Somatosensory input to the spinal cord provides information about both the external environment and internal state, driving reflex responses and complex perceptual experiences including the sensation of pain. Nociception provides animals with crucial protection from mechanical, thermal, and chemical threats, but many other types of sensory input can become painful after injury, during inflammation, or in disease. An extensive body of work has explored how networks of cells in the dorsal spinal cord process noxious versus innocuous sensory input and the context-dependent plasticity in these circuits that results in pain. Many different classes of excitatory and inhibitory interneurons have been shown to be required for processing nociceptive input and the transformation of normally innocuous signals into pain. These include cells expressing select combinations of neuropeptides and their receptors, including Substance P (encoded by Tac1) and its receptor NKR1 (encoded by Tacr1). Ultimately, these spinal circuits activate projection neurons that transmit the highly processed input to the brain.

Spinal projection neurons are sparse and heterogenous in their gene expression and their anatomical location (Koch et al., 2018; Peirs and Seal, 2016; Todd, 2010) but again include cells expressing both Substance P and NKR1 (Huang et al., 2019; Chiang et al., 2020 and Choi et al., 2020). For example, one group resides in the most superficial part of the dorsal horn (Lamina I) and is thought to receive input from peripheral nociceptors, itch neurons, and thermoreceptors (Bester et al., 2000; Wercberger et al., 2020; Wercberger and Basbaum, 2019). A second set of projection neurons is located in the deep dorsal horn (Laminae II-VI) where they integrate noxious and low-threshold sensory inputs (Wall, 1967; Wercberger and Basbaum, 2019; Willis, 1983). Both groups of spinal projection neurons target multiple sites in the brainstem and thalamus, with each site proposed to have unique roles in driving different aspects of sensation, learning and behavior (Choi et al., 2020, Huang et al., 2019; Rpc, 1986; Todd, 2010). The prevailing view is that these parallel outputs combine to elicit the full pain experience. If true, then direct activation of the correct ensemble of projection neurons should produce behaviors indistinguishable from those evoked by actual noxious stimuli. By contrast, activating or silencing individual central targets might be expected to selectively affect particular aspects of pain and potentially reveal substrates that encode specific attributes of this complex state.

The lateral parabrachial nucleus (lPBN), a brainstem nucleus of a few thousand neurons in mice, receives nociceptive inputs from the projection neurons in the dorsal spinal cord (Todd, 2010) and plays a central role in pain. Two subregions of the lPBN have been defined by their distinctive gene expression patterns as well as their forebrain projections. Neurons in the dorsal PBN (dPBN) expressing dynorphin (PDyn) project predominantly to the periaqueductal gray (PAG) and ventromedial hypothalamus (VMH), while external lateral parabrachial neurons (elPBN) expressing the neuropeptides CGRP or Substance P project to the central amygdala (CeA), bed nucleus stria terminalis (BNST), insular cortex (INS), and the medullary formation (MdD) (Rodriguez et al., 2017; Palmiter, 2018; Barik et al., 2018). Recent evidence suggests that these different output pathways have differing functional specifications. For example, CGRP projections to the amygdala appear particularly important for somatic and visceral distress (Carter et al., 2013; Han et al., 2015; Campos et al., 2018; Bowen et al., 2020) and also play a role in affective touch (Choi et al., 2020). By contrast, Tac1-positive neurons projecting to the MdD (Barik et al., 2018) participate in nocifensive responses, whereas the Pdyn neurons in the dPBN are important for sensing gut stretch (Kim et al., 2020) and forming aversive memories (Chiang et al., 2020).

Here, we studied projections from the spinal cord to the PBN and discovered a population of lPBN neurons expressing Tacr1 that receive input from Tacr1-expressing spinal projection neurons. We demonstrate that this circuit is both necessary and sufficient for the heightened behavioral responses observed during ongoing noxious stimulation. Direct activation of the spinal Tacr1 neurons causes striking behaviors that closely match pain responses. Interestingly, activation of their parabrachial Tacr1 targets demonstrates that this branch of the ascending pathway is responsible for controlling the magnitude of the behavioral response and appears to be required for affective aspects of pain sensation. Our data complement and extend recent reports highlighting the roles of both spinal (Choi et al., 2020) and parabrachial (Deng et al., 2020) Tacr1 neurons as important drivers of pain behavior.

