Compositions and methods for treating pain

Abstract

The present invention relates to methods of preventing, ameliorating, slowing the progression of, or treating diseases, disorders, and / or conditions associated with pain in human subjects, by inhibiting non-opioid G protein-coupled receptors (GPCRs) in the amygdala region of the brain.

Description

Attorney Docket No. 5470.984.WOCOMPOSITIONS AND METHODS FOR TREATING PAINSTATEMENT OF PRIORITY

[0001] The application claims the benefit of U.S. Provisional Application No. 63 / 696,698, filed on September 19, 2024, the entire contents of which are incorporated by reference herein.GOVERNMENT INTEREST

[0002] This invention was made with government support under Grant Numbers NS106301 and NS 118504 awarded by the National Institutes of Health. The government has certain rights regarding inventions.FIELD OF THE INVENTION

[0003] The present invention relates to methods of preventing, ameliorating, slowing the progression of, or treating diseases, disorders, and / or conditions associated with pain in human subjects, by inhibiting non-opioid G protein-coupled receptors (GPCRs) in the amygdala region of the brain.BACKGROUND OF THE INVENTION

[0004] Chronic pain is a debilitating disease that affects nearly two billion individuals worldwide ■A Current medications against pain, including opioids, show limited efficacy against many types of pain such as chronic neuropathic pain -- and produce substantial detrimental side effects. Specifically, although opioids remain indispensable medications, particularly for managing postoperative pain, opioids' addictive properties have led to an epidemic of opioid abuse and overdose deaths -. Measures to battle this opioid crisis include a global reduction in opioid prescribing for chronic pain leaving more pain patients suffering and without therapeutic solutions. The need for novel and safer medications to treat pain is evident and urgent.

[0005] In the past 30 years, approaches to develop new pain medicines have built on the discovery of molecules that detect, transmit, or process pain information at the primary afferent nociceptor or spinal cord dorsal horn levels / These drug discovery efforts have begun showing some success notably for specific pain types. For example, the identification in nociceptors of calcitonin gene-Attorney Docket No. 5470.984.WO related peptide (CGRP)5and of tetrodotoxin-resistant voltage-gated sodium channels4led to the development of anti -CGRP antibodies for migraine treatment and of a Navi.8 voltage-gated sodium channel inhibitor candidate therapy for acute postoperative painrespectively. A complementary approach would involve developing drugs that can interfere with the flow of nociceptive information in brain circuits that produce pain negative emotions (i.e., the unpleasant quality of pain). Pain is a multidimensional experience with sensory-discriminative, affective- motivational, and cognitive-evaluative dimensionsCrucially, pain affective-motivational dimension is a major source of suffering for individuals living with chronic pain as pain's unpleasant quality generates an emotional burdenthat can lead to other disorders including anxiety and depression —■■. Given the convergent organization of pain ascending circuits, whereby nociceptive information from the diverse primary afferents innervating all organs is represented in the same brain circuits that encode pain unpleasantness -l-~, an analgesic drug acting within these circuits could be broadly useful to treat pain unpleasantness across pain types.

[0006] Opioids such as morphine remain the most broadly used drugs to manage severe pain, because they can, for many patients, reduce pain unpleasantness. Opioids elicit this analgesic effect by binding to p-opioid receptors, a type of G protein-coupled receptor (GPCR), expressed by numerous peripheral and central neurons that process pain information, and at particularly high levels in brain regions implicated in pain affective-motivational dimension, such as the amygdala, the anterior cingulate cortex, or the medial thalamus “ -. However, the p-opioid receptor is also broadly expressed in the brain outside of the pain neural circuitry, p-opioid receptors in midbrain and forebrain reward circuits are thought to underlie opioids’ addictive properties44;;4-4, whereas p-opioid receptors in brainstem breathing circuits produce opioid-induced respiratory depression fheprimaiy cause of opioid overdose death. In contrast to our understanding of pain molecular mechanisms in peripheral and spinal pain systems, a comparable therapeutically- relevant knowledge of the molecular architecture of pain processing in the brain pain circuits is not yet available.

[0007] The amygdala is a critical node of the brain’s emotion circuits that assigns a negative emotional valence to nociceptive information during pain experience “? :. Lesion of the amygdala in humans can reduce or eliminate pain unpleasantnessBoth the central and basolateral subnuclei of the amygdala (CeA and BLA, respectively) contribute to emotional pain processingThe CeA is a predominantly GABAergic, striatal-like, structure that receives nociceptiveAttorney Docket No. 5470.984.WO information via the spino-parabrachial ascending pain pathway (i.e., via anterolateral tract)" and a major input from the BLA. In contrast, the BLA is cytoarchitectural similar to the cerebral cortexi 4and exchanges pain-relevant information via reciprocal connections with the prefrontal cortex and thalamus. In addition to these connections, the amygdala has highly complex microcircuits containing both anti- and pro-nociceptive neurons connected with numerous ascending and descending pain modulation pathways -■■■■■■ Although the functional organization of amygdalar cell types during pain has begun to be determined, the neural heterogeneity of the amygdala and lack of molecular information on druggable targets has hindered the development of precise pain therapeutics.SUMMARY OF THE INVENTION

[0008] Pain suffering is a growing medical problem for which opioid-based treatments have limited efficacy against certain types of pain and can produce harmful side effects. Here, we devised a comprehensive cellular and molecular atlas of the nociceptive amygdala to identify nonopioid GPCR pain targets.

[0009] Accordingly, the present invention is based on ligands acting at non-opioid GPCRs that reduce pain across different pain-types.

[0010] Thus, one aspect of the invention relates to a method of treating and / or preventing pain in a subject, comprising administering to the subject a therapeutically effective amount of a GPCR ligand or pharmaceutically acceptable salt thereof, wherein the GPCR ligand targets a non-opioid GPCR in the amygdalar region of the brain, and wherein pain is alleviated and / or minimized in the subject.

[0011] Another aspect of the invention relates to a method of treating unpleasantness related to pain in a subject, comprising administering to the subject a therapeutically effective amount of a GPCR ligand or pharmaceutically acceptable salt thereof, wherein the GPCR ligand targets a non- opioid GPCR in the amygdalar region of the brain, and wherein unpleasantness related to pain is alleviated and / or minimized in the subject.

[0012] These and other aspects of the invention are set forth in more detail in the description of the invention below.Attorney Docket No. 5470.984.WODESCRIPTION OF THE DRAWINGS

[0013] FIGS. 1A-1K Transcriptomic classification, activity, and connectivity of amygdalar neuron-types. (1A) Experimental strategy to label nociceptive neurons in TRAP2;Ail4 mice injected with formalin, resolve the transcriptome of amygdalar neurons, and identify GPCR targets to develop novel analgesics against pain unpleasantness. (IB) UMAP projection of cell clusters and cell types from 9695 cells isolated from the amygdala. Unsupervised cell clustering and annotation identifies 12 distinct glial cell clusters and 3 broad types of neurons; glutamatergic Slcl7a7+, glutamatergic Slcl7a6+, and GABAergic Gadl+. (1C) Dot plot representation of glial and neuronal marker genes. (ID) Identification of 17 major amygdalar neuron-types and 28 subtypes by UMAP reduction and clustering of all Snap25+ neurons (5092 total neurons). (IE) Dot plot of canonical amygdalar neuron marker genes for each cluster. (IF) Heatmaps representing the volumetric absolute density of tdTomato fluorescence in TRAP2;Ail4 mice injected with formalin. (1G) Quantification of volumetric tdTomato fluorescence density in different amygdalar nuclei. (1H) Differentially expressed lEGs between control mice, mice injected with a single dose of formalin one week prior to euthanasia (Formalin), and mice in which formalin injection was repeated formalin prior to euthanasia (FormalinX2). (II) Volcano plot of differentially expressed genes in Prkcd,Calcrl+ neuron-types (i.4b) showing genes with greater than 0.75 fold-change and 0.05 p-value. (1J) Schematic of the viral strategy used to trace the inputs to BLA nociceptive neurons. (IK) Schematic of the viral strategy used to trace the output regions of BLA nociceptive neurons. Dot size in C, E, H represents the percentage of cells expressing the gene(s) in the corresponding cell cluster, and intensity represents the mean gene expression level in cells of this cluster.

[0014] FIGS. 2A-2G. Spatial organization of amygdalar neuron-types. (2A) Spatial distribution of Slcl7a7 Rspo2. Gadl Ppplrlb, GadR Foxp2 gene expression in an example of coronal slice and annotation of amygdalar subregions based on the distribution of marker genes and histological landmarks (bottom right). (2B) Cell type relationships identified by unsupervised clustering of neurons from coronal slices visualized by UMAP. (2C) Cell type labels transferred from our single-cell RNA-seq classification study. (2D) Representative caudal spatial map of individual cells coded by cluster identity. (2E) Cell type relationships identified by unsupervised clustering of amygdalar neurons from coronal slices visualized by UMAP. Cell type labels transferred from our single-cell RNA-seq classification study. (2F) Subregion origin identified for cell types basedAttorney Docket No. 5470.984.WO on spatial distribution of coronal slice. (2G) Dot plots of canonical amygdalar neuron marker genes for each cluster. Dot size represents the percentage of cells in each class expressing the genes and intensity represents the mean gene expression level as indicated by the legend.

[0015] FIGS. 3A-3E. Opioid system organization in the amygdala. (3A) Spatial expression map of genes encoding opioid receptors and peptides from an example coronal amygdala brain slice. (3B) Dot plots of opioid system gene expression for each cluster correlated across spatial (sp) and single-cell RNA-seq (sc) transcriptomics studies. (3C) Injection of the AAV Efla-CreOnFOn- oScarlet into the CeA of 0prmJCre;SstYipOmice. Representative image of oScarlet showing viral transduction in the CeA target region and projections to the posterior thalamus (PIL, SPF, PP), BNST, and PB. (3D) Injection of the AAV encoding Efla-CreOnFOn-oScarlet transgene into the CeA of ( / ?rA7Cre;J a / FlpOmice. Representative image of oScarlet showing viral transduction in the CeA target region and projections to the bed nucleus of the stria terminalis (BNST), insular cortex (INS), the periaqueductal gray (PAG), the parabrachial nucleus (PB), raphe magnus (RM), Parvicellular reticular nucleus (PARN), and nucleus of the solitary tract. (3E) Injection of the AAV encoding Efla-CreOnFOn-oScarlet transgene into the BLA of OprklCK;VghitFpQmice. Representative image of oScarlet showing viral transduction in the BLA target region and projections targets in the BNST, insular cortex (INS), claustrum (CLA), nucleus accumbens (Acb), medial prefrontal cortex (PrL, ACA), and motor cortex (M2).

[0016] FIGS. 4A-4I. Non-opioid analgesic GPCR targets. (4A) Pain-related cell types in the amygdala associated with pro-nociceptive or anti-nociceptive function with predicted analgesic association to inhibitory Gi / o-coupled GPCR types or excitatory Gs / q-coupled GPCR types, respectively. (4B) Dot plot of genes enriched in pain-related cell types that encode for GPCRs from single-cell RNA-seq dataset (sc). (4C) Dot plot of genes enriched in pain-related cell types that encode for GPCRs from spatial transcriptomics dataset (sp). (4D) Spatial co-expression map of 7 / / / 7 / GPCR drug target with correlated expression of Rspo2, Ppplrlb, and Otof marker genes. (4E) UpSet plot summarizing the overlapping gene expression of Htrlf Rspo2, Ppplrlb, and Otof. Bar charts (left) highlight select co-expression patterns with gray representing no co-expression. (4F) Spatial co-expression map of Ntsrl GPCR drug target with correlated expression of Prkcd, Oprkl, and Calcrl marker genes. (4G) UpSet plot summarizing the overlapping gene expression of Ntsrl, Oprkl, Prkcd, and Calcrl. Bar charts (left) highlight select co-expression patterns with gray representing no co-expression. (4H) Spatial co-expression map of Vipr2 GPCR drug targetAttorney Docket No. 5470.984.WO with correlated expression of Sst, Pdyn, and Crh marker genes. (41) UpSet plot summarizing the overlapping gene expression of Vipr2, Sst, Pdyn, and Crh. Bar charts (left) highlight select coexpression patterns with blank area representing no co-expression.

[0017] FIGS. 5A-5H. In vivo pharmacology of amygdalar analgesics. (5A) Circuit diagram of amygdalar cell types considering prior studies and inference of associated analgesic GPCR drug targets. (5B-5H) Twenty minutes after intraperitoneal injection of GPCR candidate ligands or morphine (3 mg / kg), behavioral pharmacology was assessed. (5B-5E) Mice were tested with the 52°C hot plate assay for affective-motivational pain and escape behavior. (5B and 5C)The amount of times that mice attended (5B) and latency to attend (5C) to the rear hindpaw were quantified. (5D and 5E) The amount of times that mice reared (5D) and latency to rear (5E) were quantified. (5F) Reflexive pain behavior was tested by a tail-withdrawal assay in a 52°C water bath. (5G) Effect of putative amygdalar analgesics on motor coordination assessed by rotarod assay. (5H) Exploratory behavior and spontaneous movement assessed by an open field assay.

