2018). As for other populations of excitatory interneurons, we found minimal overlap with the population that transiently expresses VGLUT3 (examined at P7) (Peirs et al., 2015) or others that express Nptx2, BDNF or the NK1 receptor, a marker of many projection neurons. Similar results were found in the dorsal horn of the spinal cord and in the trigeminal nucleus caudalis (viz., dorsal horn of the medulla). We conclude that a substantial portion of the CGRP interneuron population overlaps with a subset of the Cck-negative RORα population of lamina III interneurons.

CGRP interneurons have dorsally directed dendritic arbors and are innervated by VGLUT1-expressing terminals

Despite the very intense tdTomato labeling of the cell bodies of the dorsal horn neurons, it was difficult to distinguish axonal processes from the dense primary sensory neuron-derived CGRP innervation. This was particularly the case when an antibody to tdTomato was used to detect the dorsal horn CGRP neurons. And unfortunately, although the cell body of the intracellularly recorded cells was readily filled with biotin dextran in electrophysiological slice preparations (see below), we never successfully filled dendrites or axons. Therefore, in a separate set of experiments, we first reduced the complement of primary afferent-derived CGRP-derived by making an intrathecal injection of capsaicin, 7 days prior to perfusing the mice (Cavanaugh et al., 2009). In addition, tdTomato-immunoreactivity was revealed with immunoperoxidase staining so that sections could be analyzed by either light or electron microscopy (EM). The results from this approach were both striking and especially informative. Figure 4 illustrates that the CGRP interneurons have many dorsally-directed, spine-laden dendrites. These dendritic arbors often penetrated lamina II, and some labeled processes appeared to reach lamina I. Nevertheless, despite the capsaicin treatment, the latter were rare and difficult to distinguish from residual primary afferent-derived CGRP.

Figure 4

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Based on their remarkably uniform dendritic morphology, the dorsal horn CGRP neurons appear to represent a subpopulation of excitatory, so-called radial interneurons (Grudt and Perl, 2002); however, the morphology of the CGRP-expressing radial interneurons differ considerably from those previously described in lamina II. First, the majority of lamina II radial cells have dendrites that arborize ventrally and axons that, if anything, project and collateralize dorsally, occasionally targeting presumptive projection neurons in lamina I. In contrast, not only do the CGRP interneurons have dorsally-directed dendrites, but almost all of their axons project ventrally and/or ventrocaudally. In some instances, we could trace the axons well into the neck of the dorsal horn, including lamina V (Figure 5 and Figure 5—figure supplement 1). Furthermore, EM analysis of these interneurons (Figure 5) illustrates that there is significant synaptic input to the soma, dendrites, and spines of the CGRP interneurons. Finally, given the concentration of the CGRP interneurons in lamina III, we assumed that they receive primary afferent input from large myelinated afferents. Indeed when we double-immunostained for tdTomato and VGLUT1, a glutamate transporter that is highly expressed in large myelinated afferents (Oliveira et al., 2003), we observed many close appositions of VGLUT1-immunoreactive axon terminals onto the cell bodies and dendrites of the CGRP interneurons (Figure 4f–j).

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CGRP-tdTomato interneurons receive low threshold primary afferent input

To confirm that the VGLUT1 appositions indeed mark a monosynaptic input from Aβ afferents to the CGRP-tdTomato interneurons, we prepared transverse lumbar and caudal medullary slices (350–400 µm) from 3-week-old mice for whole-cell patch-clamp recordings. The slices contained large numbers of fluorescent tdTomato-labeled CGRP neurons (Figure 6a–c). We first characterized the intrinsic properties of the CGRP-tdTomato neurons by inducing depolarizing current steps. The CGRP-tdTomato neurons in the dorsal horn and nucleus caudalis showed mostly delayed firing patterns, consistent with their excitatory and radial phenotype (delayed 19, tonic 1, reluctant 2, single 2, no response 3, Figure 6—source data 1 table). In some preparations we stimulated an attached dorsal root. At near threshold stimulation intensities (10 Hz), we recorded a very short latency component, which likely corresponds to a monosynaptic Aβ-fiber input. Of five cells recorded in three mice, all received monosynaptic Aβ input. In two additional mice, we recorded from four cells that responded to dorsal root stimulation, but we could not unequivocally establish whether they received a monosynaptic input (Figure 6d–e). Overall, the intrinsic properties of neurons recorded from lumbar dorsal horn (22 cells, eight mice) and nucleus caudalis (5 cells, two mice) were comparable (see Figure 6—source data 1 table). Taken together, we conclude that the predominant (monosynaptic) input to the CGRP interneurons derives from low threshold (Aβ) mechanoreceptors.