Tacr1 has been widely used as a marker for projection neurons in the spinal cord (Todd, 2010); however, not all projection neurons are marked by Tacr1 (Choi et al., 2020) and interneurons also express this gene (Sathyamurthy et al., 2020; Sathyamurthy et al., 2018) Nonetheless, the spinal^Tacr1 neurons are well known to play a crucial role in normal nociceptive behaviors (Mantyh et al., 1997). We initially characterized mice where Tacr1^Cre was used to drive GFP-expression to assess the proportions of projection neurons and interneurons targeted (Figure 1—figure supplement 2A, B). Our results confirmed that Tacr1^Cre mediates recombination both in the superficial and deep dorsal horn with about 40% of targeted neurons having soma diameters of projection neurons (Al Ghamdi et al., 2009). Next, we examined how activation of spinal^Tacr1 neurons contributes to pain sensation by directly stimulating a small circumscribed group of these cells using chemogenetics. Tacr1^Cre mice were transduced with an AAV encoding the excitatory DREADD receptor hM3Dq fused to an mCherry reporter (Barik et al., 2018; Krashes et al., 2011) on the right side of superficial lumbar dorsal horn (Figure 1A). As expected, immunohistochemical analysis confirmed expression by neurons in Lamina I and Laminae III-V on the side ipsilateral to the injection location (L4; Figure 1B). Importantly, administration of the hM3Dq ligand clozapine-N-oxide (CNO) resulted in widespread Fos expression on the injected side (Figure 1B), validating this stimulation paradigm (see Figure 1—figure supplement 1A, B for quantitation). Our results showed that both hM3Dq-mCherry positive and negative neurons express Fos after CNO delivery. A likely explanation is that CNO activates Tacr1+ neurons, and that Tacr1+ neurons in turn activate other non-Tacr1 neurons.

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Each side of a lumbar spinal cord segment receives sensory inputs from the ipsilateral hindlimb (Basbaum et al., 2009). Within minutes of CNO application, animals with spinal^Tacr1 neurons expressing hM3Dq began spontaneously lifting, shaking and licking the hindlimb ipsilateral to the AAV injection (Figure 1C and D; Videos 1 and 2). These responses were highly reminiscent of those caused by extremely noxious and persistent stimuli such as chemical irritants (Kwan et al., 2006), lasted for over an hour and suppressed normal homecage behaviors (e.g. grooming; Figure 1E). Remarkably, animals on CNO became so focused on the DREADD-induced ‘fictive’ pain associated with the ipsilateral hindlimb that they ignored other highly salient stimuli. In particular, they were completely unresponsive to chloroquine, a potent puritogen (Han et al., 2013; Mishra and Hoon, 2013) that normally causes robust and long-lasting scratching (Figure 1F). Notably, chloroquine was applied to the nape of the neck, a completely different body area, and still induced Fos expression in the cervical spinal cord (Figure 1—figure supplement 3). Thus, in addition to directing nocifensive behavior to a somatotopically conscribed area, the activation of a small group of lumbar spinal^Tacr1 neurons produced the well-known phenomenon of itch suppression by pain (Ikoma et al., 2006). Moreover, our data strongly suggest that itch suppression by pain must involve a central mechanism since peripheral signaling persists. Finally, as expected, the effects of CNO were fully reversible and did not cause any long-lasting sensitization (Figure 1G and H).

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We were surprised that artificial stimulation of the spinal^Tacr1 neurons so accurately phenocopied behaviors caused by genuine noxious stimulation since various types of dorsal horn projection neurons are considered necessary for pain behavior (Koch et al., 2018; Todd, 2010). Interestingly, a recent study showed that ablating spinal neurons expressing Tac1, the gene encoding the neuropeptide Substance P, significantly impaired licking responses to sustained noxious stimuli (Huang et al., 2019). In the spinal cord, Tac1 is also broadly expressed both in projection neurons and interneurons (Gutierrez-Mecinas et al., 2017; Huang et al., 2019). Our in-situ hybridization data (Figure 1—figure supplement 4A, B) revealed cells with high levels of Tacr1 transcripts in the superficial dorsal horn almost always co-express Tac1. We reasoned that activation of spinal^Tac1 neurons might produce similar behavioral effects as above (Figure 1). As predicted, chemogenetic stimulation of lumbar spinal^Tac1 neurons (Figure 1—figure supplement 4C, E) resulted in robust nocifensive behaviors directed to the ipsilateral hindlimb while at the same time suppressing grooming (Figure 1—figure supplement 4F) and itch responses (Figure 1—figure supplement 4G). There were minor differences in the behaviors with stimulation of Tac1^Cre animals being more robust than Tacr1^Cre mice (compare Videos 2 and 3), perhaps in part due to activation of Tac1 (Substance P but not NKR1) expressing sensory neurons in the dorsal horn and dorsal root ganglion (Figure 1—figure supplement 4H). These data support the existence of key populations of dorsal horn neurons mediating pain responses that can be defined by the expression of Tac1 and Tacr1. Importantly, our results demonstrate that activity of the spinal cells expressing these genes carries the information needed to elicit a pain sensation localized to a specific body area.

