Selective targeting of mu opioid receptors to primary cilia.

Opioid receptors are therapeutically important G protein-coupled receptors (GPCRs) with diverse neuromodulatory effects. The functional consequences of opioid receptor activation are known to depend on receptor location in the plasma membrane, but mechanisms mediating selective localization of receptors to any particular membrane domain remain elusive. Here, we demonstrate the targeting of the mu opioid receptor (MOR) to the primary cilium, a discrete microdomain of the somatic plasma membrane, both in vivo and in cultured cells. We further show that ciliary targeting is specific to MORs, requires a 17-residue sequence unique to the MOR cytoplasmic tail, and additionally requires the Tubby-like protein 3 (TULP3) ciliary adaptor protein. Our results reveal the potential for opioid receptors to undergo selective localization to the primary cilium. We propose that ciliary targeting is mediated through an elaboration of the recycling pathway, directed by a specific C-terminal recycling sequence in cis and requiring TULP3 in trans.

Human assembloid model of the ascending neural sensory pathway.

The ascending somatosensory pathways convey crucial information about pain, touch, itch, and body part movement from peripheral organs to the central nervous system. Despite a significant need for effective therapeutics modulating pain and other somatosensory modalities, clinical translation remains challenging, which is likely related to species-specific features and the lack of in vitro models to directly probe and manipulate this polysynaptic pathway. Here, we established human ascending somatosensory assembloids (hASA)- a four-part assembloid completely generated from human pluripotent stem cells that integrates somatosensory, spinal, diencephalic, and cortical organoids to model the human ascending spinothalamic pathway. Transcriptomic profiling confirmed the presence of key cell types in this circuit. Rabies tracing and calcium imaging showed that sensory neurons connected with dorsal spinal cord projection neurons, which ascending axons further connected to thalamic neurons. Following noxious chemical stimulation, single neuron calcium imaging of intact hASA demonstrated coordinated response, while four-part concomitant extracellular recordings and calcium imaging revealed synchronized activity across the assembloid. Loss of the sodium channel SCN9A, which causes pain insensitivity in humans, disrupted synchrony across the four-part hASA. Taken together, these experiments demonstrate the ability to functionally assemble the essential components of the human sensory pathway. These findings could both accelerate our understanding of human sensory circuits and facilitate therapeutic development.

Comprehensive mapping of sensory and sympathetic innervation of the developing kidney.

The kidney functions as a finely tuned sensor to balance body fluid composition and filter out waste through complex coordinated mechanisms. This versatility requires tight neural control, with innervating efferent nerves playing a crucial role in regulating blood flow, glomerular filtration rate, water and sodium reabsorption, and renin release. In turn sensory afferents provide feedback to the central nervous system for the modulation of cardiovascular function. However, the cells targeted by sensory afferents and the physiological sensing mechanisms remain poorly characterized. Moreover, how the kidney is innervated during development to establish these functions remains elusive. Here, we utilized a combination of light-sheet and confocal microscopy to generate anatomical maps of kidney sensory and sympathetic nerves throughout development and resolve the establishment of functional crosstalk. Our analyses revealed that kidney innervation initiates at embryonic day (E)13.5 as the nerves associate with vascular smooth muscle cells and follow arterial differentiation. By E17.5 axonal projections associate with kidney structures such as glomeruli and tubules and the network continues to expand postnatally. These nerves are synapsin I-positive, highlighting ongoing axonogenesis and the potential for functional crosstalk. We show that sensory and sympathetic nerves innervate the kidney concomitantly and classify the sensory fibers as calcitonin gene related peptide (CGRP)+, substance P+, TRPV1+, and PIEZO2+, establishing the presence of PIEZO2 mechanosensory fibers in the kidney. Using retrograde tracing, we identified the primary dorsal root ganglia, T10-L2, from which PIEZO2+ sensory afferents project to the kidney. Taken together our findings elucidate the temporality of kidney innervation and resolve the identity of kidney sympathetic and sensory nerves.

Interleukin-10 signaling in somatosensory neurons controls CCL2 release and inflammatory response.