[0018] FIGS. 6A-6R. Biased modulation or combinatorial pharmacology for effective and safer analgesia. (6A) Distribution of NTSR1 throughout the amygdala. (6B) The NTSR1 biased allosteric modulator, SBI-553 was determined to exhibit positive allosteric modulation and agonism of beta-arrestin recruitment (left) and negative allosteric modulation of Gq protein activation (right) through in vitro signaling assays (data are presented as mean values ± SEM with a minimum of two technical replicates and n= 3 biological replicates). (6C-6G) SBI-553 was systemically administered via intraperitoneal (i.p.) injections at 0, 12, 30, and 48 mg / kg. (6C and 6D) Affective-motivational and escape behaviors in the hotplate test 20 minutes post-i.p. injection of SBI-553, quantified as attending of the hindpaw (6C) and rearing (6D), respectively. (6E) Reflexive pain behavior in the tail flick assay. (6F and 6G) Rotarod and open field assays to assess motor coordination (6F) and spontaneous locomotor activity (6G), respectively. (6H) Combination therapy rationale considering prediction that identified amygdalar analgesics modulate multiple pain-related pathways, and may synergize to enhance analgesia. (6I-6M) Amygdalar analgesic mix at doses subthreshold for each of the three drugs individually was systemically administered via i.p. injections. (61 and 6 J) Affective-motivational and escape behaviors in the hotplate test 20 minutes post-i.p. injection of ATTA, quantified as attending of the hindpaw (61) and rearing (6J), respectively. (6K) Reflexive pain behavior in the tail flick assay. (6L and 6M) Rotarod and open field assays to assess spontaneous motor coordination (6L) andAttorney Docket No. 5470.984.WO locomotor activity (6M), respectively. (6N-6R) To test sufficiency of the amygdala for the antinociceptive effects of the three-drug ATTA, we locally administered the ATTA to the amygdala via cannulas at two concentrations with similar ratios of drug concentrations as systemic administration. (6N) Targeting strategy for amygdala target injection. (60 and 6P) Affective- motivational and escape behaviors in the hotplate test after cannula injection of ATTA into the amygdala, quantified as attending of the hindpaw (60) and rearing (6P), respectively. (6Q) Open field assessment of spontaneous movement after amygdalar injection of ATTA. (6R) Activity assessment during hot plate assay with 15 sec bins. Data were presented as Mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0019] FIGS. 7A-7I. Efficacy of amygdalar analgesics in preclinical rodent pain models. (7 ) Measurement of hindpaw guarding duration after formalin injection during phase I (0-15 minutes) in wild type mice receiving an i.p. injection of Vehicle or 30 mg / kg SBI-553. (7B) Mechanical nociceptive threshold determination with von Frey monofilaments following paw incision and Vehicle or 30 mg / kg SBI-553 administration. (7C) 30 mg / kg SBI-553 attenuated place preference of 5 mg / kg in the CPP assay. (7D) Measurement of hindpaw attending after formalin injection during phase I (0-15 minutes) and phase II (15-60 minutes) in wild type mice receiving intraperitoneal injection of Vehicle or ATTA. (7E) Representative confocal projections of the amygdala showing double staining for Fos mRNA (HCR; light gray) and Sst mRNA (HCR; gray) in mice receiving hindpaw formalin injection with or without administration of ATTA, compared to control mice receiving vehicle. (7F) Quantification of the number of Fos-positive cells in central amygdala (CeA), anterior BLA (BLAa), posterior BLA (BLAp), and lateral amygdala (LA) formalin-induced Fos expression. (7G) Mechanical nociceptive threshold determination with von Frey monofilaments following paw incision and ATTA or vehicle administration. (7H) To test the effect of ATTA on alleviating chronic pain, we tested the orofacial pain model in mice and assessed nociceptive response of forepaw attending to the face in response to light touch (0.16g Von Frey filament). (71) Conditioned place preference to the ATTA drug-paired chamber was assessed before and after 3 days of i.p. injections.

[0020] FIGS. 8A-8I. Sequencing quality metrics and expanded classification marker assessment. (8A) Violin plots of the number of reads (nReads) and number of genes (nGenes) per cluster from all high-quality cells. (8B) Violin plots of nReads and nGenes per cluster from neuronal population. (8C) Venn diagram showing co-expression Prkcd and Drd2 across CeA neuron types.Attorney Docket No. 5470.984.WO(8D) Images are from the Allen Mouse Brain Atlas (mouse.brain-map.org / ) showing coronal sections for Gabre in situ hybridization. (8E) Differentially expressed marker genes for glutamatergic amygdala neuron types (e.l-e.8). (8F) Differentially expressed marker genes for BLA interneuron types (i.l and i.2) and intercalated cells (i.3). (8G) Differentially expressed marker genes for CeA types (i.4-i.8). (8H) Differentially expressed marker genes for MEA types (i.9-i.10). (81) Composition of tdTomato cells expressed across neuronal types for control and formalin-induced pain conditions.

[0021] FIGS. 9A-9C. Spatial transcriptomic analysis and distribution of neuronal cell types. (9A) Dot plots of canonical amygdalar neuron marker genes for each cluster. Dot size represents the percentage of cells in each class expressing the genes and color intensity represents the mean gene expression level as indicated by the legend. (9B) Subregion origin identified for cell types based on spatial distribution of coronal slice. (9C) Rostro-caudal axis origin identified for cell types based on spatial distribution of coronal slice.

[0022] FIGS. 10A-10B. Co-expression analysis of Oprml, Oprdl, and Oprkl. (10A) Percent of individual cell types co-expressing Oprkl±Oprml (left), Oprkl±Oprdl (middle), and Oprkl±Oprdl (right). (10B) Percent of individual cell types co-expressing Oprkl±Oprdl±Oprml.

[0023] FIGS. 11A-11B. Catalog of GPCR and G protein effector expression across amygdalar neuron-types. (11A) Dot plot showing genes encoding GPCRs. (11B) Dot plot showing genes encoding G protein effectors.

[0024] FIG. 12. Additional behavior metrics of affective-motivational screening of candidate analgesics. Hotplate behavioral analysis of latency to jump. Each candidate drug was tested with vehicle control (0 mg / kg), a low dose that was subthreshold for analgesia, and a high dose determined to be analgesic.

[0025] FIGS. 13A-13N. Additional behavior metrics for amygdalar analgesic combination and different dosage combination of the amygdalar analgesics. (13A-13C) Hotplate latencies for ATTA drug mixture, including latency to attend (13A), latency to rear (13B), and latency to jump (13C). (13D) Latency to attend analysis for different concentrations of MIX administered systemically via intraperitoneal injection (i.p ). (13E) Spontaneous movement analysis from 20 minutes Open Field assay after administration of different concentrations of MIX (i.p.) or vehicle control. (13F) Representation of individual drug concentrations for different MIX compositions. (13G and 13H) Hot Plate behavior analysis of different MIX compositions for attending latencyAttorney Docket No. 5470.984.WO(13G) and number of attending occurrences in a session (13H). (131) Open Field assay assessment of ATTA-2 and ATTA-3 compared to vehicle control. (13 J) Spontaneous movement analysis of 2-drug combinations of amygdalar analgesic compounds. (13K) Core body temperature of individual compounds or in combination (MIX) as differential of pre-administration compared to 20-minutes post-administration (i.p.). (13L-13N) Hot Plate behavior analysis 20 minutes after direct amygdala cannula injection for latency to attend (13L), latency to rear (13M), and latency to jump (13N). Data were presented as Mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to vehicle control.

[0026] FIG. 14. Additional behavior metrics for preclinical screening of amygdalar analgesics. Mechanical nociceptive threshold determination with von Frey monofilaments before and after paw incision and ATTA or vehicle administration, out to 180 minutes

[0027] FIG. 15 shows the structure and in vitro signaling properties of SB 1-553 (arrestin agonistic positive allosteric modulator (PAM), Gq negative allosteric modulator (NAM)),

[0028] FIG. 16 shows the structure and in vitro signaling properties of AET-13-12 (Gq NAM).

[0029] FIG. 17 shows the structure and in vitro signaling properties of CZ-14-008 (arrestin PAM).

[0030] FIG. 18 shows hotplate analgesia for each of compounds SBI-553, AET-13-12 and CZ- 14-008, as quantified by total affective-motivational behaviors. CZ-14-008, the arrestin-biased PAM, does not decrease affective-motivational pain on the hotplate.

[0031] FIG. 19 shows that AET-13-12 provides a decrease in pain in a model of post-operative pain.

[0032] FIG. 20 shows that AET-13-12 provides a decrease in pain in a model of chronic neuropathic pain.

[0033] FIG. 21 shows that AET-13-12 decreases morphine preference / reward in conditioned place preference (CPP) testing.DETAILED DESCRIPTION

[0034] The present invention will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosureAttorney Docket No. 5470.984.WO will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. In addition, any references cited herein are incorporated by reference in their entireties.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, patent publications and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0036] Amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three-letter code, both in accordance with 37 C.F.R. §1.822 and established usage.

[0037] Except as otherwise indicated, standard methods known to those skilled in the art may be used for cloning genes, amplifying and detecting nucleic acids, and the like. Such techniques are known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 4th Ed. (Cold Spring Harbor, NY, 2012); Ausubel et al. Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0038] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.

[0039] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0040] To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0041] As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0042] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).Attorney Docket No. 5470.984.WO

[0043] The term “about,” as used herein when referring to a measurable value such as an amount of compound, antibiotic dose, time, temperature, or purity and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

[0044] As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel character! stic(s) of the claimed invention. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0045] “Pharmaceutical composition” means a mixture of substances suitable for administering to a subject. For example, a pharmaceutical composition may comprise a therapeutic of the invention in a pharmaceutically acceptable carrier.

[0046] “Pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) refers to a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms “carrier” or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents. As used herein, the term “carrier” encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.

[0047] The term “modulate,” “modulates,” or “modulation” refers to enhancement (e.g., an increase) or inhibition (e.g., a decrease) in the specified level or activity.

[0048] The term “enhance” or “increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold and / or can be expressed in the enhancement and / or increase of a specified level and / or activity of at least about 1%, 5%, 10%, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more.

[0049] “Inhibit” or “reduce” or grammatical variations thereof as used herein refers to a decrease or dimini shment in the specified level or activity of at least about 1, 5, 10, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).Attorney Docket No. 5470.984.WO

[0050] The term “contact” or grammatical variations thereof as used with respect to a GPCR ligand and a cell, refers to bringing the GPCR ligand and the cell in sufficiently close proximity to each other for one to exert a biological effect on the other.

[0051] The term “agonism” as used herein refers to the activation of a receptor or enzyme by a modulator, or agonist, to produce a biological response. Agonist as used herein refers to a modulator that binds to a receptor or enzyme and activates the receptor to produce a biological response. In some embodiments, agonist includes full agonists or partial agonists. A full agonist refers to a modulator that binds to and activates a receptor with the maximum response that an agonist can elicit at the receptor. A partial agonist refers to a modulator that binds to and activates a given receptor, but has partial efficacy, that is, less than the maximal response, at the receptor relative to a full agonist.

[0052] A biased allosteric modulator or BAM refers to a molecule that binds to a G protein- coupled receptor (GPCR) at a site different from its endogenous ligand, but in a way that selectively increases or decreases the receptor's activation of one signaling pathway over another. For example, an arrestin-biased allosteric modulator of neurotensin (NTS) receptor 1 (NTSR1) can promote |3-arrestin recruitment to NTSR1 while concurrently acting as a negative allosteric modulator for Gq and GoA signaling

[0053] The term “antagonism,” as used herein, refers to the inactivation of a receptor or enzyme by a modulator, or antagonist. Antagonism of a receptor, for example, is when a molecule binds to the receptor and does not allow activity to occur. Antagonist as used herein refers to a modulator that binds to a receptor or enzyme and blocks a biological response.

[0054] Methods as used herein may be used with a subject in need thereof, including a subject in need of ameliorating, treating, or reducing conditions or disorders related to opioid use, including decreasing opioid reward, opioid addiction / dependence, and / or opioid use disorder (OUD). “Opioid reward” refers to the pleasurable and rewarding sensations produced by opioid drugs that activate the brain's reward system, leading to increased dopamine release in the nucleus accumbens. Addiction can result with an attenuated dopamine increase in brain reward regions, where drug consumption is associated with a difference between the expected reward and the actual experience of it, which may contribute to drug-taking behavior to compensate for the difference, and ultimately contributing to further opioid dependence and addiction. “Opioid addiction,” “opioid use disorder” and / or “OUD” refers to the chronic use of opioids that causesAttorney Docket No. 5470.984.WO clinically significant distress or impairment. Symptoms of this disease include an overpowering desire to use opioids, increased opioid tolerance, and withdrawal syndrome when opioids are discontinued.

[0055] A “subject” may be any vertebrate organism in various embodiments. A subject may be individual to whom an agent is administered, e.g., for experimental, diagnostic, and / or therapeutic purposes or from whom a sample is obtained or on whom a procedure is performed. In some embodiments a subject is a mammal, e.g., a human, non-human primate, lagomorph e.g., rabbit), or rodent (e.g., mouse, rat). In some embodiments a human subject is a neonate, child, adult or geriatric subject. In some embodiments a human subject is at least 50, 60, 70, 80, or 90 years old.

[0056] Grammatical variations of “administer,” “administration,” and “administering” to a subject include any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrastemal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like. “Concurrent administration,” “administration in combination,” “simultaneous administration,” or “administered simultaneously” as used herein, means that the compounds are administered at the same point in time, overlapping in time, or one following the other. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time. “Systemic administration” refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject’s body (e g., greater than 50% of the body), for example through entrance into the circulatory or lymph systems. By contrast, “local administration” refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject's body. Administration includes self-administration and the administration by another.Attorney Docket No. 5470.984.WO

[0057] “ Treat,” “treating” and similar terms as used herein in the context of treating a subject refer to providing medical and / or surgical management of a subject. Treatment may include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically undertaken in an effort to alter the course of a disease (which term is used to indicate any disease, disorder, syndrome or undesirable condition warranting or potentially warranting therapy) in a manner beneficial to the subject. The effect of treatment may include reversing, alleviating, reducing severity of, delaying the onset of, curing, inhibiting the progression of, and / or reducing the likelihood of occurrence or recurrence of the disease or one or more symptoms or manifestations of the disease, e.g., pain unpleasantness. A therapeutic agent may be administered to a subject who has a disease or is at increased risk of developing a disease relative to a member of the general population. In some embodiments a therapeutic agent may be administered to a subject who has had a disease but no longer shows evidence of the disease. The agent may be administered e.g., to reduce the likelihood of recurrence of evident disease. A therapeutic agent may be administered prophylactically, i.e., before development of any symptom or manifestation of a disease. “Prophylactic treatment” refers to providing medical and / or surgical management to a subject who has not developed a disease or does not show evidence of a disease in order, e.g., to reduce the likelihood that the disease will occur, delay the onset of the disease, or to reduce the severity of the disease should it occur. The subject may have been identified as being at risk of developing the disease (e.g., at increased risk relative to the general population or as having a risk factor that increases the likelihood of developing the disease).

[0058] The term “ameliorating” or “ameliorate” refers to any indicia of success in the treatment of a pathology or condition, including any objective or subjective parameter such as abatement, remission or diminishing of symptoms or an improvement in a patient's physical or mental wellbeing. Amelioration of symptoms may be based on objective or subjective parameters including the results of a physical examination and / or a psychiatric evaluation.

[0059] Pain unpleasantness or affective pain refers to the emotional and psychological reactions that accompany the physical sensation of pain. Pain unpleasantness or affective pain is distinct from the sensory aspect of pain, which focuses on the intensity and location of the pain, and may include affective-motivational and / or cognitive-evaluative dimensions. Examples of emotional and psychological experiences associated with pain unpleasantness may include experiencing fear,Attorney Docket No. 5470.984.WO anxiety, or depression in response to pain, or feeling anguish and suffering due to chronic pain. Pain unpleasantness may manifest as a fear of movement, anger, frustration, and / or depression.