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Pain has many dimensions including location, quality, intensity, and duration. It also affects physiology, emotion, attention, and memory (Bushnell et al., 2013). Output from the dorsal horn of the spinal cord targets multiple brain areas that work in concert to generate this complex state (Basbaum et al., 2009; Todd, 2010). We transduced lumbar spinal^Tacr1 neurons with an AAV-DIO-GFP to visualize their supraspinal projections (Figure 1I). Consistent with their ability to coordinate a full-blown pain response, anterograde tracing revealed GFP⁺ axons in many regions of the brain (Figure 1J-M). We focused on the PBN because of its importance in pain behavior (Barik et al., 2018; Chiang et al., 2020; Huang et al., 2019). Outputs from two PBN areas, the lateral external and dorsal region, are known to have diverse impacts on pain responses, threat learning and escape behaviors (Bowen et al., 2020; Campos et al., 2018; Chiang et al., 2020; Han et al., 2015). Although GFP⁺ axons targeted both these regions, the highest density of spinal^Tacr1 projections was at the very top of the superior lateral PBN (PBN-SL; Figure 1K), a small and anatomically distinct area with unknown function and is sometimes referred to as the internal lateral PBN (Choi et al., 2020). As expected, chemogenetic activation of spinal^Tacr1 (Figure 2A) and spinal^Tac1 (Figure 1—figure supplement 4I) neurons induced robust Fos expression in the PBN-SL.

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Which cells in the PBN-SL receive the major input from the spinal dorsal horn and how do they contribute to pain sensation? To answer these questions, we sought molecular markers that could be used to genetically manipulate PBN-SL neurons so we could study their anatomy and function. Since both the neuropeptide (Tac1) and one of its receptors (Tacr1) were expressed by the spinal projection neurons, (Figure 1—figure supplement 4A) we predicted that tachykinin signaling molecules might mark this sensory circuit. Indeed, multiplex fluorescent in-situ hybridization revealed Tacr1 to be highly expressed in a tight cluster of excitatory cells in the PBN-SL (Figure 2B,C and Figure 2—figure supplement 1A). The expression of Tacr1 in both the spinal and PBN-SL neurons meant their anatomical connectivity could be probed by differentially transducing each region with AAVs in the same Tacr1^Cre animal (Figure 2D). We labeled the presynaptic specializations of spinal^Tacr1 axons with Synaptophysin-GFP and the postsynaptic specializations of the PBN-SL^Tacr1 dendrites and soma using PSD95-tagRFP (Figure 2E). Near super-resolution imaging of single Z planes from stained tissue sections from the PBN-SL showed very close apposition of red and green varicosities indicative of functional synapses (Figure 2F,G).

Despite its compact structure, the PBN appears central to many different sensory pathways with the various anatomic regions selectively targeting distinct higher centers and controlling different aspects of visceral sensation (Bernard et al., 1994), thermoregulation (Yahiro et al., 2017; Yang et al., 2020), itch (Campos et al., 2018; Mu et al., 2017), and pain (Bernard and Besson, 1990; Campos et al., 2018; Menendez et al., 1996). Therefore, we next used anterograde tracing to determine the projections of PBN-SL^Tacr1 neurons by targeting the PBN of Tacr1^Cre animals with an AAV-DIO-tdTomato (Figure 2H,I). Two forebrain regions were prominently innervated by PBN-SL^Tacr1 neurons (Figure 2J): (1) the midline thalamus (MTh) and (2) the lateral hypothalamus/ parasubthalamic nucleus (LH/PSTN). Both these regions appear to be selective targets of PBN-SL excitatory projections that are not innervated by other lateral PBN subnuclei (Barik et al., 2018). Moreover, expression of synaptophysin-GFP in PBN-SL^Tacr1 neurons provided evidence for presynaptic terminal specializations in both areas (Figure 2—figure supplement 2).