Figure 6

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Figure 6—source data 1

Electrophysiological properties of CGRP-tdTomato interneurons in the dorsal horn and nucleus caudalis.

Most CGRP-tdTomato neurons showed delayed firing patterns (delayed 19, tonic 1, reluctant 2, single 2, no response 3). Based on electrical stimulation of dorsal roots, we conclude that CGRP interneurons in the lumbar cord predominantly receive monosynaptic input from Aβ primary afferent fibers.

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Blocking tonic inhibition increases excitability of the CGRP interneurons

In a separate set of experiments, we specifically sought evidence that the neurons, under baseline conditions, are under inhibitory control. To this end, cells were patched and then rheobase determined, before and after application of a combination of bicuculline and strychnine. Figure 7a–d illustrate that concurrent blocking of the GABA and glycine receptors significantly reduced rheobase, from 46.0 ± 7.4 pA before antagonist treatment to 31.0 ± 4.9 pA after antagonist treatment (two-tailed, paired T-test; p=0.0005, n = 25). Of the 25 neurons studied, rheobase decreased in 21, increased in one and did not change in 3. Figure 7c shows that application of bicuculline and strychnine to neurons in which current was maintained 10 pA below rheobase also generated action potentials. Figure 7e shows that resting membrane potential also showed a significant depolarization after application of the GABA and glycine receptor antagonists, from −53.8 ± 1.8 mV before antagonist treatment to −49.1 ± 1.7 mV after antagonist. Taken together, these results demonstrate that the CGRP interneurons, under resting conditions, are under tonic inhibitory control.

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Mechanical stimuli only activate CGRP neurons (induce Fos expression) in a nerve injury setting

To provide a global activity measure of the stimuli that engage the CGRP interneurons, we first monitored Fos expression using a battery of noxious and innocuous stimuli. As expected, a unilateral injection of dilute formalin into the cheek (10 µl of 2% formalin, Figure 8—figure supplement 1c) or a unilateral hindpaw injection of capsaicin (Figure 8—figure supplement 2a–b), produced considerable Fos immunolabeling of dorsal horn neurons, but not of the CGRP-tdTomato interneurons (Figure 8—figure supplements 1c and 2a–b). Unexpectedly, however, selectively engaging non-nociceptive afferents by having the animal walk for 90 min on a rotarod, which provokes considerable Fos in laminae III and IV (Neumann et al., 2008), did not induce Fos expression in the CGRP interneurons (Figure 8—figure supplement 1a). The same was true for brushing of the cheek, another innocuous stimulus that activates Aβ afferents (Figure 8). Finally, although CGRP is strongly implicated in the generation of migraine, largely but not exclusively via its peripheral vasodilatory action (Brain et al., 1985), systemic injection of nitroglycerin, which triggers migraine in humans and profound mechanical hypersensitivity in animals (Bates et al., 2010), did not induce Fos in the CGRP interneurons (Figure 8—figure supplement 1b).

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We conclude that despite our electrophysiological evidence that Aβ afferents engage the CGRP interneurons, there does not appear to be sufficient input to activate these cells under natural innocuous mechanical stimulus conditions in uninjured mice (5.3%, 5/88 tdTomato cells; Figure 8a). We, therefore, next asked whether an injury state would render the CGRP interneurons more responsive to an innocuous stimulus. In fact six days after inducing the spared nerve injury (SNI) model of neuropathic pain, we found that brushing the ipsilateral paw evoked Fos expression in 50% (53/110 tdTomato cells) of the dorsal horn CGRP interneurons (Figure 8c and d). Importantly, although we recorded significant dorsal horn Fos expression in nerve-injured mice without brushing (Figure 8b), no Fos expression occurred in the CGRP interneurons (3%; 6/205 tdTomato cells). We conclude that activation of the CGRP interneurons only occurs when the innocuous input, which could include contact of the plantar surface of the paw with the ground (Liu et al., 2018), engages the interneurons in the setting of nerve injury.