Appropriate regulation of the inflammatory response is essential for survival. Interleukin-10 (IL-10), a well-known anti-inflammatory cytokine, plays a major role in controlling inflammation. In addition to immune cells, we previously demonstrated that the IL-10 receptor (IL-10R1) is expressed in dorsal root ganglion sensory neurons. There is emerging evidence that these sensory neurons contribute to immunoregulation, and we hypothesized that IL-10 signaling in dorsal root ganglion (DRG) neurons facilitates the regulation of the inflammatory response. We showed that mice that lack IL-10R1 specifically on advillin-positive neurons have exaggerated blood nitric oxide levels, spinal microglia activation, and cytokine upregulation in the spinal cord, liver, and gut compared to wild-type (WT) counterparts in response to systemic lipopolysaccharide (LPS) injection. Lack of IL-10R1 in DRG and trigeminal ganglion (TG) neurons also increased circulating and DRG levels of proinflammatory C-C motif chemokine ligand 2 (CCL2). Interestingly, analysis of published scRNA-seq data revealed that Ccl2 and Il10ra are expressed by similar types of DRG neurons; nonpeptidergic P2X purinoceptor (P2X3R + ) neurons. In primary cultures of DRG neurons, we demonstrated that IL-10R1 inhibits the production of CCL2, but not that of the neuropeptides substance P and calcitonin-gene related peptide (CGRP). Furthermore, our data indicate that ablation of Transient receptor potential vanilloid (TRPV)1 + neurons does not impact the regulation of CCL2 production by IL-10. In conclusion, we showed that IL-10 binds to its receptor on sensory neurons to downregulate CCL2 and contribute to immunoregulation by reducing the attraction of immune cells by DRG neuron-derived CCL2. This is the first evidence that anti-inflammatory cytokines limit inflammation through direct binding to receptors on sensory neurons. Our data also add to the growing literature that sensory neurons have immunomodulatory functions.

Graph theory analysis reveals an assortative pain network vulnerable to attacks.

The neural substrate of pain experience has been described as a dense network of connected brain regions. However, the connectivity pattern of these brain regions remains elusive, precluding a deeper understanding of how pain emerges from the structural connectivity. Here, we employ graph theory to systematically characterize the architecture of a comprehensive pain network, including both cortical and subcortical brain areas. This structural brain network consists of 49 nodes denoting pain-related brain areas, linked by edges representing their relative incoming and outgoing axonal projection strengths. Within this network, 63% of brain areas share reciprocal connections, reflecting a dense network. The clustering coefficient, a measurement of the probability that adjacent nodes are connected, indicates that brain areas in the pain network tend to cluster together. Community detection, the process of discovering cohesive groups in complex networks, successfully reveals two known subnetworks that specifically mediate the sensory and affective components of pain, respectively. Assortativity analysis, which evaluates the tendency of nodes to connect with other nodes that have similar features, indicates that the pain network is assortative. Finally, robustness, the resistance of a complex network to failures and perturbations, indicates that the pain network displays a high degree of error tolerance (local failure rarely affects the global information carried by the network) but is vulnerable to attacks (selective removal of hub nodes critically changes network connectivity). Taken together, graph theory analysis unveils an assortative structural pain network in the brain that processes nociceptive information. Furthermore, the vulnerability of this network to attack presents the possibility of alleviating pain by targeting the most connected brain areas in the network.

A suite of engineered mice for interrogating psychedelic drug actions.

Psychedelic drugs like lysergic acid diethylamide (LSD) and psilocybin have emerged as potentially transformative therapeutics for many neuropsychiatric diseases, including depression, anxiety, post-traumatic stress disorder, migraine, and cluster headaches. LSD and psilocybin exert their psychedelic effects via activation of the 5-hydroxytryptamine 2A receptor (HTR2A). Here we provide a suite of engineered mice useful for clarifying the role of HTR2A and HTR2A-expressing neurons in psychedelic drug actions. We first generated Htr2a-EGFP-CT-IRES-CreERT2 mice (CT:C-terminus) to independently identify both HTR2A-EGFP-CT receptors and HTR2A-containing cells thereby providing a detailed anatomical map of HTR2A and identifying cell types that express HTR2A. We also generated a humanized Htr2a mouse line and an additional constitutive Htr2A-Cre mouse line. Psychedelics induced a variety of known behavioral changes in our mice validating their utility for behavioral studies. Finally, electrophysiology studies revealed that extracellular 5-HT elicited a HTR2A-mediated robust increase in firing of genetically-identified pyramidal neurons--consistent with a plasma membrane localization and mode of action. These mouse lines represent invaluable tools for elucidating the molecular, cellular, pharmacological, physiological, behavioral, and other actions of psychedelic drugs in vivo.

Tonic Meningeal Interleukin-10 Upregulates Delta Opioid Receptor to Prevent Relapse to Pain.