Do the same neurons target both MTh and LH/PSTN? Or are there two distinct types of PBN-SL^Tacr1 neurons with different higher brain targets and potential roles? To answer these questions, we adopted an intersectional strategy where we labeled PBN-SL^Tacr1 neurons projecting to the LH/PSTh. Our approach used a Cre-dependent mCherry to label the cell bodies of Tacr1-expressing neurons in the PBN. We also injected the PSTN with retroAAV-FlpO and used a PBN injected Flp-dependent synaptophysin-GFP (Sathyamurthy et al., 2020) to label the synaptic termini of these cells (Figure 3A,B; see Materials and methods for details). We confirmed that the PBN-SL neurons expressed both labels (Figure 3A) meaning that PBN-SL^Tacr1 neurons project to the LH/PSTN, validating our approach. Importantly, Synaptophysin-GFP puncta were also abundant in the MTh (Figure 3B) demonstrating collateralization of PBN cells that we presume express Tacr1. Together, these anatomical experiments reveal two previously unappreciated targets of PBN-SL^Tacr1 neurons in the forebrain. Therefore, we anticipated that these neurons may play a specialized role related to pain and next set out to record their responses as well as determine their function.

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Tacr1 expression marks a small group of neurons restricted to the PBN-SL. Fiber photometry provides a simple yet sensitive approach for measuring the population responses of genetically defined and anatomically restricted neurons (Gunaydin et al., 2014). Therefore, to study how PBN-SL^Tacr1 neurons are tuned to respond to sensory stimuli, we engineered mice expressing the calcium sensor GCaMP6f (Chen et al., 2013) in these neurons and implanted optical fibers to record population-level activity (Figure 4A,B). Consistent with our anatomical studies (Figure 2), fiber-photometry recordings demonstrated that many types of noxious stimulus strongly activate PBN-SL^Tacr1 neurons. An example recording (Figure 4C) highlights the typical type of long-lasting calcium transient that was elicited by a single pinch of the hindpaw of a lightly anesthetized mouse. Activation of these neurons was observed irrespective of the pinch location (Figure 4—figure supplement 1A). Next, we recorded activity in non-anesthetized mice using two types of noxious but non-damaging sustained mechanical stimulation: pinch with blunt forceps (Figure 4D) and pinch with the clip assay (Figure 4E). Again, PBN-SL^Tacr1 neurons exhibited robust population calcium responses to this type of stimulation (Figure 4D,E). By contrast, punctate mechanical stimuli did not evoke calcium responses in PBN-SL^Tacr1 neurons (Figure 4F). This was surprising since we used stiff von Frey filaments (0.6 g) that reliably cause nocifensive reflexes (Abdus-Saboor et al., 2019). Additionally, pinprick also failed to evoke a calcium response (Figure 4—figure supplement 1B). Together, these data may indicate that PBN-SL^Tacr1 neurons preferentially respond to sustained stimuli.

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Painful mechanical, thermal, and chemical stimuli are detected by separate nociceptive channels (Basbaum et al., 2009; Gatto et al., 2019; Le Pichon and Chesler, 2014). We next asked whether these pathways converged on PBN-SL^Tacr1 neurons by recording evoked activity in this population to high temperatures and chemical irritants. In keeping with these neurons playing a general role in pain sensation they exhibited robust activity to hot-plate exposure (Figure 4G) and topical application of the pungent component of mustard oil, allyl isothiocyanate (AITC; Figure 4—figure supplement 2A and B). Like pinch, both of these are long-lasting painful stimuli that activate distinct sets of peripheral nociceptors (Basbaum et al., 2009). However, PBN-SL^Tacr1 neurons were not activated by short-term noxious heat (Hargreaves test; Figure 4H). Interestingly, although AITC is known to cause allodynia and hyperalgesia (Albin et al., 2008), this type of skin irritation did not sensitize PBN-SL^Tacr1 responses to acute touch or heat stimuli (Figure 4—figure supplement 2C-F). In combination, the photometry data strongly support PBN-SL^Tacr1 neurons as playing a major role in an ascending pathway from spinal dorsal horn that is activated by sustained noxious inputs.