Dorsal horn CGRP interneurons contribute to mechanical hypersensitivity in vivo

As electrical stimulation of the dorsal root at Aβ intensity readily excites the CGRP interneurons, the inability of brush stimulation to activate the neurons in the absence of injury was surprising. The discrepancy may reflect the fact that dorsal root stimulation involves a synchronous activation of many primary sensory neurons. In contrast, natural stimuli (e.g. brushing or walking on a rotarod) trigger an asynchronous afferent drive. However, as brushing was effective in the nerve injury setting, we hypothesized that a central sensitization rendered the CGRP neurons hyperexcitable. To test this hypothesis, we asked whether a different mode of activation, namely chemogenetic (direct) activation of the CGRP interneurons, could generate behaviors indicative of mechanical allodynia, comparable to what is observed in response to innocuous mechanical stimuli in the setting of nerve injury.

In these studies, we used an intersectional approach to target expression of a Designer Receptor Exclusively Activated by Designer Drugs (DREADD) selectively in the CGRP interneurons. To this end, we crossed the Calca^creER mice to a FLPo mouse line, driven by the Lbx1 gene. The latter gene is only expressed in neurons of dorsal spinal cord and hindbrain, but not in sensory neurons of the DRG (Bourane et al., 2015b). We then made a unilateral microinjection of an adenoassociated virus (AAV) expressing a Cre and FLPo-dependent DREADD (hM3Dq) into the dorsal horn of the Calca^CreER/FLPo mice. Four weeks later, we evaluated the behavioral effects of a systemic injection of CNO, which activates the DREADD.

We first established that there was no constitutive effect of virus infection. Thus, CNO injection, compared to saline, did not alter the latency to fall from an accelerating rotarod (Figure 9c). Furthermore, baseline von Frey mechanical thresholds of the DREADD-expressing mice, measured prior to injection of CNO, did not differ from mice injected with the AAV-GFP virus. In distinct contrast, Figure 9 shows that CNO injection in the experimental group produced a significant reduction of von Frey threshold of the ipsilateral hindpaw, compared to baseline or to saline-injected mice (Figure 9b). Mechanical thresholds did not change from baseline in the AAV-GFP control animals, whether they received saline or CNO (Repeated Measures Two-way ANOVA, F_(1,20)=6.964, p=0.012, interaction effect between DREADD group and CNO treatment). The groups contained the same numbers of males and females (DREADD animals: 8 of each; GFP controls: 3 of each), but there was no significant interaction between sex and treatment (CNO versus saline). Nor did factoring in sex reduce the error (R²) in the full Repeated Measures Two-way ANOVA. Consistent with a contribution of CGRP-expressing interneurons to mechanical sensitivity, mice in which we ablated selectively the CGRP-expressing spinal cord interneurons with a virally derived caspase (Figure 10a and Figure 10—figure supplement 1) exhibited significantly higher mechanical thresholds than did control mice (Figure 10b). On the other hand, and somewhat unexpectedly, the mechanical hypersensitivity produced in the SNI model of neuropathic pain was not altered by the ablation.

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Lastly, we evaluated heat and cold responsiveness after CNO injection. Neither latency to withdraw the hindpaw to noxious heat in the Hargreaves test (n = 16; Figure 9d) nor time spent paw lifting after exposure of the plantar surface of the hindpaw to a cold (acetone) stimulus (n = 11; Figure 9e), differed when comparing CNO and control saline injection (p>0.05, Students T-test and Wilcoxon Signed Ranks Test, respectively). Similarly, responses to noxious heat (Hargreaves; Figure 10c) or cold (acetone; Figure 10d) stimuli were unchanged after caspase-mediated ablation of the CGRP-expressing interneurons. We conclude that direct and likely synchronous activation of the CGRP interneurons produces a selective mechanical hypersensitivity, mimicking the mechanical allodynia observed in response to low threshold (Aβ) mechanical stimulation (brush) in the setting of nerve injury.