[0060] In some embodiments, a method of treating and / or preventing pain in a subject is provided, comprising administering to the subject a therapeutically effective amount of a G protein-coupled receptor (GPCR) ligand or pharmaceutically acceptable salt thereof, wherein the GPCR ligand targets a non-opioid GPCR in the amygdalar region of the brain, and wherein pain is alleviated and / or minimized in the subject.

[0061] In some embodiments, a method of treating unpleasantness related to pain in a subject is provided, comprising administering to the subject a therapeutically effective amount of G protein- coupled receptor (GPCR) ligand or pharmaceutically acceptable salt thereof, wherein the GPCR ligand targets a non-opioid GPCR in the amygdalar region of the brain, and wherein unpleasantness related to pain is alleviated and / or minimized in the subject.

[0062] In some embodiments the therapeutically effective amount of the GPCR ligand is a dose of about 0.1 mg / kg / day to about 100 mg / kg / day. In some embodiments, two or more GCPR ligands are administered to the subject. In some embodiments, two or more GCPR ligands are administered to the subject to thereby collectively activate two or more GCPRs. In some embodiments, one or more of the ligands is administered at subthreshold doses. A subthreshold dose as used herein is a dose amount less than that which would trigger a detectable biological effect in a subject. In some embodiments, the subthreshold dose is 5 mg / kg LY344864 or less, 0.1 mg / kg PD149163 or less, and / or 1 mg / kg BAY55-9837 or less.

[0063] In some embodiments, the GPCR ligand specifically binds to a non-opioid GPCR. In some embodiments, the non-opioid GPCR is selected from the group consisting of Ackrl, Ackr2, Ackr3, Ackr4, Adcyaplrl, Adgral, Adgra2, Adgra3, Adgrbl, Adgrb2, Adgrb3, Adgrdl, Adgrd2-ps, Adgrel, Adgre4, Adgre5, Adgrfl, Adgrf2, Adgrf3, Adgrf4, Adgrf5, Adgrgl, Adgrg2, Adgrg3, Adgrg4, Adgrg5, Adgrg6, Adgrg7, Adgrll, Adgrl2, Adgrl3, Adgrl4, Adgrvl, Adoral, Adora2a, Adora2b, Adora3, Adrala, Adralb, Adrald, Adra2a, Adra2b, Adra2c, Adrbl, Adrb2, Adrb3, Agtrla, Agtrlb, Agtr2, Agtrap, Aplnr, Avprla, Avprlb, Avpr2, Bdkrbl, Bdkrb2, Brs3, C3arl, C5arl, C5ar2, Calcr, Calcrl, Casr, Cckar, Cckbr, Ccrl, CcrlO, Ccrlll, Ccr2, Ccr3, Ccr4, Ccr5, Ccr6, Ccr7, Ccr8, Ccr9, Ccrl2, Celsrl, Celsr2, Celsr3, Chrml, Chrm2, Chrm3, Chrm4, Chrm5, Cmklrl, Cnrl, Cnr2, Crcp, Crhrl, Crhr2, Cx3crl, Cxcrl, Cxcr2, Cxcr3, Cxcr4, Cxcr5, Cxcr6, Cysltrl, Cysltr2, Drdl, Drd2, Drd3, Drd4, Drd5, Ednra, Ednrb, F2r, F2rll, F2rl2, F2rl3, Ffarl,Attorney Docket No. 5470.984.WOFfar2, Ffar3, Ffar4, Fprl, Fpr2, Fpr3, Fpr-rs3, Fpr-rs4, Fpr-rs6, Fpr-rs7, Fshr, Fzdl, Fzdl O, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, Gabbrl, Gabbr2, Galrl, Galr2, Gcgr, Ghrhr, Ghsr, Gipr,Glplr, Glp2r, Gml4496, Gm5127, Gm9268, Gnrhr, Gpbarl, Gperl, Gprl, GprlOl, Gprl07, Gprl l9, Gprl2, Gprl32, Gprl35, Gprl37, Gprl39, Gprl41, Gprl42, Gprl43, Gprl46, Gprl49, Gprl5, Gprl5O, Gprl51, Gprl52, Gprl53, Gprl56, Gprl57, Gprl58, Gprl60, Gprl61, Gprl62, Gprl7, Gprl71, Gprl73, Gprl74, Gprl76, Gprl79, Gprl8, Gprl82, Gprl83, Gprl9, Gpr20, Gpr21, Gpr22, Gpr25, Gpr26, Gpr27, Gpr3, Gpr31b, Gpr33, Gpr34, Gpr35, Gpr37, Gpr3711, Gpr39, Gpr4, Gpr45, Gpr5O, Gpr55, Gpr6, Gpr61, Gpr62, Gpr63, Gpr65, Gpr68, Gpr75, Gpr82, Gpr83, Gpr84, Gpr85, Gpr87, Gpr88, Gprc5a, Gprc5b, Gprc5c, Gprc5d, Gprc6a, Grik3, Grml, Grm2, Grm3, Grm4, Grm5, Grm6, Grm7, Grm8, Grpr, Hcarl, Hcar2, Hcrtrl, Hcrtr2, Hpgd, Hrhl, Hrh2, Hrh3, Hrh4, Htrla, Htrlb, Htrld, Htrlf, Htr2a, Htr2b, Htr2c, Htr4, Htr5a, Htr5b, Htr6, Htr7, Inpp5k, Kisslr, Lgr4, Lgr5, Lgr6, Lhcgr, Lparl, Lpar2, Lpar3, Lpar4, Lpar5, Lpar6, Ltb4rl, Ltb4r2, Masi, Mclr, Mc2r, Mc3r, Mc4r, Mc5r, Mchrl, Mrgpral, Mrgpra2a,Mrgpra2b,Mrgpra3, Mrgpra4, Mrgpra6, Mrgpra9, Mrgprbl, Mrgprb2, Mrgprb3, Mrgprb4, Mrgprb5, Mrgprb8, Mrgprd, Mrgpre, Mrgprf, Mrgprg, Mrgprh, Mrgprxl, Mrgprx2, Mtnrla, Mtnrlb, Nlrp6, Nmbr, Nmurl, Nmur2, Npbwrl, Npffrl, Npffr2, Npr3, Npsrl, Npylr, Npy2r, Npy4r, Npy5r, Npy6r, Ntsrl, Ntsr2, Ogfr, Ogfrll, Opnlmw, Opnlsw, Opn3, Opn4, Opn5, Oprdl, Oprkl, Oprll, Oprml, Oxgrl, Oxtr, P2ryl, P2ryl0, P2ryl2, P2ryl3, P2ryl4, P2ry2, P2ry4, P2ry6, Pgrl51, Ppard, Prlhr, Prokrl, Prokr2, Ptafr, Ptgdr, Ptgdr2, Ptgerl, Ptger2, Ptger3, Ptger4, Ptgfr, Ptgir, Pthlr, Pth2r, Qrfpr, Rho, Rorb, Rrh, Rxfpl, Rxfp2, Rxfp3, Rxfp4, Slprl, Slpr2, Slpr3, Slpr4, Slpr5, Sctr, Smo, Sstrl, Sstr2, Sstr3, Sstr4, Sstr5, Sucnrl, Taarl, Taar2, Taar3, Taar4, Taar5, Taar6, Taar8a, Taar9, Tacrl, Tacr2, Tacr3, Tbxa2r, Tpral, Trhr, Trhr2, Tshr, Uts2r, Viprl, Vipr2, and Xcrl .

[0064] In some embodiments, the non-opioid GPCR is selected from the group consisting of NTSR1, VIPR2, SSTR4, mGluR2, mGluR3, and 5HT1F.

[0065] In some embodiments, the GPCR ligand is a NTSR1 ligand. In some embodiments, the NTSR1 ligand is a biased allosteric modulator of NTSR1, a negative allosteric modulator activity at Gq, a positive allosteric modulator (PAM) activity, and / or an agonist. In some embodiments, the NTSR1 ligand is the NTS peptide, NTS(8-13) fragment, SRI-9829, SBI810, PD149163, or SBI- 553.

[0066] In some embodiments, the GPCR ligand is a Vasoactive intestinal polypeptide receptor 2 (VIPR2) ligand. In some embodiments, the VIPR2 ligand is a receptor agonist. In someAttorney Docket No. 5470.984.WO embodiments, the ligand is VIP(125-152) or BAY 55-9837, or KS-133 (Sakamoto et al, PLoS ONE 18(7): e0286651).

[0067] In some embodiments, the GPCR ligand is a somatostatin receptor subtype 4 (SSTR4) ligand. In some embodiments, the SSTR4 ligand is a receptor agonist. In some embodiments, the SSTR4 ligand is J-2156 or NNC-269100. Additional example SSTR4 ligands are described, for example, in Kantas, et al. Front Pharmacol. 2021 Jan 27; 11 :601887. doi: 10.3389 / fphar.2020.601887, incorporated herein by reference in its entirety.

[0068] In some embodiments, the GPCR ligand is a metabotropic glutamate receptor 2 (mGluR2) ligand and / or metabotropic glutamate receptor 3 (mGluR3) ligand. In some embodiments, the mGluR2 and / or GluR3 ligand is an agonist. In some embodiments, the mGluR2 and / or mGluR3 ligand is TASP 0433864, VU6023326, DCG-IV, LY379268 or LY354740. See, Strauss, et al. Nat Commun 15, 6498 (2024); doi: 10.1038 / s41467-024-50548-x, incorporated herein by reference in its entirety.

[0069] In some embodiments, the GPCR ligand is a 5-hydroxytryptamine (serotonin) receptor IF (5HT1F) ligand. In some embodiments, the 5HT1F ligand is a receptor agonist. In some embodiments, the 5HT1F ligand is LY344864, LY334370, Lasmiditan. See, Clemow et al., J Headache Pain 21, 71 (2020); doi: 10.1186 / sl0194-020-01132-3, incorporated herein by reference in its entirety.

[0070] In some embodiments, the GPCR ligand is selected from the group consisting of PD149163 (N2-[(2S)-2,6-Diaminohexyl]-L-lysyl-L-prolyl-L-tryptophyl-3-methyl-L-valyl-L-Leucine ethyl ester tetrahydrochloride hydrate), SBI-553 (2-[[2-(l-fluorocyclopropyl)-4-[4-(2-methoxyphenyl)- l-piperidinyl]-6-quinazolinyl]methylamino]-ethanol), BAY 55-9837, LY379268 (1R,4R,5S,6R)- 4-Amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid, NNC-269100 (l-[3-[N-(5-Bromopyridin-2-yl)-N-(3,4-dichlorobenzyl)amino]propyl]-3-[3-(lH-imidazol-4- yl)propyl]thiourea), or LY344864 (N-[(3R)-3-(Dimethylamino)-2,3,4,9-tetrahydro-lH-carbazol- 6-yl]-4-fluoro-benzamide).

[0071] Some embodiments comprise repeating the administering of the ligand to the subject. In some embodiments, the subject is administered two or more GPCR ligands, e.g., at subthreshold doses. In some embodiments, the subject is administered ligands for 5HT1F, NTSR1, and VIPR2. In some embodiments, administering the ligand comprises oral administration, intravenousAttorney Docket No. 5470.984.WO administration, intrathecal administration, intraperitoneal administration, intra-articular administration, epidural administration, or any combination thereof.

[0072] In some embodiments, unpleasantness related to pain may be triggered from a variety of injury and diseases such as trauma, metabolic diseases (e.g., diabetic neuropathy), structural defects (e.g., back pain), sickle cell disease) or has unclear etiology (e.g., fibromyalgia). Patients can present with allodynia, hyperalgesia, and / or spontaneous pain, and pain intensity, where pain quality (e.g., sharp, stabbing, throbbing), and triggers may vary considerably. In some embodiments, the pain is peripheral or central neuropathic pain, inflammatory pain, migraine- related pain, headache-related pain, irritable bowel syndrome-related pain, fibromyalgia-related pain, arthritic pain, skeletal pain, joint pain, gastrointestinal pain, muscle pain, angina pain, facial pain, pelvic pain, claudication, postoperative pain, post traumatic pain, tension-type headache, obstetric pain, gynecological pain, or chemotherapy-induced pain. In some embodiments of the invention, the subject has acute pain, chronic pain, or both. In some embodiments, the subject has sustained one or more injuries and / or one or more physical injuries, including, for example, surgical injuries.

[0001] In some embodiments, the subject has an opioid dependence, opioid addiction, and / or opioid use disorder. In some embodiments, methods can comprise decreasing opioid reward in a subject in need thereof.

[0073] In some embodiments, the methods of the invention further comprise administering to the subject a therapeutically effective amount of one or more therapeutic agents, e.g., analgesic agents. In some aspects of the invention, the one or more therapeutic agents comprise of opioids, narcotic analgesics, Mu receptor agonists, Kappa receptor antagonists, non-narcotic analgesics, monoamine uptake inhibitors, adenosine regulating agents, cannabinoid derivatives, Substance P antagonists, neurokinin- 1 receptor antagonists, calcitonin gene-related peptide (CGRP) and its receptor inhibitors, sodium channel blockers, non-steroid anti-inflammatory drugs (NSAIDs), and any combination thereof.

[0074] These and other aspects of the invention are set forth in more detail in the description of the invention below.Attorney Docket No. 5470.984.WOEXAMPLESExample 1. Molecular Profiling of Nociceptive Brain Neurons Enables Precision Pharmacology Against Pain Unpleasantness

[0075] It was hypothesized that by resolving the molecular architecture of pain-relevant cell types in the amygdala, druggable non-opioidergic GPCRs to treat pain unpleasantness could be identified. Here, transcriptomic analysis of amygdalar neurons revealed 17 types and 28 subtypes of neurons with distinct spatial organizations, connectivity, GPCR repertoires, and activity profiles during pain. Within pro- and anti-nociceptive amygdalar circuits, we found a dozen non-opioid GPCRs with analgesic potential. Remarkably, engagement of each of the six lead candidate GPCRs with repurposed ligands attenuated acute pain unpleasantness. Furthermore, activating amygdalar GPCRs via biased allosteric modulation or a combination therapy of three ligands used at subthreshold also provided analgesia in models of postoperative and chronic orofacial neuropathic pain, with limited side effects, while opposing opioid-induced reward to prevent transition to addiction. Together, these findings establish the cellular and molecular structure of the nociceptive amygdala and identify highly druggable targets to treat pain unpleasantness across pain types.