We next set out to determine how this circuit influences pain behaviors by generating mice where PBN-SL^Tacr1 neurons could be chemogenetically activated (Figure 5A,B). Although activation of PBN-SL^Tacr1 neurons did not induce spontaneous directed pain responses, CNO delivery transformed behavior: mice became skittish, avoided handling and repeatedly attempted to escape from the test chambers (possibly a general pain state). This heightened arousal and anxiety were quantified using an open-field arena as increased movement and avoidance of the center (Figure 5—figure supplement 1A). Mice now withdrew from the slightest touch with thin von Frey filaments (0.07 g; Figure 5—figure supplement 1C), something not seen in saline controls. Moreover, in a sustained pinch assay using a clip applied to the paw, activation of PBN-SL^Tacr1 neurons increased nocifensive behavior and almost doubled the time a mouse spent shaking the clipped paw (Figure 5C,D). Even more strikingly, behavioral changes persisted after the clip was removed (Figure 5E). Although saline -treated mice quickly returned to normal resting behaviors, after CNO the same animals continued to lick and investigate the hindpaw for many minutes after the pinch was terminated (Figure 5E). Equally robust effects of stimulating PBN-SL^Tacr1 neurons were observed in responses to AITC, a chemical irritant which normally causes licking and attending to the affected area (Figure 5F). The time a CNO-treated mouse spent licking the injected site more than doubled. CNO-injected mice also exhibited heightened escape responses when exposed to heat in a hotplate assay (Figure 5G). Therefore, this circuit plays a role in controlling the magnitude of behavioral responses to a range of noxious stimuli that activate different peripheral sensory neurons. Interestingly, the latency in a mouse’s response to heat, thought to involve simple spinal reflexes, was not influenced indicating that peripheral sensitization is not involved (Figure 5—figure supplement 1D). Thus, activation of PBN-SL^Tacr1 neurons induces a ‘hyper-vigilant’ state resembling the effects of long-term noxious stimulation (e.g. inflammation) as seen in assays like the formalin test (Figure 5—figure supplement 1B). Interestingly, activating the PBN-SL^Tacr1 neurons using CNO did not enhance responses to formalin, presumably reflecting the potency of this inflammatory stimulus. In a related study, the Sun lab has demonstrated that inhibiting PBN-SL^Tacr1 neurons reduces behavioral responses to peripheral inflammation (Deng et al., 2020).

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Recent work from other groups has suggested that inhibiting PBN activity suppresses scratching (Campos et al., 2018; Mu et al., 2017); however, our data show that stimulating the Tacr1-expressing spinal neurons strongly suppressed itch-related behaviors. Therefore, we next investigated how activation of PBN-SL^Tacr1 neurons affected behavioral responses to pruritogens. To induce itch, we injected chloroquine, a potent non-histaminergic puritogen that activates a select type of peripheral itch neuron and evokes vigorous scratching (Han et al., 2013). Injecting this compound into the nape of the neck of control mice reliably induced localized bouts of scratching starting a few minutes after injection and lasting for about 30 min. Remarkably, chemogenetic stimulation of the small group of PBN-SL^Tacr1 neurons completely suppressed scratching (Figure 5—figure supplement 2A and B), mimicking the effects both of painful stimuli and the fictive pain induced by activating Tacr1-expressing spinal projection neurons. Therefore, the PBN-SL^Tacr1 neurons serve as a central substrate that is sufficient to suppress itch.

Chemogenetic activation and functional recordings demonstrate that PBN-SL^Tacr1 neurons respond to painful stimuli and can induce hypervigilance and increased behavioral responses to sustained types of pain. However, it is well known that the central pathways controlling pain sensation and responses involve many regions of the brain (Bushnell et al., 2013). Therefore, we next used directed expression of tetanus toxin (TeNT-GFP) (Han et al., 2015; Li et al., 2019) to silence synaptic output and determine the role of this PBN-SL^Tacr1 circuit in pain behaviors (Figure 5I,J). We again examined responses to different types of sustained stimuli that in control animals induce robust behaviors: the clip assay of extended pinch, and topical application of AITC. Remarkably, in both cases, silencing PBN-SL^Tacr1 neurons almost completely eliminated shaking and licking normally induced by these stimuli (Figure 5K–O). Moreover, at a qualitative level, mice with silenced PBN-SL^Tacr1 neurons effectively ignored these normally painful stimuli. Thus, this small and localized cluster of PBN-SL^Tacr1 neurons appears to play a profound role in pain and is necessary for normal behavioral responses.