Chronic pain often alternates between transient remission and relapse of severe pain. While most research on chronic pain has focused on mechanisms maintaining pain, there is a critical unmet need to understand what prevents pain from re-emerging in those who recover from acute pain. We found that interleukin (IL)-10, a pain resolving cytokine, is persistently produced by resident macrophages in the spinal meninges during remission from pain. IL-10 upregulated expression and analgesic activity of δ-opioid receptor (δOR) in the dorsal root ganglion. Genetic or pharmacological inhibition of IL-10 signaling or δOR triggered relapse to pain in both sexes. These data challenge the widespread assumption that remission of pain is simply a return to the naïve state before pain was induced. Instead, our findings strongly suggest a novel concept that: remission is a state of lasting pain vulnerability that results from a long-lasting neuroimmune interactions in the nociceptive system.

Cell-type specific molecular architecture for mu opioid receptor function in pain and addiction circuits.

Opioids are potent analgesics broadly used for pain management; however, they can produce dangerous side effects including addiction and respiratory depression. These harmful effects have led to an epidemic of opioid abuse and overdose deaths, creating an urgent need for the development of both safer pain medications and treatments for opioid use disorders. Both the analgesic and addictive properties of opioids are mediated by the mu opioid receptor (MOR), making resolution of the cell types and neural circuits responsible for each of the effects of opioids a critical research goal. Single-cell RNA sequencing (scRNA-seq) technology is enabling the identification of MOR-expressing cell types throughout the nervous system, creating new opportunities for mapping distinct opioid effects onto newly discovered cell types. Here, we describe molecularly defined MOR-expressing neuronal cell types throughout the peripheral and central nervous systems and their potential contributions to opioid analgesia and addiction.

Graph theory analysis reveals an assortative pain network vulnerable to attacks.

The neural substrate of pain experience has been described as a dense network of connected brain regions. However, the connectivity pattern of these brain regions remains elusive, precluding a deeper understanding of how pain emerges from the structural connectivity. Here, we use graph theory to systematically characterize the architecture of a comprehensive pain network, including both cortical and subcortical brain areas. This structural brain network consists of 49 nodes denoting pain-related brain areas, linked by edges representing their relative incoming and outgoing axonal projection strengths. Sixty-three percent of brain areas in this structural pain network share reciprocal connections, reflecting a dense network. The clustering coefficient, a measurement of the probability that adjacent nodes are connected, indicates that brain areas in the pain network tend to cluster together. Community detection, the process of discovering cohesive groups in complex networks, successfully reveals two known subnetworks that specifically mediate the sensory and affective components of pain, respectively. Assortativity analysis, which evaluates the tendency of nodes to connect with other nodes with similar features, indicates that the pain network is assortative. Finally, robustness, the resistance of a complex network to failures and perturbations, indicates that the pain network displays a high degree of error tolerance (local failure rarely affects the global information carried by the network) but is vulnerable to attacks (selective removal of hub nodes critically changes network connectivity). Taken together, graph theory analysis unveils an assortative structural pain network in the brain processing nociceptive information, and the vulnerability of this network to attack opens up the possibility of alleviating pain by targeting the most connected brain areas in the network.

Distinct and sex-specific expression of mu opioid receptors in anterior cingulate and somatosensory S1 cortical areas.

ABSTRACT: The anterior cingulate cortex (ACC) processes the affective component of pain, whereas the primary somatosensory cortex (S1) is involved in its sensory-discriminative component. Injection of morphine in the ACC has been reported to be analgesic, and endogenous opioids in this area are required for pain relief. Mu opioid receptors (MORs) are expressed in both ACC and S1; however, the identity of MOR-expressing cortical neurons remains unknown. Using the Oprm1-mCherry mouse line, we performed selective patch clamp recordings of MOR+ neurons, as well as immunohistochemistry with validated neuronal markers, to determine the identity and laminar distribution of MOR+ neurons in ACC and S1. We found that the electrophysiological signatures of MOR+ neurons differ significantly between these 2 areas, with interneuron-like firing patterns more frequent in ACC. While MOR+ somatostatin interneurons are more prominent in ACC, MOR+ excitatory neurons and MOR+ parvalbumin interneurons are more prominent in S1. Our results suggest a differential contribution of MOR-mediated modulation to ACC and S1 outputs. We also found that females had a greater density of MOR+ neurons compared with males in both areas. In summary, we conclude that MOR-dependent opioidergic signaling in the cortex displays sexual dimorphisms and likely evolved to meet the distinct function of pain-processing circuits in limbic and sensory cortical areas.