[0076] Results

[0077] Transcriptomic classification of amygdalar neuron-types

[0078] To discover novel analgesic drug targets in the amygdala, we profiled the gene expression of individual amygdalar neurons active during nociception using single-cell RNA sequencing (scRNA-seq) (FIG. 1 A). To generate activity in nociceptive circuits, we used an established model of tissue injury-induced pain, injecting the algogen formalin into the left hindpaw. This procedure induces sustained activity in nociceptors innervating the paw and central nervous system (CNS) pain circuits, and highly stereotyped nocifensive affective-motivational behaviors consisting of attending behaviors such as paw licking We performed these experiments using TRAP2;Ail4 mice which permits labeling of active neurons with the fluorescent protein tdTomato. We then microdissected the amygdala, dissociated amygdalar cells, selected individual tdTomato+ neurons via fluorescence-activated cell sorting (FACS), and processed cells for single-cell mRNA sequencing (FIG. 1A). We sequenced the transcriptome of 9,695 high-quality cells, including neurons (5092) and glia (4603), distinguished based on cluster annotation and known marker genes (FIG. IB, FIGS. 8A,8B). Focusing on neurons (FIGS. 1B-1E), we used unsupervised clusteringAttorney Docket No. 5470.984.WO and known marker genes from the literature to identify 16 major neuron types: four glutamatergic types expressing the Slcl7a7 gene that encodes the vesicular glutamate transporter 1 (VGLUT1, e.1 -e.4), two additional glutamatergic types expressing the Slcl 7a6 gene that encodes the vesicular glutamate transporter 2 (VGLUT2, e.7 and e.8), and ten GABAergic neuron types expressing the Gadl gene encoding the enzyme glutamate decarboxylase 1 (GAD1) permitting GABA synthesis (i.l-i.10) (FIGS.1B-1G). A subclustering analysis identified a total of 28 neuronal subtypes (FIG. IE; FIGS. 8C-8E).

[0079] In the BLA, previous studies revealed the presence of two functionally distinct glutamatergic neuronal populations, primarily differentiated by their expression of either Rspo2 or Ppplrlb. These two populations encode the negative and positive emotional valence of stimuli, respectively . However, we found that A.s / w2-expressing neurons include two major Rspo2+ cell types (e.l and e.5). The e.l subtype is enriched for multiple novel marker genes including Ptprc, Gliprl, Oprkl, Thsd4, Sema3e, Bink, and Prss23. In contrast, e.5 neurons selectively express Adam33 and Col23al. Based on the relatively high expression of Ppplrlb, we identified the putative positive-valence BLA cells as the e.2 type and report the novel markers Rnd2, Slc29a4, Lpl, Otof, Meis2, Hspala, and Hspalb. Additionally, Ppplrlb is weakly expressed across most Slcl7a7+ neuron types K We defined lateral amygdala (LA) neurons as cell-type e.3 based on the expression of Rorb wAMyl4 -■■, and further identified multiple novel markers associated with e.3 including Neb, Car8, Pamrl, and Trpc3. Finally, we report the novel markers for the Slcl7a7+ type e.4 Zfp57, Rbms3, and Qrjpr. BLA interneurons have thus far been classified into four populations defined by expression of the neuropeptides somatostatin (Sst) or vasoactive intestinal peptide (J Ip), or of the calcium-binding protein genes parvalbumin (Pvalb), calbindin (Calbl), or Calretinin (Calb2). By comparison, we found that the BLA includes seven types of molecularly defined interneurons: Sst;Npy without Nosl expression are local interneurons (i. la), Sst;Npy with Nosl expression are long-range projections neurons (i. lb), small Vip;Calb2 neuronal types (i.2a)

[0080] and large Cck;Calbl neurons

[0081] In the CeA, neuronal diversity has been intensely studied in the lateral central amygdala (CeL) and capsular subdivision of the CeA (CeC) divisions, while only a handful of CeA general marker genes have been described. Prkcd and Sst delimit the two major cell types of the CeL, which interact through local mutual inhibitory circuitsWe detected Prkcd expression in type i.4, along with correlated markers Pltp, Slc4all, Prss23, Ezr, and Gprl50. Previously, theAttorney Docket No. 5470.984.WOCGRP receptor encoded by Calcrl has been determined to be expressed in the anatomically- distinct CeC that expresses Prkcd as well as a subset of Drd2 neurons -A Accordingly, we defined the CeC Prkcd type as the i ,4b subtype in our study due to restricted Calcrl expression compared to i.4a. Importantly, we identified the novel i.4b markers Bean, Ephx2, Chrm5, Rasdl, Upklb, and Brcal to gain genetic access to these cells. The i.4a type, which are comprised of r / c / 7-negative Prkcd+ cells, are enriched for expression of Cartpt, Igfbp2, Clql2, and Slc29a4. We also determined that the rare cell population i.7 shows high level of co-expression between Sst, Nts, Crh, Pdyn, and Tac2 in the CeA as previously reported -5 fi- .

[0082] Further, we identified multiple novel i.7 marker genes, including Itgal, Vipr2, Rhpnl, Syndigll, Car8, Ebfl, Slc4all, and Dnah5. Drd2 is known to be co-expressed with Adora2a in a distinct CeA population, with a subpopulation expressing CalcrlOur data further demarcate the Ca / crZ-expressing Drd2 cell type as subcluster i.5b and provide additional novel markers Ctxn3, GprlOl, 1700003M02Rik, Trhr, and Pax6. In contrast, we differentiated the Calcrl- negative Drd2 subcluster i.5a by expression of novel markers Scn4b, Cd4, Rarb, Serpina9, Syndigll, and Gabrd. To date, reports differ regarding the co-expression of Drd2 and Prkcd in the central amygdalaHere, we detect only rare cases of co-expression of Drd2 and Prkcd(Prkcd: 19 / 436, Drd2: 19 / 232) (FIG. 8C). CeA neurons expressing the kappa (K)-opioid receptor (encoded by OprkP) have been extensively studiedTo date, there have been no known molecular markers of these Oprkl CeA neuron types. Here we determined Oprkl to be selectively expressed in CeA type i.7 and we discovered multiple novel markers including Sp8, Tspanl8, Gfral, Dlkl, Adm, and Crabpl. The medial central amygdala (CeM) is known to be loosely segregated by non-overlapping expression of Nts, Sst, and Tac2 - - . We detected these three marker genes across cell types i.8a and i.8b, but were unable to resolve distinct subpopulations from our scRNA-seq results via unsupervised clustering. We determined that the marker genes Gabre, Htrld, Ucp2, Lmol, Moab, Ctsz, Zarl, and Ucp2 are shared between i.8a and i.8b. Accordingly, the Allen Institute ISH database demonstrates that expression of Gabre is confined to the CeM (FIG. 8D). Furthermore, we found several marker genes restricted to either i.8a or i.8b. Novel markers for 1.8a include Isll, Ebfl, GprlOl, Sgpp2, Chstl5, Tacr3, and Abi3bp, while novel markers for i.8b include Vdr, Avprla, Mc4r, Pnoc, BC030867, Glplr, Rasa4, and Galrl.

[0083] Finally, our study also resolves the molecular identity of neurons in two other subregions of the amygdala: the amygdalar intercalated cell masses and the medial extended amygdalaAttorney Docket No. 5470.984.WO(MEA). Intercalated cells (ITCs) are three tightly packed masses of neurons adjacent to the BLA. Neurons in the main intercalated nucleus of the amygdala (Im) are reciprocally connected to BLA principal cells and receive additional inputs from the thalamus and cortexThe lateral paracapsular ITC cluster contributes feedforward inhibition to the BLA while the medial paracapsular ITC cluster mediates communication between the BLA and CeAWe found that ITCs correspond to the neuron type i.3 given that these cells express established ITC marker Foxp2. Our analysis reveals two distinct molecular subtypes of ITCs, i.3a and i.3b (FIG. 8F). Notably, i.3b is enriched for Npy expression, which based on prior studies was thought to be restricted to the Im '-1. In contrast, ITC type i .3 a is defined by the selective markers Mgp, Tnfaip6, and Gml4637. The MEA comprises the excitatory neuron types e.7 and e.8, as well as the inhibitory types i.9a-c and i.lOa-c. We were unable to determine markers for e.6 and i.l 1, possibly due to the limited transcriptomic information for these cells considering the relatively low number of profiled genes for e.6 (average nGenes 4707 ± 2068), i.l 1 (nGenes 3212 ± 1466) compared to other cell types (FIG. 8B). For each cell type identified from our scRNA-seq study, we provide a resource of additional novel marker genes (FIGS. 8E-8H). Overall, our single-cell RNA-seq analysis determined the molecular signatures for all previously recognized neuronal cell types in the amygdala at unprecedented depth and clarity.

[0084] Activity signatures, connectivity, and spatial organization of amygdalar neurons

[0085] To quantify the activation of amygdalar neurons during pain experience, we first used whole-brain tissue clearing and light-sheet microscopy. We determined the density of tdTomato+ neurons across 700+ structures of the brain in TRAP2;Ail4 mice following the intraplantar injection of formalin (FIGS. IF, 1G). In the amygdala, we found that the number of labeled neurons increased significantly (sAmy, FIG. 1G). The magnitude of the increased neuronal labeling varied across amygdalar subregions (CeA, BLA, ITC, MEA, LA, BLA, FIG. 1G). At the transcriptomic level, we did not detect a significant increase in IdTomato RNA in amygdalar neuron-types between control and formalin-injected mice FIG. 81). This result could be explained by technical factors such as incomplete sampling of rare cell types, differences in populations sampled due to microdissection variation, and enrichment of pain-unrelated active neurons by sequencing similar numbers of tdTomato-positive versus -negative neurons for both control and formalin conditions. To test this possibility, we directly assessed formalin-induced c-fos expression by performing scRNA-seq on samples from mice in which we repeated the formalin injection 90 minutes prior toAttorney Docket No. 5470.984.WO euthanasia. Consistent with our finding of unchanged tdTomato expression, c-fos expression was not significantly different in the repeated-formalin condition compared to control or to the singleformalin condition for which mice did not receive intraplantar formalin on the day of dissection (FIG. 1H). Interestingly, however, this experiment uncovered the expression profile of six other immediate early genes (lEGs), Egr2, Egr3, Nr4al, Nr4a3, Fosb, Fosl2, that suggest activation of distinct neuron types during pain, including the pro-nociceptive Prkcd+;Calcrl+ neuronal population (FIG. II). These data suggest that monitoring expression of these lEGs instead of c-fos more faithfully report CeA neuron activity during nociception.

[0086] Next, we conducted neural circuit tracing studies to understand how nociceptive information may reach and be processed in the amygdala during pain. Given that previous studies described the pain pathways by which parabrachial and thalamic neurons can engage the CeAwef0CUSed here on the connectivity of BLA neurons. We injected Cre-dependent recombinant adeno-associated virus (rAAV) helpers (rAAV-CAG-FLEx-mKate-TVA and rAAV- CAG-FLEx-G) and the CVS-N2c(AG)-GFP mutant rabies virus (MRV) with enhanced retrograde synaptic transfer and neuronal viability in the BLA of TRAP2 mice, and induced Cre-mediated recombination in BLA nociceptive neurons. We found that this approach successfully labeled mono-synaptically connected neurons in various brain regions (FIG. 1J). Notably, these studies revealed that BLA nociceptive neurons receive inputs from the anterior cingulate cortex (ACC), anterior insular cortex (Al), paraventricular nucleus of the thalamus (PVT), caudate-putamen (CPu) and pre-optic area (POA), demonstrating the distinct connectivity and function of BLA versus CeA neurons during. To identify the outputs of BLA nociceptive neurons, we injected in the BLA an AAV-DJ-hSyn-FLEx-mGFP-2A-Synaptophysin-mRuby, which labeled axons with GFP and their terminals with both GFP and mRuby in target regions, including the ACC and CPu, highlighting the BLA-ACC and BLA-CPu reciprocal connectivity (FIG. IK). We also found that BLA nociceptive neurons project to the CeA, anterior amygdala (AA), parabrachial nucleus (PB), CPu, nucleus accumbens (NAc). These studies establish divergent connectivity and function of BLA versus CeA neurons during pain, particularly BLA reciprocal connectivity with cortical regions involved in the emotional and cognitive dimensions of pain.

[0087] scRNA-seq datasets have inherent defects, such as inclusion of cells outside of the area of interest from insufficiently precise dissection, potential misrepresentation of cell-type proportions due to differences in viability, and gene expression changes during tissue processing. To overcomeAttorney Docket No. 5470.984.WO these limtations and capture spatial patterns of gene expression in amygdalar neurons, we next performed highly multiplexed single-molecule RNA-fluorescence in situ hybridization (smFISH) (FIG. 2A; FIG. 9). We spatially resolved the expression of 97 genes encoding cell-type-selective marker genes, including novel gene markers identified from our scRNA-seq study (FIG. 2A). We applied Seurat label transfer from our scRNA-seq reference dataset to map all amygdalar cell types to spatial locations within the amygdala, identifying all neuron types in the smFISH dataset (FIGS. 2B,2C). Each annotated cluster recapitulated comparable expression patterns of predicted novel and established markers (FIG. 2D, FIG. 9 A).

[0088] Next, we focused our analysis on the finer-grain neuron types spatially enriched in the LA, BLA, ITC, and CeA subregions of the amygdala (FIG. 2; FIG. 9). These classes of neurons expressed amygdalar regional markers such as Slcl7a7 in the BLA, Slcl7a6 in the LA, Foxp2 in the ITCs (FIG. 2A). We identified 14 glutamatergic and 28 GABAergic subtypes and proceeded to assign regions of origin to each neuron-type (FIGS. 2E-G; FIG. 9). We spatially demarcated and annotated the anterior BLA (BLAa) and posterior BLA (BLAp) neurons based on the enrichment of the Rspo2+ negative-valence neuron or the Ppplrlb+ positive-valence-neuron gene markers, respectively. To date, these BLA valence-associated neuron types have been considered to otherwise be molecularly homogeneous. Spatial transcriptomics results corroborate that Rspo2 expression is restricted to the e. l BLAa type and Ppplrlb expression is enriched in e.2 in the BLAp, while also resolving transcriptionally finer-grain subtypes. Rspo2+ type e.l stratified into the e.la subpopulation expressing Bink that is distinct from e.lb, which co-expresses Ptprc, (jliprR Thsd4, Bink, Prss23, and Sema3e. Additionally, we determined that Ppplrlb+ type e.2b is defined by selective Lpl expression while e.2a expresses the markers Olof Rnd2, Col23al, and Slc29a4. Notably, we identified that the distinct Rspo2+ cell type sc.e.5 is spatially enriched in the endopiriform cortex (EC) based on the combined expression of EC regional markers such as Bace2, Abca8ci, Pcsk5, and Col23al (FIGS. 9B, 9C).