Together our results identify a circuit involving Tacr1-expressing neurons in the spinal cord and the PBN-SL that controls how mice respond to a wide range of persistently painful stimuli. Interestingly, more than 30 years ago McMahon and Wall described a loop from lamina I dorsal horn to the parabrachial nucleus that influences the activity of lamina I cells (McMahon and Wall, 1988). Our results here show that PBN-SL neurons expressing Tacr1 are important for responses to persistent noxious stimuli, exactly as predicted (Wall et al., 1988). Importantly, activating the lumbar spinal^Tacr1 neurons closely mimics persistent pain by inducing pronounced, somatotopically directed behaviors. Very recently and in keeping with our data, other groups have demonstrated that spinal^Tacr1 neurons are quite diverse (Sheahan et al., 2020; Choi et al., 2020) and include a population of neurons that selectively innervate the PBN-SL to cause strong nocifensive responses (Choi et al., 2020). Interestingly, chemogenetic activation of spinal^Tacr1 neurons using a different genetic strategy induced itch-related behavior (Sheahan et al., 2020). In combination, these data highlight the role of spinal^Tacr1 neurons in nociception but suggest that the different mouse lines and approaches likely targeted slightly different subsets or numbers of neurons to elicit distinct sensations. Nonetheless our results are entirely concordant with results from the Ginty lab (Choi et al., 2020) that implicate projections of spinal^Tacr1 neurons to a small subnucleus of the PBN as crucial for affective aspects of pain.

We show that a primary target of spinal^Tacr1 projection neurons are PBN-SL neurons that also express the NKR1-receptor. These PBN-SL^Tacr1 neurons are both necessary and sufficient to drive a subset of pain-related behaviors. PBN-SL^Tacr1 neurons are tuned to long-lasting noxious input regardless of location and are selectively important for behaviors that only occur when pain is persistent. When activated, PBN-SL^Tacr1 neurons dramatically potentiate complex behavioral responses but not simple reflexes. Interestingly, when this group of neurons is stimulated in the absence of noxious peripheral input, mice become ‘jumpy’ and hypervigilant, now recoiling vigorously from the gentlest of touches that normally would be ignored in the absence of stimulation. For example, response to a first von Frey filament often evoked locomotion and escape from further attempts at stimulation even prior to touch. Comorbidities such as anhedonia and suppression of feeding have been reported to occur during ongoing pain and involve other nuclei in the PBN and their connections with the central amygdala (Carter et al., 2013; Chiang et al., 2020). Therefore, in the future it will be interesting to determine if PBN-SL^Tacr1 neurons are also crucial for these types of behavioral changes since PBN-SL^Tacr1 neurons do not directly target the amygdala but project to distinct regions of the forebrain.

The stimuli that we showed PBN-SL^Tacr1 neurons respond to are all persistent types of pain. Since both substance P (Tac1) and its receptor NKR1 (Tacr1) are present both in the spinal projection neurons and in the PBN, it seems likely that neuropeptide signaling may modulate aspects of pain through this pathway. However, given that NKR1-antagonists have limited efficacy in human subjects, we suspect that painful stimulation must normally activate additional pathways that elicit negative valence. Finally, unlike spinal projection neurons, PBN-SL^Tacr1 neurons do not themselves evoke pain responses or provide positional information but instead appear to drive affective aspects of pain. This is particularly apparent in the clip assay where mice with silenced PBN-SL^Tacr1 neurons almost completely ignore this type of sustained, and normally painful, pinch. In the future, determining how other central targets of the spinal^Tacr1 projection neurons control behavior should help reveal if pain responses are simply the sum of distinct parallel circuits or require interactions between distributed regions of the brain.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data have been uploaded.

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Acceptance summary:

The authors of this paper show that chemogenetic activation of spinal cord neurons that expression the Tacr1 receptor, which responds to substance P, evokes many nocifensive behaviors that correlate with the experience of pain. The effects are exerted via connections with the parabrachial nucleus of the brainstem, which in turns connects with brain circuits that underlie emotional components of the pain experience.