Spinal cord retinoic acid receptor signaling gates mechanical hypersensitivity in neuropathic pain.

Central sensitization caused by spinal disinhibition is a key mechanism of mechanical allodynia in neuropathic pain. However, the molecular mechanisms underlying spinal disinhibition after nerve injury remain unclear. Here, we show in mice that spared nerve injury (SNI), which induces mechanical hypersensitivity and neuropathic pain, triggers homeostatic reduction of inhibitory outputs from dorsal horn parvalbumin-positive (PV+) interneurons onto both primary afferent terminals and excitatory interneurons. The reduction in inhibitory outputs drives hyperactivation of the spinal cord nociceptive pathway, causing mechanical hypersensitivity. We identified the retinoic acid receptor RARα, a central regulator of homeostatic plasticity, as the key molecular mediator for this synaptic disinhibition. Deletion of RARα in spinal PV+ neurons or application of an RARα antagonist in the spinal cord prevented the development of SNI-induced mechanical hypersensitivity. Our results identify RARα as a crucial molecular effector for neuropathic pain and a potential target for its treatment.

Hyperexcitable arousal circuits drive sleep instability during aging.

Sleep quality declines with age; however, the underlying mechanisms remain elusive. We found that hyperexcitable hypocretin/orexin (Hcrt/OX) neurons drive sleep fragmentation during aging. In aged mice, Hcrt neurons exhibited more frequent neuronal activity epochs driving wake bouts, and optogenetic activation of Hcrt neurons elicited more prolonged wakefulness. Aged Hcrt neurons showed hyperexcitability with lower KCNQ2 expression and impaired M-current, mediated by KCNQ2/3 channels. Single-nucleus RNA-sequencing revealed adaptive changes to Hcrt neuron loss in the aging brain. Disruption of Kcnq2/3 genes in Hcrt neurons of young mice destabilized sleep, mimicking aging-associated sleep fragmentation, whereas the KCNQ-selective activator flupirtine hyperpolarized Hcrt neurons and rejuvenated sleep architecture in aged mice. Our findings demonstrate a mechanism underlying sleep instability during aging and a strategy to improve sleep continuity.

Sympathetic yet painful: Autonomic innervation drives cluster firing of somatosensory neurons.

In this issue of Neuron, Zheng et al. (2021) report synchronized cluster firing of dorsal root ganglion (DRG) neurons that correlates with spontaneous pain in the setting of nerve injury. The authors' findings further suggest that sympathetic sprouting in the DRG plays a key role in this phenomenon.

Brain circuits for pain and its treatment.

Pain is a multidimensional experience with sensory-discriminative, affective-motivational, and cognitive-evaluative components. Pain aversiveness is one principal cause of suffering for patients with chronic pain, motivating research and drug development efforts to investigate and modulate neural activity in the brain's circuits encoding pain unpleasantness. Here, we review progress in understanding the organization of emotion, motivation, cognition, and descending modulation circuits for pain perception. We describe the molecularly defined neuron types that collectively shape pain multidimensionality and its aversive quality. We also review how pharmacological, stimulation, neurofeedback, surgical, and cognitive-behavioral interventions alter activity in these circuits to relieve chronic pain.

Delta opioid receptor regulation of calcitonin gene-related peptide dynamics in the trigeminal complex.

ABSTRACT: Migraine is highly prevalent and is the sixth leading cause worldwide for years lost to disability. Therapeutic options specifically targeting migraine are limited, and delta opioid receptor (DOP) agonists were recently identified as a promising pharmacotherapy. The mechanisms by which DOPs regulate migraine are currently unclear. Calcitonin gene-related peptide (CGRP) has been identified as an endogenous migraine trigger and plays a critical role in migraine initiation and susceptibility. The aim of this study was to determine the behavioral effects of DOP agonists on the development of chronic migraine-associated pain and to investigate DOP coexpression with CGRP and CGRP receptor (CGRPR) in the trigeminal system. Chronic migraine-associated pain was induced in mice through repeated intermittent injection of the known human migraine trigger, nitroglycerin. Chronic nitroglycerin resulted in severe chronic cephalic allodynia which was prevented with cotreatment of the DOP-selective agonist, SNC80. In addition, a corresponding increase in CGRP expression in the trigeminal ganglia and trigeminal nucleus caudalis was observed after chronic nitroglycerin, an augmentation that was blocked by SNC80. Moreover, DOP was also upregulated in these head pain-processing regions following the chronic migraine model. Immunohistochemical analysis of the trigeminal ganglia revealed coexpression of DOP with CGRP as well as with a primary component of the CGRPR, RAMP1. In the trigeminal nucleus caudalis, DOP was not coexpressed with CGRP but was highly coexpressed with RAMP1 and calcitonin receptor-like receptor. These results suggest that DOP agonists inhibit migraine-associated pain by attenuating CGRP release and blocking pronociceptive signaling of the CGRPR.