[0089] We identified GABAergic (Gadl+) neuron subtypes spatially enriched in the BLA, ITC, and CeA that exhibited the molecular signatures expected from scRNA-seq data, with additional resolution. Regarding parvalbumin-expressing inhibitory interneurons, although scRNA-seq data included cells expressing Pvalb and other marker genes associated with these neurons, limited sampling of this population precluded a full account of Pvalb+ neuron molecular diversity. In contrast, spatial transcriptomics clearly separated two subtypes of BLA Pvalb+ neurons:Attorney Docket No. 5470.984.WOPvalb,Calbl basket neurons (type i.lc. l) and Pvalb,Vipr2 chandelier neurons (type i. lc.2)Similarly, for somatostatin-expressing inhibitory interneurons, we found that the CeA Sst;Pdyn type was split into two distinct subtypes, i.7.1 and i.7.2, via unsupervised clustering of spatial transcriptomics data despite being a single homogeneous type based on scRNA-seq data. More precisely, i.7.1 and i.7.2 were distinguished by the absence or presence of Crh;Nts;Tac2 coexpression, respectively. Further, we determined that i.7 is limited to the caudal amygdala, being nearly absent in rostral CeA (FIG. 9C). We also clarified that the Drd2+ subpopulation of sc.i.5b belongs to the nearby caudate putamen while the Drd2+ subpopulation expressing Ctxn3 belongs to the nearby amygdala-striatal transition area (ASt) (FIG. 9B). Overall, these results resolve at single-cell resolution the spatial organization of both known and novel amygdalar neuron-types (FIG. 2G).

[0090] Organization of neurotransmitter systems in the amygdala

[0091] Next, we resolved the architecture of intercellular communication in the amygdala by determining, at the single cell level, the distribution of neuropeptide and small molecule neurotransmitter receptors, the source of their ligands within the amygdala, and intracellular effectors for GPCR signaling (FIGS. 1-3; FIG. 11). Among these ligand-receptor systems, the opioid system in the amygdala plays an important role in the regulation of pain, mood, anxiety, stress, and rewardWe and others have provided evidence that opioid receptors are expressed in the amygdala and that opioid signaling in the amygdala can produce antinociception - . A recent study revealed that opioid action in the CeA may also underlie some of the addictive properties of opioids, particularly physical dependence and withdrawalUi. However, the amygdalar cell types through which endogenous and exogenous opioids can modulate pain and addiction remain poorly resolved. Next, we therefore comprehensively characterized the amygdalar neuron-types that express the genes encoding opioid peptides and receptors: Met / Leu- enkephalin, P-endorphin, dynorphin A / B, encoded by Penk, Pome, Pdyn, respectively, and the p- , 8-, and K-opioid receptors, encoded by Oprm I, Oprdl , Oprkl, respectively. Met / Leu-enkephalin, -endorphin, and dynorphin A / B are preferential agonists of p- and 8-opioid receptors, of p- opioid receptors, and of K-opioid receptors, respectively.

[0092] The p-opioid receptor is responsible for both the analgesic and addictive effects of opioids used clinically and abused. We found that Oprml is extensively expressed across multiple GABAergic neuron-types, including BLA Sst+ interneurons (i.l), CeC / L (i.4, i.5, and i.6), CeMAttorney Docket No. 5470.984.WO(i.8), and ITC (i.3) (FIGS. 3A,B). Additionally, we identified Oprml expression on LA glutamatergic neuron Rorb+ subtype e.3, an association that had not been identified previously. By contrast, Oprml expression was absent in BLA Vip+ neurons (i.2a type) or Vipr 2 -expressing Sst;Pdyn CeA neuron type i.7. This Oprml expression pattern is corroborated between our scRNA- seq and spatial transcriptomics results. In particular, we confirmed that Oprml is almost absent from Sst+ neurons in the CeC and CeL subdivisions of the CeA, but present in the Sst+ i.8d type in the CeM. These results greatly extend previous co-expression analyses that globally reported an enrichment of Oprml in Prkcd+ neurons compared to Sst+ neurons in the CeAL! l. Among these types of Oprml+ neurons, the connectivity of CeM Oprml;Sst neurons (i .8d) is unknown. To map the projections of CeM Oprml;Sst neurons, we used an intersectional viral strategy and injected an AAV-Efla-FLExcre-FLExHp-oScarlett virus in the CeA of Oprm / Crc;.S's7l lpmice (FIG. 3C). We primarily observed oScarlet+ cells in the CeM injected region, with relatively few cells in the neighboring CeL. We found that the most densely innervated area is the posterior thalamus nucleus, which includes the posterior intralaminar thalamic nucleus (PIL) and parvocellular subparafascicular nucleus of the thalamus (SPFp). Notably, oScarlet+ axon terminal labeling was absent from the neighboring medial geniculate nucleus. We observed additional oScarlet+ fibers in the bed nucleus of the stria terminalis (BNST), lateral parabrachial nucleus (PB1), and parvocellular reticular nucleus. This intersectional mapping of Oprml, Sst CeA neuron-subtype outputs identified at least four target regions, in contrast to prior studies that identified the BNST and PB1 as the primary outputs of all Oprml -expressing CeA neurons -tu.

[0093] 8-opioid receptor signaling in the amygdala is thought to modulate anxiety, including during painJIn contrast to the p-opioid receptor, we previously showed that the 8-opioid receptor is mostly expressed in the BLA Intriguingly, we could only identify weak Oprdl expression in sparse cellular populations from our scRNA-seq data (FIG. 3B, left). However, spatial transcriptomics data revealed extensive presence of Oprdl in the BLA (FIG. 3B, right) and elucidated the molecular identity of the cell types that express 8-opioid receptor, revealing enriched expression in A.s / >2-expressing e.1 cell types compared to Ppplrlb-expressmg e.2. Additionally, Oprdl was moderately expressed in Oprkl+ CeA type i.6 and CeM type i.8, but absent in BLA interneurons and in Prkcd+ or Sst+ CeA types expressing Oprml.

[0094] Opioid peptides that bind to the p- and 8-opioid receptors are also abundant in the amygdala. P-endorphin primarily binds to the p-opioid receptor. We identified only moderateAttorney Docket No. 5470.984.WO expression of Pome in the amygdala across multiple types including Rorh+ neurons of the LA, multiple CeCL neuron types, and Gabre;Nts neurons in the CeM (FIG. 3B). In contrast, Penk, which binds to both p- and 8-opioid receptors, is extensively expressed across CeA and ITC cell types.

[0095] Activation of the K-opioid receptor in the amygdala by dynorphin has been implicated in stress and affective behaviors in injury-induced pain conditions Our scRNA-seq data revealed Pdyn expression in Foxp2,Drdl i.3b ITC cell type as well as Sst;Pdyn;Crh i.7 type and CeM type i.8. Our spatial transcriptomics validated high Pdyn expression in ITCs, Sst+ CeA types, but also clarified moderate expression in the remaining CeA types i.4, i.5, and i.6 (FIG. 3B). OprH-expressing BLA principal neurons projecting to the BNST and mPFC constitute an anxiolytic pathway ';: :Further, K-opioid receptor activation may inhibit BLA outputs to theWe uncovered the molecular identity of these cells as the BLA glutamatergic neuron type e. l, which selectively expressed Oprkl and co-expressed the negativevalence neuron marker Rspo2. To clarify the projections of Oprkl -expressing BLA pyramidal neurons, we injected AAV-Efla-FLExcre-FLExFip-oScarlett virus in the BLA of Oprkl(FSIcl7a7v'pmice (FIG. 3D).

[0096] We found that glutamatergic Oprkl+ neurons in BLA predominantly project to the ACC layer 2 / 3, BNST, the ventral striatum, claustrum, and insular cortex. Inhibition of CeA neurons following K-opioid receptor activation by dynorphin is pro-nociceptive through the modulation of descending pain control pathwaysAdditionally, the CeA Dyn / K-opioid receptor system regulates anxiety-like behavior and the negative affect during alcohol withdrawalIn the CeA, we found that Oprkl is selectively expressed by the ii.6 neuron-type. To distinguish the projection targets of Oprkl -expressing cell types in the GABAergic CeA, we injected AA Jr-CreON,FlpON- oScarlet virus in the CeA of ()prkl<'-K. lc32alv^ mice (FIG. 3E). Prior work has inferred Oprkl+ inhibitory input to the BNST from the CeA . In addition to CeA Oprkl+ outputs to BNST, we also identified major outputs to the ventrolateral periaqueductal gray (vlPAG), PB, locus coeruleus, parvocellular reticular nucleus, and nucleus of the solitary tract. There were minor direct CeA Oprkl+ inputs to the RVM region (FIG. 3E).

[0097] The nociceptin opioid peptide (NOP) receptor, also known as opioid-receptor-like 1 (ORL- 1), is highly homologous to p-, 6-, and K-opioid receptors but has distinct pharmacology. In the CeA, NOP modulates the rewarding properties of palatable foodOur transcriptomic resultsAttorney Docket No. 5470.984.WO indicate broad expression of Oprll, which encodes the NOP receptor, across most amygdalar cell types with notable exceptions, including lack of expression in Vip type i.2a, Pvalb type i. lc, and Sst;Crh;Pdyn type i.7 types. Additionally, we found that Pnoc, which encodes nociceptin, is enriched in the Gabre+ CeM i.8 types, as well as BLA interneuron i.2 types (FIG. 3B). Spatial transcriptomics data also clarified that Pnoc is expressed at low levels across most amygdalar cell types, with the highest expression level in Gabre;Nts i.8 CeM neuron-types (FIG. 3 A).

[0098] Functional interactions between distinct opioid receptor types within pain neural circuits, including via co-expression and direct physical interactions, has been a subject of major interest to develop safer opioid analgesicsWe f'ounc[ that Oprkl and Oprml expression is segregated across distinct groups of neurons in the BLA, whereas Oprkl is co-expressed with Oprml in the i.6 type of the CeA. Oprdl was moderately coexpressed across both Oprkl and O / vv?? / -expressing populations in the BLA (FIG. 10A). Further, we found that Oprdl is coexpressed with Oprkl in CeC / Li.6 and CeM / i.8 neuron-types (FIG. 10A). Strikingly, the BLA interneuron type i.2b and Sst;Pdyn CeA type i.7 lack expression of Oprdl, Oprkl, and Oprml (FIG. 10B). Together, these results resolve the complex functional organization of the endogenous opioid system within the amygdala.

[0099] Identification of amygdalar non-opioid GPCRs with analgesic potential

[0100] GPCRs are highly druggable proteinsthat alter neurotransmission by modulating neuronal excitability and neurotransmitter releaseDespite their harmful side effects, p- opioid receptor agonists such as morphine remain indispensable medicines due to their unparalleled ability to reduce the unpleasant quality of pain. We reasoned that engaging other, non-opioid, GPCRs in amygdalar pathways may disrupt the flow of nociceptive information that underlies the encoding of pain unpleasantness. Next, we leveraged this transcriptomic atlas to establish a comprehensive catalog of GPCRs expressed in the amygdala (FIG. 4, FIG. 11 A).

[0101] We first investigated GPCR expression in three pronociceptive amygdalar neuron-types: Rspo2 BLA neurons, Prkcd;Calcrl CeA neurons, and Avprla+ CeM neurons (FIG. 4A, left). We prioritized the discovery of Gi / o-coupled GPCRs that may not only alter the flow of nociceptive information in the amygdala but also directly inhibit these pro-nociceptive neurons (FIG. 4B). The JN / ?o2-expressing BLA neurons (e.l type) serve well-established functions in behaviors related to negative valence, including during pain perception ‘. We identified several genes encoding Gi / o- coupled GPCRs that are primarily expressed by Rspo2+ e.1 BLA neurons, including the serotoninAttorney Docket No. 5470.984.WO receptor 5HT1F or HTR1F (Htrlf), somatostatin receptors SSTR2 and SSTR4 (Sstr2, Sstr4 metabotropic glutamate receptors mGluR2, mGluR3, mGluR8 Grm2, Grm3, Grm8 and K-opioid receptor (Oprkl) (FIG. 4B). We tested Htrlf amygdalar expression with spatial transcriptomics and confirmed relatively high expression of Htrlf in BLA Rspo2+ cell types e.1 in contrast to the low Htrlf expression in the positive-valence Ppplrlb+ cell types e.2, especially the Otof- expressing subtype e.2a (FIGS. 4C-4E). These results suggest that 5HT1F activation could inhibit Rspo2+ neuron-types associated with negative-valence without affecting the activity of positivevalence-associated Ppplrlb+ neurons.

[0102] In the CeA, activation of Prkcd+ neurons can promote nocifensive behaviorAdditionally, Prkcd,Calcrl+ CeA neurons receive pronociceptive input from CGRP+ PB1 neuronsWe identified i .4b as the pronociceptive CeA neuron type that co-expresses Prkcd;Calcrl (FIG. 4B). We did not identify Gi / o-coupled GPCRs with enriched expression in the i 4b neurons. However, we found that the gene Chrm5 that encodes the excitatory muscarinic acetylcholine receptor M5 (Gq-coupled GPCR) is selectively expressed by Priced, Calcrl+ i.4b neurons, as well as by Drd2+ i.5a neurons, and could potentially be blocked or inhibited with an antagonist, inverse agonist, or negative allosteric modulator (NAM). In addition, we found that A vprla+ CeM type i.8b is pronociceptive given that the activation of this excitatory Gq-coupled GPCR vasopressin receptor, AVPR1, increases the affective-motivational, rather than the sensory-discriminative, dimension of pain “Furthermore,, we established that i.8b selectively expresses the excitatory Gs-coupled GPCR MC4R encoded by Mc4r, which represents an attractive target given that intra- CeA administration of an MC4R antagonist reduces mechanical and thermal hypersensitivity in a rodent model of chronic inflammatory pain •4~-. Finally, we also identified i.8b-selective expression of the Galrl gene encoding the Gi / o-coupled GPCR GALR1 (FIGS. 4B, 4C), which selective agonist M617 shows antinociceptive activity in normal and neuropathic rats upon intra-CeA administration:" .

[0103] We also extrapolated that the Oprkl+ CeA neuron-type is antinociceptive considering that neuronal inhibition by K-opioid receptor Gi / o signaling is pronociceptive via modulation of descending pain pathwaysWe found that the excitatory GPCR genes Ntsrl, Hrhl, and Qrfpr, encoding the neurotensin (NTS) receptor 1 (NTSR1), histamine receptor Hl (H1R), and pyroglutamylated RF-amide peptide receptor (QRFPR), respectively, are enriched in i.6 neurons (FIG. 4B). Further, we clarified using spatial transcriptomics that Ntsrl is also enriched inAttorney Docket No. 5470.984.WOPrkcd;Calcrl-neg?A\Ne CeL neurons (i.4a) (FIGS. 4C, 4F, 4G). Prkcd CeL neurons are known to share reciprocal connections with pronociceptive Prkcd;Calcrl+ neurons in the CeC (i.4b) Therefore, activation of VIPR2 and NTSR1 in the amygdala may provide analgesia by driving GABAergic inhibition of pronociceptive neurons in both ascending and descending pain pathways.