Decision letter after peer review:

Thank you for submitting your article "A spinoparabrachial circuit defined by Tacr1 expression drives pain" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Richard Aldrich as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Richard D Palmiter (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Summary:

This is a concise paper describing a projection from spinal cord to Tacr1-expressing neurons in the PBN. The authors conclude that activation of this spinal cord > PBN Tacr1 circuit "does not trigger pain responses but instead serves to dramatically heighten nocifensive behaviors and suppress itch" and that mice with "silenced Tacr1 neurons ignore long-lasting noxious stimuli”. These are interesting and important findings that complement a recent paper (Deng et al., 2020). The conclusions reached by the authors are generally substantiated, but considerable revision is required, but some additional experiments are essential. These experiments will not only strengthen the authors' conclusions, but may provide some insight as to underlying mechanism as to the very interesting finding relating pain to itch inhibition.

Essential revisions:

Major concerns several requiring additional experimental information:

1) This paper appears to have been written for a different journal. As the authors have more space in eLife they should move some of the more interesting supplementary figures to main figures. They should also expand on the Discussion and take advantage of the Deng/Sun paper to compare results of the two studies to create an integrated picture.

2) The Fos induction after activating Tacr1 neurons in spinal cord is not impressive and does not appear to be colocalized with red cells expressing the DREADD (Figure 1—figure supplement 4C). Quantification Fos overlap with Tacr1 and Fos in non-Tacr1 cells is needed. Likewise, quantification of overlap of Tacr1 and Tac1 in lamina 1 of spinal cord is needed.

3) A poke (von Frey) and a pinch (squeeze) are different, so the conclusion that the PBN Tacr1 neurons only respond to sustained stimuli needs better validation. A revision should definitely include a different painful stimulus; foot shocks that may be easier to precisely time. Alternatively a dry ice application test could be used.

4) The authors say that "PBN-Tacr1 neurons induces a "hyper-vigilant" state resembling the effects of long-term noxious stimulation (e.g. inflammation)." Which raises the question of how the mice respond to an inflammatory stimulus (e.g. carrageenan or formalin)?

5) Most importantly, what percentage of spinal Tacr1-expressing neurons are projection neurons? The authors gave the impression that Tacr1-expressing cells in spinal cord are all projection neurons, but as Todd has reported some are undoubtedly interneurons. This should be quantified.

6) The question of the nature of the NK1R neurons is particularly relevant given the recent contribution of the NK1R neurons from the Sarah Ross lab, which has a Biorxiv paper indicating NK1R(Tacr1)-expressing spinal interneurons are involved in itch (https://www.biorxiv.org/content/10.1101/2020.07.14.199471v1.full). Of course, this paper indicated that activating Tacr1-neurons suppresses itch. This should be discussed.

7) Here also is where an additional experiment could not only focus on the difference in the results from the Ross lab, but also provide some insight into mechanism:

8) The inhibition of nape of the neck chloroquine-induced scratching is perhaps the most remarkable and exciting finding. Does the mechanism involve inhibition at the level of the spinal cord (via an ascending-descending loop), or is it an effect that occurs supraspinally? If the latter, then one would predict that nape of the neck chloroquine-evoked cervical cord Fos expression would persist after lumbar cord DREADD stimulation. This is an easy experiment, somewhat complicated perhaps by the fact that the loss of scratching would inevitably reduce Fos in the cervical cord. But any Fos attributed to the chloroquine activation of pruritoceptors should either persist, if the inhibition occurs supraspinally, or disappear, if the inhibition occurs at the level of the cord. This is an experiment that should absolutely be included in a revision.

9) Lastly, it would great if the authors could provide videos of activating Tacr1 neurons showing pain like behaviors.

Essential revisions:

Major concerns several requiring additional experimental information:

1) This paper appears to have been written for a different journal. As the authors have more space in eLife they should move some of the more interesting supplementary figures to main figures. They should also expand on the Discussion and take advantage of the Deng/Sun paper to compare results of the two studies to create an integrated picture.

We have rewritten the paper and included multiple citations and discussion of all requested literature.

2) The Fos induction after activating Tacr1 neurons in spinal cord is not impressive and does not appear to be colocalized with red cells expressing the DREADD (Figure 1—figure supplement 4C). Quantification Fos overlap with Tacr1 and Fos in non-Tacr1 cells is needed. Likewise, quantification of overlap of Tacr1 and Tac1 in lamina 1 of spinal cord is needed.