Neuronal interleukin-1 receptors mediate pain in chronic inflammatory diseases.

Chronic pain is a major comorbidity of chronic inflammatory diseases. Here, we report that the cytokine IL-1ß, which is abundantly produced during multiple sclerosis (MS), arthritis (RA), and osteoarthritis (OA) both in humans and in animal models, drives pain associated with these diseases. We found that the type 1 IL-1 receptor (IL-1R1) is highly expressed in the mouse and human by a subpopulation of TRPV1+ dorsal root ganglion neurons specialized in detecting painful stimuli, termed nociceptors. Strikingly, deletion of the Il1r1 gene specifically in TRPV1+ nociceptors prevented the development of mechanical allodynia without affecting clinical signs and disease progression in mice with experimental autoimmune encephalomyelitis and K/BxN serum transfer-induced RA. Conditional restoration of IL-1R1 expression in nociceptors of IL-1R1-knockout mice induced pain behavior but did not affect joint damage in monosodium iodoacetate-induced OA. Collectively, these data reveal that neuronal IL-1R1 signaling mediates pain, uncovering the potential benefit of anti-IL-1 therapies for pain management in patients with chronic inflammatory diseases.

Targeting Morphine-Responsive Neurons: Generation of a Knock-In Mouse Line Expressing Cre Recombinase from the Mu-Opioid Receptor Gene Locus.

The mu-opioid receptor (MOR) modulates nociceptive pathways and reward processing, and mediates the strong analgesic and addictive properties of both medicinal as well as abused opioid drugs. MOR function has been extensively studied, and tools to manipulate or visualize the receptor protein are available. However, circuit mechanisms underlying MOR-mediated effects are less known, because genetic access to MOR-expressing neurons is lacking. Here we report the generation of a knock-in Oprm1-Cre mouse line, which allows targeting and manipulating MOR opioid-responsive neurons. A cDNA encoding a T2A cleavable peptide and Cre recombinase fused to enhanced green fluorescent protein (EGFP/Cre) was inserted downstream of the Oprm1 gene sequence. The resulting Oprm1-Cre line shows intact Oprm1 gene transcription. MOR and EGFP/Cre proteins are coexpressed in the same neurons, and localized in cytoplasmic and nuclear compartments, respectively. MOR signaling is unaltered, demonstrated by maintained DAMGO-induced G-protein activation, and in vivo MOR function is preserved as indicated by normal morphine-induced analgesia, hyperlocomotion, and sensitization. The Cre recombinase efficiently drives the expression of Cre-dependent reporter genes, shown by local virally mediated expression in the medial habenula and brain-wide fluorescence on breeding with tdTomato reporter mice, the latter showing a distribution patterns typical of MOR expression. Finally, we demonstrate that optogenetic activation of MOR neurons in the ventral tegmental area of Oprm1-Cre mice evokes strong avoidance behavior, as anticipated from the literature. The Oprm1-Cre line is therefore an excellent tool for both mapping and functional studies of MOR-positive neurons, and will be of broad interest for opioid, pain, and addiction research.

Synapse-specific opioid modulation of thalamo-cortico-striatal circuits.

The medial thalamus (MThal), anterior cingulate cortex (ACC) and striatum play important roles in affective-motivational pain processing and reward learning. Opioids affect both pain and reward through uncharacterized modulation of this circuitry. This study examined opioid actions on glutamate transmission between these brain regions in mouse. Mu-opioid receptor (MOR) agonists potently inhibited MThal inputs without affecting ACC inputs to individual striatal medium spiny neurons (MSNs). MOR activation also inhibited MThal inputs to the pyramidal neurons in the ACC. In contrast, delta-opioid receptor (DOR) agonists disinhibited ACC pyramidal neuron responses to MThal inputs by suppressing local feed-forward GABA signaling from parvalbumin-positive interneurons. As a result, DOR activation in the ACC facilitated poly-synaptic (thalamo-cortico-striatal) excitation of MSNs by MThal inputs. These results suggest that opioid effects on pain and reward may be shaped by the relative selectivity of opioid drugs to the specific circuit components.