[0104] Next, we selected anti-nociceptive cell types from this amygdalar cell atlas and searched for selectively expressed GPCRs coupled to excitatory G proteins (Gs or Gq) that could produce analgesia. Sst;Crh;Pdyn CeA neurons send long-range inhibitory input to the PB. Activation of this pathway can reduce transmission of nociceptive information in ascending pain circuitsRelatedly, CeL Sst;Crh;Tac2+ neurons positively influence appetitive behaviors and receive direct monosynaptic input from BLA Ppplrlb+ principal neurons We uncovered that Sst;Crh;Pdyn+ (i.7 type) neurons selectively express Vipr2, which encodes the excitatory Gs- coupled vasointestinal peptide receptor 2 (VIPR2 or VPAC2), and Npbwrl which encodes the inhibitory Gi o-coupled GPCR neuropeptides B and W receptor 1 (NPBWR1) (FIG. 4B). Our spatial transcriptomics data validate the selective expression of Vipr2 in Sst;Pdyn;Crh+ (sp.i.7a), while clarifying the absence of expression in the Sst;Pdyn;Crh-negative subpopulation (sp.i.7b) (FIGS. 4C, 4H, 41). Further, we confirm Vipr2 expression in BLA chandelier neurons (sp.i.lc) (FIG. 4C). Collectively, our findings thus far establish the cellular and molecular structure of the nociceptive amygdala and identify highly druggable amygdalar targets for the development of analgesics against pain unpleasantness (FIG. 5A).

[0105] In vivo pharmacology of amygdalar analgesics

[0106] To test the hypothesis that amygdalar GPCRs can be targeted to develop novel analgesics against pain unpleasantness, we monitored pain affective-motivational behaviors that mice stereotypically demonstrate in response to noxious stimulation to make pain unpleasantness cease. These pain affective-motivational behaviors include attending behaviors, such as paw licking or guarding, and escaping behaviors, such as rearing and jumping, that mice displayWe repurposed commercially available selective agonists to independently activated five of our GPCR analgesic candidates: NTSR1 with PD149163VIPR2 with BAY 55-98375HT1F with LY34486415;:, SSTR4 with NNC-2691002 ;-, and metabotropic glutamate receptors mGluR2 and mGluR3 with LY379268(FIGS. 5B-5H). We used the hotplate test to screen for compounds that may reduce pain unpleasantness: For each compound, we determined 20 min after administration the effect of two doses or vehicle on attending (hindpaw licking) and escaping painAttorney Docket No. 5470.984.WO behaviors (rearing and jumping). Remarkably, it was found that for at least one of the doses administered, all compounds significantly decreased the number of attending behaviors compared to vehicle administration (PD149163: P = 0.0006; BAY55-9837: P = 0.03; LY344864: P = 0.04; NNC269100: P = 0.04; LY379268: P = 0.004) (FIG. 5B). The number of rearings was also significantly reduced by PD149163 (0.1 mg / kg, P = 0.02; 1 mg / kg, P = 0.002), LY344864 (25 mg / kg, P = 0.02), NNC269100 (5 mg / kg, P = 0.02), LY379268 (3 mg / kg, P = 0.001; 10 mg / kg, P = <0.0001) (FIG. 5D). Furthermore, with the exception of LY344864, all compounds also increased the latency to display hindpaw attending (PD149163: 0.1 mg / kg, P = 0.02 and Img / kg, P = 0.0083; BAY55-9837: 5 mg / kg, P = 0.006; NNC269100: 5 mg / kg, P = 0.01; LY379268: 10 mg / kg, P = 0.0006) (FIG. 5C) and / or rearing behaviors (PD149163: Img / kg, P < 0.0001; BAY55- 9837: 5 mg / kg, P = 0.006; LY344864: 25 mg / kg, P = 0.02; NNC269100:5 mg / kg, P = 0.004; LY379268: 10 mg / kg, P = 0.0006) (FIG. 5E). As a positive control, we used systemic administration of morphine. Morphine reduces both withdrawal reflexes and affective- motivational pain behaviors by acting on p-opioid receptors broadly distributed along pain circuits at the primary afferent, spinal and brain levelsi ?. As expected, morphine reduced hindpaw attending (P = 0.04) (FIG. 5B) and rearing (P = 0.0008) (FIG. 5D), with similar analgesic efficacy as PD 149163 (1 mg / kg) and LY379268 (10 mg / kg).

[0107] In a previous study, we showed that the chemogenetic inhibition of nociceptive amygdalar neurons can selectively decrease affective-motivational pain behaviors, without altering withdrawal reflex behaviorsWe therefore tested whether our amygdalar analgesics modulate the spinal reflex responses to a noxious thermal stimulus using the tail-withdrawal assay. Strikingly, we found that at doses that reduce affective-motivational pain behaviors, most amygdalar analgesic compounds did not prolong tail withdrawal latency, with the exception of PD149163 (P = 0.0006) (FIG. 5F). Predictably, morphine significantly delayed reflexive withdrawal at both 3 mg / kg and 10 mg / kg (P = 0.0003 and P = 0.0002, respectively). These results suggest that the compounds tested induced analgesia primarily by reducing the unpleasantness of pain, without altering the detection of noxious stimuli or processing of nociceptive information, at the primary afferent and spinal levels, respectively. We refer to these compounds as amygdalar analgesics in the rest of the study.

[0108] Next, we examined the side effect profile of amygdalar analgesics. Thus, the clinical utility of each of these compounds may be limited by side-effects that have been described in previousAttorney Docket No. 5470.984.WO preclinical studies, including reduced locomotor activity, motor coordination, or core body temperature (CBT): :We found that motor coordination was normal at lower doses of the NTSR1 agonist PD149163 (0.1 mg / kg, P > 0.99), but significantly reduced at the analgesic dose of 1 mg / kg (P = 0.004) (FIG. 5G). However, the other amygdalar analgesics acting at VIPR2, 5HT1F, SSTR4 or mGluR2 / 3 had no effect on motor coordination at any of the doses tested. We tested the locomotor activity with the open field test and found that VIPR2, 5HT1F, and SSTR4 agonists did not alter locomotor activity, even at high analgesic doses (FIG. 5H). In contrast, high doses of PD149163 or of the mGluR2 / 3 agonist LY379268 almost completely eliminated locomotor activity. Finally, the CBT was slightly reduced by low, subanalgesic, doses of BAY 55- 9837 (-1.4°C, 1 mg / kg) and of LY344864 (-0.4°C, 5 mg / kg), while PD149163 increased CBT (+0.93°C, 0.1 mg / kg). At analgesic doses, all three drugs individually reduce CBT by approximately two degrees (FIG. 13K), an effect comparable to that of morphine. These results demonstrate the potential of amygdalar GPCR drug targets for the development of novel central analgesics, and the challenges associated with developing pain drugs acting in the brain with no or limited side effects.

[0109] Biased modulation or synergistic agonism delivers safer analgesia

[0110] To ameliorate the therapeutic index of amygdalar analgesics, we next devised two approaches: biased modulation, and synergistic poly-agonism. To test the possibility that biased agonism may elevate the safety of amygdalar analgesics, we focused on NTSR1, for several reasons. First, both transcriptomic data and histology data in 7sr7X enusreporter miceindicate that NTSR1 is not only expressed at multiple loci within amygdalar nociceptive circuits (FIGS. 4F-4G, FIG. 6A), but also in several other brain regions involved processing of pain information, as well as in classes of primary afferent and spinal neurons implicated in mechanical hypersensitivity. Second, the NTS / NTSR1 system can oppose addictive behaviorsmaking this GPCR particularly attractive to develop non-addictive analgesics. Third, an NTSR1 biased ligand, SBI-553, already exists - ", and has been shown to retain anti-addictive properties, while attenuating PD 139163 -induced side effects like decreased locomotor activity Although SBI- 553 was initially described as a NTSR1 arrestin-biased agonistwe confirmed here that SBI- 553 presents a considerably more complex pharmacological profile, with negative allosteric modulator activity at Gqand positive allosteric modulator (PAM) activity and agonism at arrestin (FIG. 6B), as shown previously by Krumm, Roth and collaborators ■■■-.Attorney Docket No. 5470.984.WO

[0111] We next tested whether SBI-553 could reduce affective-motivational pain behaviors like the NTSR1 agonist PD149163, but without producing motor side effects. Remarkably, we found that SBI-553 profoundly and dose-dependently decreased both attending and escape behaviors in the hotplate test. At the dose of 12 mg / kg, SBI-553 significantly decreased the number of rearings (P = 0.0269) and the latency to rear (P = 0.0329) without affecting attending behaviors, while at the doses of 30 and 48 mg / kg, SBI-553 administration significantly diminished both attending (P < 0.0001 for both 30 and 48 mg / kg) (FIG. 6C) and rearing behaviors (P < 0.0001 for both 30 and 48 mg / kg) (FIG. 6D). SBI-553 did not alter reflexive pain behaviors at the 12 mg / kg and 30 mg / kg doses, (vehicle vs. 12 mg / kg: P = 0.7121; vehicle vs. 30 mg / kg: P =0.2378), while prolonging the latency for tail withdrawal at 48 mg / kg (P < 0.0001) (FIG. 6E). Moreover, in stark contrast with the effect of PD149163, SBI-553 had no effect on mouse performance in the rotarod test, at any dose (30 mg / kg vs. 48 mg / kg: P = 0.3040; all other comparisons: P> 0.9999) (FIG. 6F). In contrast to the complete elimination of locomotor activity induced by the high dose of PD149163 necessary to generate analgesia (FIGS. 5B, 5F), SBI-553 only modestly reduced locomotor activity (vehicle vs. 12 mg / kg: P = 0.0019; vehicle vs. 30 mg / kg and vehicle vs. 48 mg / kg: P < 0.0001) (FIG. 6G). Together, these data reveal the potential of NTSR1 as a target to develop analgesics that may reduce pain unpleasantness with limited side effects.

[0112] Second, combination therapy with multiple drugs is a major approach for areas of medicine such as HIV-1 infection and cancerbujs not yet fu|]y deployed in the pain field. To reduce the distinct side effects attributed to 5HT1F, NTSR1, and VIPR2 activationwereasoned that we could combine their agonists at low doses to produce safer and enhanced analgesia. We predicted that the collective activation of 5HT1F, NTSR1, and VIPR2 at subthreshold doses (5 mg / kg LY344864, 0.1 mg / kg PD149163, and 1 mg / kg BAY55-9837, respectively) could promote combinatorial analgesia by simultaneously acting at three nodes within amygdalar pain circuits (FIG. 6H). Therefore, we called this combination therapy Amygdalar Triple Therapy Analgesic (ATTA).

[0113] Accordingly, we found that ATTA reduced and delayed affective-motivational pain behaviors, including attending (number of attending behaviors: P = 0.04; latency to attend: P < 0.0001) (FIG. 61; FIG.13A) and escape behaviors (number of rearings: P = 0.0005; latency to rear: P = 0.0001; latency to jump: P = 0.004) (FIG. 6J; FIGS. 13B, 13C). Reflexive pain behaviors were also delayed in mice injected with ATTA in comparison to vehicle (P = 0.0016), as demonstratedAttorney Docket No. 5470.984.WO in the tail-withdrawal assay (FIG. 6K). Importantly, in the rotarod assay, mice receiving ATTA performed similarly to control mice injected with vehicle (P = 0.9383) (FIG. 6L). Furthermore, in the open field test, we found that mice treated with ATTA did not show the complete lack of activity observed following PD 149163 administration (FIG. 5F) and instead explored the arena, although to a lesser extent compared to vehicle treated mice (P < 0.0001) (FIG. 6M). Diluting the ATTA solution (ATTA / saline 1 : 1 or 3: 1) eliminated its effects on affective-motivational pain behaviors (ATTA: latency to attend, P = 0.0015) and locomotor activity (ATTA: P < 0.0001) (FIGS. 13D, 13E). Additionally, we tested two different doses for the three drug combinations, which showed moderate reduction in affective-motivational pain behavior (Latency to attend: ATTA-3, P = 0.03; attending: ATTA-2, P = 0.02 and ATTA-3, P = 0.0007) and locomotor activity ATTA-2: P = 0.03; ATTA-3: P = 0.02) (FIGS. 13F-13I). To identify which drug is responsible for ATTA effect on locomotor activity, we tested two-drug combinations at subanalgesic doses. We found that all two-drug combinations reduced locomotor activity to an intermediate level compared to vehicle and ATTA administration (PD149163+BAY55-9837: P = 0.03; PD149163+LY344864: P = 0.09; BAY55-9837+LY344864: P = 0.03) (FIG. 13J), suggesting a minor contribution from all three drugs. Further, administration of ATTA resulted in a small reduction of CBT of 0.84°C (P = 0.0004) (FIG. 13K). These results demonstrate the benefits of ATTA considering that each drug individually reduced the CBT by approximately two degrees (FIG. 13K).

[0114] Next, to test whether modulation of amygdalar circuits by the ATTA can indeed produce antinociceptive effects, we locally administered the ATTA in the amygdala via cannulas (FIG. 6N). In the hotplate test, we found that the ATTA reduced affective-motivational pain behaviors at both low (lx) and high (2x) concentrations (attending: ATTA- lx, P = 0.05 and ATTA-2x, P = 0.007; rearing: ATTA-lx, P = 0.004 and ATTA-2x, P = 0.001) (FIGS. 60, 6P; FIGS. 13L-13N). Interestingly, in contrast to vehicle-injected mice that showed typical increased locomotor activity ATTA-injected mice maintained initial activity levels throughout the duration of the hot plate assay. In the open field test, we observed reduced locomotor activity (ATTA-1X: P = 0.006; ATTA-2x: P = 0.0003) (FIG. 6Q). These results are consistent with ATTA antinociceptive activity and suggest that the elimination of the pain-related increase in locomotor activity may be the manifestation of a minor sedative and calming effect that could be beneficial for the management of severe pain (30-45s: ATTA-2x, P = 0.01; 45-60s: ATTA-lx, P = 0.04 and ATTA-2x, P = 0.007)Attorney Docket No. 5470.984.WO(FIG. 6R). Together, these results show that ATTA produces analgesia, but in contrast to the individual drugs, has no effect on motor coordination and only modest, tolerable, effects on locomotor activity and CBT.