CGRP interneurons and itch

Based on their single cell transcriptome analysis, Häring and colleagues (Häring et al., 2018) concluded that several populations of dorsal horn excitatory neurons that express Calca mRNA co-express gastrin-releasing peptide (GRP), a peptide linked to dorsal horn circuits that drive itch-provoked scratching (Albisetti et al., 2019; Sun and Chen, 2007). To confirm this, we performed double in situ hybridization for Calca and Grp. Although the Grp interneurons predominated in a band just dorsal to the Calca interneurons, consistent with our previous report (Solorzano et al., 2015), we did find several instances of co-localization of Calca mRNA and Grp mRNA. Interestingly, however, when using immunohistochemistry, we found almost no overlap of GRP and CGRP in a double transgenic GRP-GFP/CGRP-tdTomato mouse line (Figure 11a–d). This difference is likely related to the fact that neurons labeled in the reporter mouse constitute less than half of the Grp mRNA-positive population (Solorzano et al., 2015; Dickie et al., 2019). Despite these discordant findings, we also examined the pattern of Fos expression provoked by injection of chloroquine (CQ), a strong pruritogen, into the cheek or hindpaw. To prevent scratching-induced Fos, the CQ injections were performed in anesthetized mice. As Figure 11e–f illustrates, despite considerable chloroquine-induced Fos expression, we found only an occasional double-labeled neuron. Furthermore, the number of scratching bouts induced by a subcutaneous calf injection of 100 μg chloroquine was comparable between control and CGRP-ablated mice (Figure 10e). We conclude that the CGRP interneurons, despite some overlap with GRP, likely do not transmit chemical itch, a finding consistent with the effects of deleting RORα (Bourane et al., 2015b). Whether the CGRP interneurons are engaged in conditions in which mechanical stimulation can trigger itch (alloknesis) remains to be determined.

Figure 11

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Despite overwhelming evidence that primary sensory neurons are the predominant source of dorsal horn CGRP, here we describe a morphologically uniform population of dorsal horn CGRP-expressing interneurons. Many of these interneurons correspond to the Cck-negative subset of the RORα population in lamina III of the dorsal horn and trigeminal nucleus caudalis, are excitatory and are activated by electrical stimulation of non-nociceptive, Aβ primary afferents. In contrast to the Cck-expressing subset of RORα neurons, and despite their location in the so-called, low threshold mechanoreceptive recipient zone of the dorsal horn (Abraira et al., 2017), the CGRP interneurons do not express Fos in response to natural Aβ-mediated, innocuous mechanical stimulation (brushing or walking on a rotarod). We hypothesize that this reflects competition with the ongoing inhibition of these neurons (see below). As for the RORα population, the CGRP interneurons do not respond to noxious chemical stimulation. Even peripheral nerve injury, without superimposed stimulation, did not activate these neurons. On the other hand, brush stimulation in the nerve injury setting did activate the CGRP interneurons. Furthermore, and consistent with a limited contribution of these neurons in the setting of nerve injury, ablation of CGRP interneurons did not influence the magnitude of mechanical allodynia that develop following peripheral nerve injury. Interestingly, however, brush stimulation in the nerve injury setting did induce Fos in the CGRP interneurons. This distinction suggests that unless these neurons are rendered hyperexcitable, as occurs after nerve injury, only synchronous afferent input or direct neuronal sensitization (e.g. by DREADD activation) is sufficient to engage the circuits in which the CGRP interneurons participate. Consistent with this conclusion, chemically provoked (chemogenetic) synchronous activation of these neurons produced a significant mechanical hypersensitivity and conversely their ablation increased mechanical thresholds. Based on the predominant ventrally directed axonal arbors of these interneurons we suggest that the dorsal horn CGRP interneurons contribute either to ascending circuits originating in deep dorsal horn or to the reflex circuits in baseline conditions, but not in the setting of nerve injury. The fact that nerve injury-induced mechanical hypersensitivity persisted after ablation of the CGRP interneurons undoubtedly reflects the major contribution of other mechanosensitive afferents and dorsal horn interneurons. Indeed, we previously reported that the MrgprD subpopulation of sensory neurons is an important driver of the nerve-injury induced mechanical sensitivity (Cavanaugh et al., 2009) and these afferents target interneurons located dorsal to the predominant band of CGRP interneurons.