New Figure 1—figure supplement 1 now more explicitly shows the correspondence; we have also added quantification as requested.

3) A poke (von Frey) and a pinch (squeeze) are different, so the conclusion that the PBN Tacr1 neurons only respond to sustained stimuli needs better validation. A revision should definitely include a different painful stimulus; foot shocks that may be easier to precisely time. Alternatively a dry ice application test could be used.

We added data for additional stimuli including pinprick to which mice showed no Ca-response in these neurons (Figure 4—figure supplement 1B); similarly dry ice did not evoke Ca-transients.

4) The authors say that "PBN-Tacr1 neurons induces a "hyper-vigilant" state resembling the effects of long-term noxious stimulation (e.g. inflammation)." Which raises the question of how the mice respond to an inflammatory stimulus (e.g. carrageenan or formalin)?

We added the formalin test (Figure 5—figure supplement 1) and observed no enhanced effect. I.e. these are not additive responses likely because the activation of Tacr1-neurons already reaches a ceiling. We also reference the Sun paper where they used inhibition of the neurons and reduced behavioral responses to inflammation.

5) Most importantly, what percentage of spinal Tacr1-expressing neurons are projection neurons? The authors gave the impression that Tacr1-expressing cells in spinal cord are all projection neurons, but as Todd has reported some are undoubtedly interneurons. This should be quantified.

We used standard methods in the field (Gramdi et al., 2009, see also the recent Ross paper on these neurons) to quantify the presumptive projection neurons based on soma size (see Figure 1—figure supplement 2).

6) The question of the nature of the NK1R neurons is particularly relevant given the recent contribution of the NK1R neurons from the Sarah Ross lab, which has a Biorxiv paper indicating NK1R(Tacr1)-expressing spinal interneurons are involved in itch (https://www.biorxiv.org/content/10.1101/2020.07.14.199471v1.full). Of course, this paper indicated that activating Tacr1-neurons suppresses itch. This should be discussed.

We added substantial discussion of this study along with recent work from the Ginty lab that is also related to our findings. Together these papers help clarify the importance of these projection neurons and their heterogeneity (in part addressing point 7)

7) Here also is where an additional experiment could not only focus on the difference in the results from the Ross lab, but also provide some insight into mechanism:

See points 6 and 7

8) The inhibition of nape of the neck chloroquine-induced scratching is perhaps the most remarkable and exciting finding. Does the mechanism involve inhibition at the level of the spinal cord (via an ascending-descending loop), or is it an effect that occurs supraspinally? If the latter, then one would predict that nape of the neck chloroquine-evoked cervical cord Fos expression would persist after lumbar cord DREADD stimulation. This is an easy experiment, somewhat complicated perhaps by the fact that the loss of scratching would inevitably reduce Fos in the cervical cord. But any Fos attributed to the chloroquine activation of pruritoceptors should either persist, if the inhibition occurs supraspinally, or disappear, if the inhibition occurs at the level of the cord. This is an experiment that should absolutely be included in a revision.

New Figure 1—figure supplement 3 directly addresses this point and shows that there is robust itch-evoked cervical Fos induction even when lumbar Tacr1-neurons are activated. Thus the inhibition of scratch is super-spinal again speaking to mechanism (point 7).

9) Lastly, it would great if the authors could provide videos of activating Tacr1 neurons showing pain like behaviors.

Now included.

Author details

1. Arnab Barik

   National Center for Complementary and Integrative Health, National Institutes of Health, Bethesda, United States

   Present address

   Center for Neuroscience, Indian Institute of Science, Bengaluru, India

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   For correspondence

   arnabbarik@iisc.ac.in

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6850-0894

2. Anupama Sathyamurthy

   National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States

   Present address

   Center for Neuroscience, Indian Institute of Science, Bengaluru, India

   Contribution

   Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

3. James Thompson

   National Center for Complementary and Integrative Health, National Institutes of Health, Bethesda, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

4. Mathew Seltzer

   National Center for Complementary and Integrative Health, National Institutes of Health, Bethesda, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

5. Ariel Levine

   National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States

   Contribution

   Resources, Supervision, Funding acquisition, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0335-0730

6. Alexander Chesler

   1. National Center for Complementary and Integrative Health, National Institutes of Health, Bethesda, United States
   2. National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States

   Contribution

   Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Visualization, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   alexander.chesler@nih.gov

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3131-0728

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