[0115] Preclinical evaluation of amygdalar analgesics

[0116] Next, we tested whether amygdalar analgesics can be effective in two preclinical models of tissue injury-induced pain, the intraplantar formalin model and the paw incision model of postsurgical pain.

[0117] In the formalin test, we found that administration of SBI-553 (30 mg / kg) significantly reduced guarding behaviors in the first phase of the formalin response (P = 0.0285) compared to vehicle administration (FIG. 7A), without affecting either guarding or licking during the second phase of the test. To model postsurgical pain, we used the paw incision procedure and evaluated mechanical sensitivity with the von Frey test. As expected, the incision caused a significant decrease in the paw withdrawal threshold (P = 0.0017) (FIG. 7B), demonstrating hypersensitivity to mechanical stimulation (i.e., mechanical allodynia), as seen postoperatively in patients. Notably, compared to vehicle administration, a single injection of SBI-553 significantly reduced incision- induced mechanical hypersensitivity (P = 0.0047) (FIG. 7B). These results suggest that targeting NTSR1 produces analgesia and that NTSR1 assets may represent a novel class of non-addictive analgesics that could replace, or be combined with, opioid analgesics to manage postoperative pain. To test the later possibility, given that SBI-553 can attenuate the consumption of other drugs of abuse including psychostimulants:and alcohol -1-, we tested whether SBI-553 could reduce morphine drug seeking in the conditioned place preference (CPP) assay. As expected, administration to mice of morphine (5 mg / kg) alone produced a strong preference for the chamber associated with morphine (P = 0.0061) (FIG. 7C), evincing the harmful rewarding properties of opioids. Remarkably, however, co-administration of SBI-553 (30 mg / kg) with morphine significantly attenuated this CPP (P = 0.1227) (FIG. 7C). This result suggests that NTSR1 assets may not only be used alone as analgesics, but could also be prescribed together with opioid analgesics to both effectively reduce pain and minimize the risk of transition to opioid addiction.

[0118] Next, we evaluated the analgesic efficacy of ATTA in these tissue-injury-induced pain models. Remarkably, in the formalin test, administration of ATTA eliminated both phases of the formalin pain response (Phase 1 : P = 0.02; Phase 2: P = 0.001) (FIG. 7D). Notably, this antinociceptive effect was accompanied by an increase in c-fos expression in the CeA (P = 0.02),Attorney Docket No. 5470.984.WO consistent with the prediction that activation of excitatory GPCRs enriched in GABAergic CeA neurons could alter neural function and pain processing in the CeA (FIGS. 7E, 7F). c-fos expression in LA and BLA were not significantly different across conditions (FIGS. 7E, 7F). In the paw incision model, administration of the ATTA significantly increased the paw withdrawal threshold (P = 0.0079), demonstrating the anti-allodynic properties of the ATTA (FIG. 7G). This ATTA-mediated anti-allodynia was maintained out to 60 minutes (P = 0.0023) and 180 minutes (P = 0.0002) post-administration (FIG. 14A). These results suggest that as shown for SBL553, ATTA or other assets engaging the same GPCRs, could represent highly valuable medicines to treat postoperative pain.

[0119] Finally, to test the utility of the ATTA to relieve chronic pain. We used a model of orofacial neuropathic pain. We induced neuropathic pain by ligating the infraorbital trigeminal nerve, which causes long-term mechanical hypersensitivity of the affected whisker pad, evidenced by face attending affective-motivational pain behaviors following innocuous mechanical stimulation with von Frey filaments. Notably, the ATTA significantly reduced face attending after a single administration (P = 0.0004) (FIG. 7H). Given that the pertinence of opioid prescription to manage non-malignant types of chronic pain is debated, especially considering their side effects, amygdalar analgesics may represent an effective non-addictive alternative to treat neuropathic pain. To test this possibility, we next asked if ATTA treatment may also pose risks of transition to addiction like opioids, by assessing the rewarding properties of ATTA in the CPP assay. We found that ATTA did not elicit place preference (P = 0.6809) (FIG. 71), suggesting that the analgesic activity of amygdalar analgesics may be devoid of rewarding properties and abuse liability. Collectively, these results reveal GPCRs in distinct components of amygdalar pain circuits that can be individually engaged or co-activated at low drug dose to relieve acute, postoperative and chronic neuropathic pain with limited side effects.

[0120] Discussion

[0121] Pain suffering is a growing medical problem for which opioid-based treatments have limited efficacy against certain types of pain and can produce harmful side effects -. Here, we devised a comprehensive cellular and molecular atlas of the nociceptive amygdala to identify non-opioid GPCR pain targets. We demonstrate that ligands acting at these GPCRs represent a novel class of analgesics, termed amygdalar analgesics, that can reduce pain unpleasantness across pain-types.Attorney Docket No. 5470.984.WO

[0122] Cellular and Molecular Architecture of Nociception Neuromodulation in the Amygdala

[0123] The mechanisms underlying emotional pain processing, including the organization of endogenous ligands-GPCR-intracellular effector signaling systems that participate in this process, remain unclear. This study considerably advances our understanding of these processes in the amygdala. We integrated scRNA-seq and high-resolution single-molecule FISH data to elucidate the cellular and molecular architecture of the nociceptive amygdala. This atlas accounts for all previously described CeA, BLA, and ITC neuron-types, evinces novel subtypes of amygdalar neurons, and provides a full repertoire of dozens of novel marker genes that enable future studies of amygdala cells and circuits.

[0124] During pain, the CeA receives nociceptive information ascending from the spinal cord from the PB1 x. We provide high-definition novel markers for the Drd2 and Prkcd subpopulations expressing CALCRL, the receptor for pro-nociceptive neuropeptide inputs from PB1. Surprisingly, we failed to identify inhibitory GPCRs enriched on Prkcd;Calcrl subpopulation aside from Oprml, which we clarified is expressed across most of the CeA except for the Sst;Pdyn type. Our molecular characterization of the anti-nociceptive Sst;Pdyn and Oprkl CeA neuronal populations also clarifies how the VIP-VIPR2 and NTS-NTSR1 signaling systems may modulate nociception. Regarding Sst;Pdyn CeA neurons, we established that Vipr2, Crh, and Nts coexpression distinguishes two subpopulations: Sst;Pdyn;Crh;Nts;Vipr2 and Sst;Pdyii;Crh-;Nts- ;Vipr2- CeA neurons (i.7a and i.7b, respectively). Previous studies indicated that Sst;Pdyn;Crh;Nts;Vipr2 neurons preferentially project to the vlPAG, a crucial region of the descending pain processing circuitry, with minimal input to the PB, while Sst;Pdyn;Crh-;Nts- ;Vipr2- neurons primarily project to the PB -?. Interestingly, other experiments showed that optogenetic activation of the CrA-expressing CeA neurons projecting to the PB pathway produces analgesia, and that NTS-expressing CeA neurons provide strong inhibitory input to the PBAs it pertains to VIP, local VIP interneurons in the BLA are well-established to communicate with Sst and PV interneurons, of which the chandelier neuron subset selectively express VIPR2. In contrast, the primary source of VIP in the CeA originates from dorsal raphe / vlPAG Vglut2+ glutamatergic inputs, which sends collaterals to the oval nucleus of the BNST (ovBNST)a structure that also expressesIn support of a J 'ipi‘2 -mediated BNST-CeA mechanism of pain modulation, chemogenetically activating vPAG / DR dopamine neurons or optogeneticallyAttorney Docket No. 5470.984.WO activating the the ascending DR / vlPAG pathway terminals in the BNST produces supraspinal antinociceptive effects Regarding Oprkl CeA neurons, we determined that the most enriched gene encoding an excitatory GPCR is Ntsrl, which is also expressed in the BLA. In this study, we describe the circuits by which nociceptive BLA neurons can be engaged during pain, showing BLA distinct connectivity map compared to the CeA, particularly its reciprocal connections with cortical regions involved in the emotional and cognitive dimensions of pain. Prior work established that Ntsrl -expressing BLA neurons projecting to the CeM play an important role in valence assignment. Our study uncovers the specific expression of NtsrJ by a subset of Ppplrlb+ pyramidal neurons that expresses Olof ' .

[0125] Further, in addition to neuropeptide-based modulation, this atlas establishes the cell-type specific distribution of the glutamatergic metabotropic receptors by which glutamate can regulate nociception in the amygdala. Notably, we clarified which amygdalar neuron-types may mediate the reduction in inflammatory and affective pain behaviors previously described to occur upon activation of glutamate metabotropic receptors signaling through Group 2 (mGluR2 / 3) and Group 3 (mGluR8) Regarding GABAergic control of nociception, a previous study showed that optogenetic excitation of a population of CeA neurons activated by general anesthetics is sufficient to produce analgesiaThese neurons, termed CCAGA neurons, were determined to express marker genes broadly expressed in the CeA, such as Prkcd and Penk. Our amygdalar analgesics include agonists of the excitatory GPCR VIPR2 selectively expressed by antinociceptive Sst;Crh;Pdyn neurons and the excitatory GPCR NTSR1 enriched on Oprkl - expressing CeA neurons. We hypothesize that these populations could be included in the small subset of neurons labeled by the induced Anesthesia-induced CeA neuronsFuture studies utilizing the marker genes described here can fully resolve the connectivity, physiology, and contributions to pain processing of these different populations of pain-related amygdalar neurons.

[0126] A Novel Class of Centrally Acting Analgesics Against Pain Unpleasantness

[0127] Current drug discovery efforts in the pain field focus on interfering with transmission of nociceptive information within nociceptors and spinal circuits. Although this approach is promising, as for the targeting of voltage-gated sodium channelspursuing other therapeutic strategies is important given the magnitude and complexity of the problem of pain. First, pain is a multidimensional experience with sensory-discriminative, affective-motivational, and cognitive- evaluative dimensions ■■■'. Thus, following activation of nociceptors, the CNS generates, for all painAttorney Docket No. 5470.984.WO types, the nocifensive behavioral responses, including withdrawal reflexes, attending, escape, more complex behaviors, associated with these three perceptual dimensions. It follows that an analgesic manipulation at the primary afferent nociceptor level is expected to evenly reduce all pain perceps and behavioral responses. This may not be ideal, given that subjects under treatment for a particular pain condition may ineffectively sense and / or withdraw from other noxious stimuli, as seen in individuals lacking functional expression of Navi.7 . Second, pain can result from a variety of injury and diseases such as trauma, metabolic diseases (e.g., diabetic neuropathy), structural defects (e.g., back pain), sickle cell disease) or has unclear etiology (e.g., fibromyalgia). Patients can present with allodynia, hyperalgesia, and / or spontaneous pain, and pain intensity, quality (e.g., sharp, stabbing, throbbing), and triggers vary considerably. A challenge to develop new treatments at the primary afferent level is that these different types of pain and presentations can involve distinct DRG / TG neurons and molecular mechanisms and may require elucidating pain-type specific mechanisms, as evidenced by the development of anti-CGRP signaling assets for migraine headache.

[0128] In other fields of medicine, numerous patients greatly benefit from the availability of several classes of drugs that target distinct mechanisms of the disease and can be combined. Examples include cancer (poly-drug chemotherapy), infection diseases (antiretroviral cocktail therapy for HIV), metabolic diseases (diabetes), cardiovascular diseases (high blood pressure). We propose here to enable a similar approach by developing, in parallel with drug development efforts at the primary afferent and spinal levels, a class of analgesics, which we termed amygdalar analgesics, that predominantly acts in the brain’s emotional circuits to reduce pain unpleasantness across pain types, while preserving withdrawal reflexes and pain sensation. This strategy is supported by both clinical and preclinical observation. Clinical reports show that damage to the amygdala, as observed in patient H.M. or in individuals with the genetic Urbach-Wiethe disease, can induce pain asymbolia, a phenomenon in which noxious stimuli are detected but are devoid of perceived unpleasantness . Regarding preclinical evidence, we previously used vivo Ca2+ imaging to record the activity of an estimated 17,000 amygdalar neurons in freely moving mice experiencing painWe found that an ensemble of neurons, which we named the nociceptive amygdalar ensemble, is activated during both acute pain across modalities (heat, cold, mechanical pain) and chronic neuropathic pain. Furthermore, chemogenetic disruption of normal processing of nociceptive information in the amygdala significantly reduced pain affective-motivationalAttorney Docket No. 5470.984.WO behaviors, including attending and escape behaviors during both acute pain and chronic neuropathic pain, and across pain modalities (heat, cold, mechanical), consistent with Ca2+ imaging data, but without altering reflexive withdrawalThat is, these mice, but not control mice, behaved as if pain was less unpleasant, corresponding to the clinical presentation of subjects with amygdala damage. However, a general concern associated with drug development efforts that target a molecule in the CNS or a molecule with relatively broad expression, as is typical for GPCRs, is safety. However, it is noteworthy that despite the very broad distribution of the mu opioid GPCR in PNS and CNS neural circuits, and their harmful side effects, opioids remain the most broadly used agent for the treatment of severe pain and an indispensable medicine. This suggests that agents acting at other GPCRs expressed in CNS pain circuits can be useful agents.

[0129] Here, we first identified GPCR targets enriched across pro- and anti-nociceptive circuits of the amygdala that may disrupt the encoding of pain unpleasantness. We then validated the analgesic efficacy of six repurposed, commercially-available ligands acting at these GPCRs. Finally, we devised two approaches towards safer analgesia. First, given the expression of NTSR1 in several types of nociceptive amygdalar neurons and the receptor’s established role in attenuating addictive behaviorswe tested if the administration of an allosteric modulator could produce the potent analgesia we observed with an orthosteric ligand, devoid of its side effects. Indeed, we demonstrated that the NTSR1 allosteric modulator, SBI-553, was efficacious in decreasing acute and post-operative pain without impacting motor coordination. Importantly, we further demonstrated that an analgesic dose of SBI-553 attenuated morphine preference in a CPP assay. This result suggests that NTSR1 assets may not only be used alone as analgesics, but could also be prescribed together with opioid analgesics to both effectively reduce pain and minimize the risk of transition to opioid addiction. Second, we further designed an innovative combination approach of three amygdalar analgesics that provided more efficacious and safe pain relief than the individual drugs alone We noted that the analgesic effect of both NTSR1 ligands and ATTA was accompanied by a moderate reduction in locomotor activity. However, mice performed normally on the rotarod and showed intact withdrawal reflexes, arguing against motor impairment. Interestingly, opioids, tricyclic antidepressants, anticonvulsants / gabapentinoids, muscle relaxants, which are used clinically for pain management, present sedation as a side effect. In the context of pain suffering with heightened anxiety and stress, these observations suggest that light sedationAttorney Docket No. 5470.984.WO may represent a beneficial calming effect that is a necessary component of the analgesia produced by brain-acting analgesics.