RNA-Seq analyses have now defined at least 15 subsets of excitatory interneurons and 15 subsets of inhibitory neurons in the dorsal horn of the spinal cord (Häring et al., 2018; Sathyamurthy et al., 2018). Ablation, optogenetic and chemogenetic studies further characterized those classes based on functional properties. Of note, an increasing number of dorsal horn interneurons that ‘gate’ mechanical pain have been identified. These include neurochemically distinct excitatory interneuron populations: transient VGLUT3, somatostatin, RORα, calretinin, and Tac1 (Bourane et al., 2015a; Cheng et al., 2017; Duan et al., 2014; Huang et al., 2019; Peirs et al., 2015; Petitjean et al., 2019) and distinct inhibitory interneuron populations: dynorphin, calretinin, parvalbumin, and enkephalin (Boyle et al., 2019; Duan et al., 2014; François et al., 2017; Petitjean et al., 2019; Petitjean et al., 2015). The CGRP-expressing interneurons define yet another population of dorsal horn interneurons that contributes to spinal cord processing of mechanical inputs. Interestingly, there is a striking laminar organization of these molecularly distinct populations of interneurons. For example, the transiently expressing VGLUT3 population is located ventral to the CGRP interneurons, receives low-threshold mechanoreceptive input and their chemogenetic activation also enhances mechanical sensitivity (Cheng et al., 2017; Peirs et al., 2015). Dorsal to the CGRP interneuron are PKCγ and calretinin excitatory interneurons that contribute to nerve injury induced mechanical allodynia (Malmberg et al., 1997; Neumann et al., 2008; Peirs et al., 2015; Petitjean et al., 2019; Smith et al., 2019).

To what extent these mechanically driven neuronal populations are interconnected or whether they represent parallel, independent circuits activated under different mechanical pain conditions (e.g. naive vs injury vs inflammation) remains to be determined. Here, the unique morphology of the CGRP interneurons is instructive. In contrast to many of the interneuron populations whose axons arborize longitudinally (e.g. PKCγ cells) or dorsally (e.g. calretinin cells), the CGRP interneurons have ventrally-directed axons. In some respects, the CGRP interneurons resemble the lamina II radial cells described by Grudt and Perl, 2002 in the mouse, many of which are nociceptive, and the lamina III interneurons demonstrated in Golgi preparations in the cat and primate (Beal and Cooper, 1978; Maxwell, 1985). The fact that the CGRP interneurons show delayed firing patterns is also consistent with the properties of excitatory lamina II radial cells (Dickie et al., 2019; Grudt and Perl, 2002; Punnakkal et al., 2014; Yasaka et al., 2010). Surprisingly, despite their dorsal dendrites, which extend into lamina II, where many nociceptive afferents terminate, we found no evidence that the CGRP interneurons are activated by acute noxious inputs (capsaicin or formalin). On the other hand, we did detect an occasional polysynaptic input following synchronous electrical stimulation of primary afferent C fibers. Most importantly, compared to the lamina II radial cells, we recorded much more extensive ventral axon trajectories of the CGRP interneurons, which suggests that these interneurons engage very different circuits in the dorsal and potentially ventral horn. In this regard, the CGRP interneurons are distinct from the calretinin interneurons that target lamina I projection neurons (Petitjean et al., 2019).

An RNA sequencing study of dorsal horn interneurons demonstrated expression of Calca, the gene that encodes CGRP, in different clusters of neurons (Häring et al., 2018), including several that express Rora, the gene that encodes RORα. Consistent with those results, our in situ hybridization studies found extensive co-expression of Calca and Rora. In fact, almost 55% of the CGRP interneurons co-express RORα message and there are significant similarities in their anatomical and functional properties (Bourane et al., 2015b). Specifically, the majority of RORα interneurons are excitatory and approximately 1/3 has a radial morphology, with ventrally arborizing axons. Furthermore, both the CGRP and RORα interneurons receive a monosynaptic Aβ afferent input and interestingly, despite the lack of response to capsaicin, some neurons in both populations receive a polysynaptic A delta and C input. Consistent with the report that deletion of the RORα population did not influence itch (Bourane et al., 2015b), and despite some overlap of the CGRP and GRP subsets of interneurons, we found that pruritogens did not activate (induce Fos) in the CGRP interneurons and ablation of CGRP interneurons did not influence scratching in response to exogenous pruritogens. There are, however, some striking differences between the RORα and CGRP interneurons. For example, although a majority of the RORα interneurons co-express Cck, the CGRP interneurons rarely do. Furthermore, whereas RORα interneurons are activated by innocuous mechanical stimuli (e.g. brushing) in both naive and injured conditions, the CGRP interneurons respond to innocuous stimuli only in the setting of nerve injury.

To our knowledge, the CGRP interneurons represent the first class of excitatory interneurons in lamina III that are unresponsive to innocuous mechanical stimulation under basal conditions despite receiving a monosynaptic Aβ input. One possibility is that the CGRP interneurons are tonically inhibited under normal conditions, which is consistent with our electrophysiological recordings showing bicuculline-mediated facilitation of these interneurons. Reduction of these inhibitory inputs in the setting of injury (Torsney and MacDermott, 2006) would render the neurons responsive to an innocuous stimulus (e.g. brush). In turn, the ventrally directed axons of these interneurons could drive reflex withdrawal circuits, which is consistent with increased mechanical thresholds in the CGRP-interneuron ablated mice, and/or engage ascending nociceptive pathways located in deep dorsal horn. The fact that DREADD-mediated direct activation of many CGRP interneurons lowered mechanical withdrawal thresholds is consistent with that hypothesis. In other words, we suggest that sensitization of these neurons is critical to mechanisms that underlie Aβ-mediated mechanical allodynia in the setting of nerve injury. Interestingly, Lu et al., 2013 provided evidence for convergence of a primary afferent-derived Aβ and a tonic glycinergic inhibitory input to PKCγ interneurons, some of which we found express CGRP. Loss of this glycinergic inhibition allowed Aβ input to access lamina I nociceptive circuits. Other studies demonstrated a comparable outcome, in this case by a presynaptic glycinergic inhibition of non-nociceptive inputs to superficial dorsal horn neurons (Sherman and Loomis, 1996). Furthermore, Imlach et al., 2016 proposed that decreased glycinergic inhibition is selective for radial cells in lamina II and likely contributes to neuropathic pain. We suggest that a comparable circuit involving the CGRP radial cells could uncover low threshold inputs to ventrally located nociceptive circuits, which in recent years have been largely ignored (Wercberger and Basbaum, 2019).

All data generated or analysed during this study are included in the manuscript and supporting files. Source data file is available for figure 6.

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Author details

1. Line S Löken

   Department of Anatomy, University California, San Francisco, San Francisco, United States

   Contribution

   conceptualized and designed the study, performed the experiments, collected the data, analyzed data and wrote the manuscript

   For correspondence

   line.loken@gu.se

   Competing interests

   No competing interests declared

   Additional information

   Present affiliation: Institute of Neuroscience and Physiology, Department of Physiology, University of Gothenburg, Gothenburg, Sweden.

   "This ORCID iD identifies the author of this article:" 0000-0001-6762-9717

2. Joao M Braz

   Department of Anatomy, University California, San Francisco, San Francisco, United States

   Contribution

   conceptualized and designed the study, performed experiments and collected data, analyzed data and wrote the manuscript

   Competing interests

   No competing interests declared

3. Alexander Etlin

   Department of Anatomy, University California, San Francisco, San Francisco, United States

   Contribution

   performed the experiments, collected the data, analyzed data and wrote the manuscript

   Competing interests

   No competing interests declared

4. Mahsa Sadeghi

   Department of Anatomy, University California, San Francisco, San Francisco, United States

   Contribution

   performed experiments, collected and analysed the data

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9769-4082

5. Mollie Bernstein

   Department of Anatomy, University California, San Francisco, San Francisco, United States

   Contribution

   performed the experiments and collected the data

   Competing interests

   No competing interests declared

6. Madison Jewell

   Department of Anatomy, University California, San Francisco, San Francisco, United States

   Contribution

   performed the experiments and collected the data

   Competing interests

   No competing interests declared

7. Marilyn Steyert

   Department of Anatomy, University California, San Francisco, San Francisco, United States

   Contribution

   performed the experiments and collected the data

   Competing interests

   No competing interests declared

8. Julia Kuhn

   Department of Anatomy, University California, San Francisco, San Francisco, United States

   Contribution

   performed the experiments and collected the data

   Competing interests

   No competing interests declared

9. Katherine Hamel

   Department of Anatomy, University California, San Francisco, San Francisco, United States

   Contribution

   performed the experiments and collected the data

   Competing interests

   No competing interests declared

10. Ida J Llewellyn-Smith

    1. Discipline of Physiology, Adelaide Medical School, University of Adelaide, Adelaide, Australia
    2. Department of Cardiology, Flinders Medical Centre, Bedford Park, Australia

    Contribution

    performed the experiments, collected the data, analyzed data and wrote the manuscript

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-4269-6846

11. Allan Basbaum

    Department of Anatomy, University California, San Francisco, San Francisco, United States

    Contribution

    conceptualized and designed the study, analyzed data and wrote the manuscript

    For correspondence

    allan.basbaum@ucsf.edu

    Competing interests

    Reviewing editor, eLife

    "This ORCID iD identifies the author of this article:" 0000-0002-1710-6333

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