[0130] Methods

[0131] Animals

[0132] All procedures followed animal care guidelines approved by the Stanford University and University of North Carolina at Chapel Hill Administrative Panel on Laboratory Animal Care, in accordance with American Veterinary Medical Association guidelines and the International Association for the Study of Pain. Mice were housed 2-5 per cage and maintained on a 12 hr light / dark cycle in a temperature controlled environment with ad lib access to food and water. Both male and female C57BL / 6 wild-type mice (JAX stock #000664) from 8 to 15 weeks old were used for behavioral pharmacology and neuroanatomy experiments.

[0133] For pain TRAP experiments, TRAP2 mice (pOs2A~,CreER; JAX stock #030323: generously gifted from Liqun Luo, Stanford University) previously described inwere crossed to to Cre- inducible Ail4 reporter mice (ROSA26tdTomatoJAX stock #007914), describedAnimals undergoing TRAPing were single housed and handled for at least three days leading up to experimentation.

[0134] KOR-Cre mice (Qprkl,mI 1(cre)Sros / J JAX stock #035045: generously gifted from Sarah Ross, University of Pittsburgh) have been described previouslyNTSR1 -Venus mice(C57BL / 6-jVNr7ZmJ A# / BouvJ; Jax stock #038290) have been described previouslyMOR-Cre mice (genotype?) were generated by the Stanford Transgenic Core and Sst-Flp mice (SOM1 res^F,p / +^ Vglutl-Flp mice (B6; 129S6-Slcl7a7emI^o)Tasic, 'J JAX stock #034422), and Vgat-Flp mice (B6.Cg-Slc32altml l(7lpo)Hze / J,' JAX stock #029591), and were generated by the Allen Institute.

[0135] Drug Administration

[0136] The following chemicals were reconstituted in water, frozen at -80 degrees, and prepared in 0.9% saline on the day of experiment: BAY 55-9837 (Tocris), LY 344864 HC1 (Tocris), LY 379268 (Tocris), Morphine sulfate (Sigma), and PD 149163 tetrahydrochloride hydrate (Sigma). NNC 26-1900 (Tocris) was dissolved in 50% DMSO. LY 344864 HC1 and LY 379268 were sonicated vigorously to dissolve the compound. SBI-553 HC1 was synthesized by the Aube lab at UNC Chapel Hill and dissolved in 5% (2-Hydroxypropyl)-P-cyclodextrin in 0.9% saline withAttorney Docket No. 5470.984.WO vigorous vortexing and heating to 30C. 4-hydroxytamoxifen (Sigma, H6278) prepared in Kolliphor EL (Sigma, # 27963) as previously described - -

[0137] SBI-553 HC1 synthesis

[0138] SBI-553 was synthesized as described by Pinkerton et al. 2019 and converted to the HC1 salt as follows. To 2-((2-(l-fluorocyclopropyl)-4-(4-(2-methoxyphenyl)piperidin-l-yl)quinazolin- 6-yl)

[0139] (methyl)amino)ethan-l-ol (279 mg, 1.00 equiv, 619 pmol) was added a 4M solution of HC1 in 1,4-dioxane (774 pL, 5.00 equiv, 3.10 mmol), which was then further diluted in 1,4 dioxane (1.59 mL, 30.0 equiv, 18.6 mmol). The reaction was allowed to stir at room temperature until a precipitate formed, at which point the solvent was removed under vacuum. Then toluene was added to azeotrope off residual moisture until the precipitate became crystalline. The target salt was obtained in 90% yield.

[0140] TRAP’ing of neurons active during pain

[0141] Our studies used TRAP2;Ail4 double transgenic mice, in which expression of the reporter tdTomato reporter is induced in CreERT2-expressing neuronsTo label amygdalar neurons active during pain with the tdTomato marker, we injected formalin into the hindpaw of TRAP2;Ail4 mice to induce activity in nociceptors '. immediately followed by subcutaneous injection of 4-OHT (Fig 3 A). Littermate control mice received 4-OHT, but without formalinhindpaw injections. For repeated formalin samples (FormalinX2), we reinjected formalin into the left hindpaw 60 minutes prior to euthanization and processed brain tissue for single-cell RNA-seq.

[0142] Briefly, 5-7 days after 4-hydroxytamoxifen (4-OHT) administration, we anesthetized the mice under isofluorane and perfused with ice-cold bubbled ACSF containing 2.5 mM KC1, 7 mM MgCh, 0.5 mM CaCh, 1.3 mM NaH2PO4, 110 mM choline chloride, 25 mM NaHCOs, 1.3 mM Na ascorbate, 20 mM glucose, and 0.6 mM sodium pyruvate. We removed the brains, submerged in ice-cold bubbled ACSF, made a marking cut in the right motor cortex to maintain laterality, sliced into 300 pm coronal sections on a vibratome (Leica VT1200S, Speed=4, Amplitude=9), and microdissected the amygdala regions.

[0143] Next, we enzymatically dissociated the amygdala tissue pieces via a modified papain-based digestion system (Worthington LK003150). Briefly, tissue chunks were incubated in 30U / ml of papain (containing 1-cysteine and EDTA), DNase and kynurenic acid for 30 minutes at 35 °C. Once enzyme digestion completed, the papain solution was replaced with a room temperatureAttorney Docket No. 5470.984.WO trituration solution (10% v / v ovomucoid inhibitor, 4.5% v / v DNase and 0.1% v / v kynurenic acid) and tissues were triturated 10 times with a Pl 000 pipette tip and 20 times with a P200 pipette tip. Hoechst stain was added (1:2,000; H3570, Life Technologies) and incubated in the dark at room temperature for 15 minutes. The cell suspensions were centrifuged at 350g for 10 min, supernatant removed, resuspended in ice-cold ACSF, and passed through a 70-pm cell strainer to remove debris. Cells were sorted via the Sony SH800 into 384-well plates (Bio-Rad) with lysis buffer containing oligodT and immediately snap-frozen until processing as described previouslyTRAP positive cells were FACS gated as being both Hoechst and tdTomato4. To gain additional contextual information, we also selected an equal number of Hoechst+ but tdTomato-negative cells. Each plate was kept on the sorter for less than 25 minutes to prevent evaporation.

[0144] Sequencing

[0145] We performed whole-cell lysis, first-strand synthesis and cDNA synthesis using the Smart - seq-2 protocol in 384-well formats as described previouslyThe cDNA libraries were prepared, pooled, and cleaned using Illumina Nextera XT kits. We sequenced a total of 13747 single cells from seven biological replicates of amygdala tissue samples from adult male and female mice on Nextseq or Novaseq (Illumina) using 75 bp paired-end reads with approximately 1.5 million reads per cell. We demultiplexed the sequences using bcl2fastq, aligned the reads to the mouse mm 10 genome using the STAR aligner toolkit, and determined gene counts using FeatureCounts

[0146] We identified diverse neural cell populations using single-cell RNA-seq analysis. From 13747 sequenced cells, we selected 9695 high quality cells for further analysis based on the threshold of number detected genes between 200-10000 and <30% of expression contribution from mitochondrial genes (supplemental). We lo -normalized the gene expression counts for each cell and the raw gene counts of each cell were transformed to account for differences in sequencing depth per cell and across replicates. Clustering and marker analysis differentiated neuronal cells (5092) from glial (4603) based on known markers including: Oligodendrocytes (Mog or Pdgfra); Microglia (Cx3crl or Cbr2); Endothelial cells (Cldn5); Astrocyte (Aqp4) (supplemental). We identified the neuronal cell types by expression of Snap25 and confirmed by co-expression of other known general markers: Rbfox3 / NeuN, Camk2a, and Thyl. The GABAergic subpopulations were identified by expression of Gadl and Gad2, which largely overlapped. The glutamatergic population was identified by expression of Slcl7a7 and / or Slcl7a6.Attorney Docket No. 5470.984.WO

[0147] We used the Seurat software package (V3) implemented in R for unbiased clustering of cell types!We filtered low-quality cells with fewer than 200 expressed genes or greater than 30% mitochondrial counts. We regressed out batch effects across seven biological replicates by implementing the Seurat integration procedure. We selected 9695 high quality cells for further analysis based on the threshold of number detected genes between 200-10000 and <30% of expression contribution from mitochondrial genes (supplemental). We log-normalized the gene expression counts for each cell and the raw gene counts of each cell were transformed to account for differences in sequencing depth per cell and across replicates. Clustering and marker analysis differentiated neuronal cells (5092) from glial (4603) based on known markers including: Oligodendrocytes (Mog or Pdgfra); Microglia (Cx3crl or Cbr2); Endothelial cells (Cldn5); Astrocyte (Aqp4) (supplemental). We identified the neuronal cell types by expression of Snap25 and confirmed by co-expression of other known general markers: Rbfox3 / NeuN, Camk2a, and Thyl. The GABAergic subpopulations were identified by expression of Gadl and Gad2, which largely overlapped. The glutamatergic population was identified by expression of Slcl7a7 and / or Slcl7a6.

[0148] We focused our analysis on transcriptionally distinct amygdalar neuronal cell types by using the SubsetData function to create a new Seurat object containing only the z 25-positive cells and reiterated clustering. We identified variable features for downstream cluster analysis selected the first 40 principal components for subsequent cluster analyses, and visualized the data using the nonlinear dimensionality-reduction technique, uniform manifold approximation and projection (UMAP). A neuron was designated ‘TRAPed’ if it had tdTomato mRNA raw count > 0. Neuron subtype marker genes and DEGs were found by using the FindAllMarkers function in Seurat (min. pct = 0.0.25, logfc.threshhold = 0.25). All visualizations of the single-cell RNA-seq data were generated and performed in R, including violin plots, feature plots, UpSet plots, and heat maps

[0149] Spatial transcriptomics

[0150] Sample Preparation

[0151] Briefly, the mouse was sacrificed at 20 min post injection by transcardinally injection of ice-cold ACSF during deep anesthesia. The brain was dissected out and the right hemisphere was immersed in OCT in a cryo-mold, then flash-frozen with isopentane dry ice bath and stored at -80 degrees. Then, the frozen brains were sectioned into coronal slices on a cryostat at -20Attorney Docket No. 5470.984.WOCelsius(section thickness: 10 pm). Brain sections containing the cytoarchitectural and stereotaxic coordinates of the amygdala were placed within the capture areas of slides provided by Resolve Biosciences and dried at -20 Celsius before packaging. Slides containing brain samples were shipped on dry ice to Resolve Biosciences for further processing and imaging for 100-plex combinatorial single molecule fluorescence in-situ hybridization.

[0152] Cell segmentation for Resolve data

[0153] We first segmented cell nuclei with CellposeProviding DAPI channel images as the input, we ran Cellpose with diameter set to 65 and mask threshold set to -0.1 and got cell nuclei segmentation masks as the output. We then used these cell nuclei masks as prior segmentation information with confidence of 0.9 to BaysorBaysor was run with scale set to 45 and scale_std set to 50%. We excluded one gene (Adra2a) with aberrant data patterns from technical issues. The final segmentation counts and cell metadata were used for downstream analysis.

[0154] Graph user interface (GUI) for brain region annotation

[0155] We developed a simple GUI using Naparito annotate brain regions and apply anatomical information to cell metadata. The GUI displays a downsampled DAPI channel image, overlaid with expression patterns of user-selected marker genes, either at cell level (as segmentation masks colored by expression level) or at single transcript level (as dots). The user may also examine co-expression patterns of two genes in the same layer. Using morphological features, marker gene expression patterns, and prior knowledge, a brain region mask is then drawn interactively on a Napari ‘label’ layer. This label layer assigns a brain region label to each pixel, either BLA, CeA, basomedial amygdala (BMA), MEA, ITC, CPu, CORTEX, or unlabeled. Finally, a brain region label is looked up by each cell by where its center coordinate falls in the label layer, and annotated to the AnnData object.

[0156] All code for the cell segmentation pipeline and brain region annotation GUI are available at http s : / / github . com / keyuxi / re sol ve_analy si s / .

[0157] Neural circuit tracing

[0158] To study the projections of CeA neuron subpopulations, AAV-Efla-Con / Fon-oScarlet (titer: 2.2 x 1012 copies / ml, Addgene, 137136-AAV8) was injected unilaterally into the CeA (AP: from -1.1, LM: - 2.9 mm, depth: -4.6 mm) of KOR-Cre;Vgat-Flp mice or MOR-Cre;Sst-Flp mice. To study the projections of BLA neuron subpopulations, we injected 200 nl of AAV-Efla- Con / Fon-oScarlet into the BLA (AP: from -1.5, LM: - 3.0 mm, depth: -4.6 mm) of KOR-Attorney Docket No. 5470.984.WOCre;Vglutl-Flp mice under ~2% isoflurane anesthesia. Injection volume was 200 nl (CeA or BLA). The mice (8-11 weeks old) were kept in their home cages for at least 3 weeks after AAV injection.

[0159] Immunohistochemistry and imaging

[0160] Mice were anesthetized and perfused with lx PBS, followed by 4% paraformaldehyde (PF A) for >12 hours. Fixed brains were then immersed in 30% sucrose solution for >4 days at 4 °C. Then brains were sectioned into coronal slices on a cryostat (section thickness: 40 pm). The slices were washed three times with PBS for 10 mins each. Then the tissues were submerged in a blocking buffer containing 5% normal donkey serum (NDS) and 0.5% Triton-X in phosphate buffer saline (PBS) for an hour at RT. Next, the tissues were incubated with primary antibodies (anti-tdTomato, goat polyclonal, 1: 1000, Sigen AB8181-200) in the blocking buffer containing 1% NDS overnight at 4 °C. The slices were then washed three times with the blocking buffer and then incubated with secondary antibodies (Donkey anti-goat IgG, Alexa Fluor™ 555, 1 : 1000, Invitrogen A32816) in the blocking buffer for 2h atRT. Cellular nuclei were stained by incubation for 7 min with DAPI (1 pM in PBS) at RT. The stained samples were mounted using Fluoromount- G™ Mounting Medium. Then observed under a confocal microscope (Leica TCS SP8 X White Light Laser Confocal Microscope) and imaged with 20x magnification dry. Images were processed with a microscope software (LAS X Life Science Microscope Software).

[0161] In situ hybridization

[0162] Hybridization chain reaction (HCR). The RNA-FISH was performed following the manufacturer's protocol with minor modification. Probes targeting the corresponding genes (Ntsrl-B4, Crh-B2, and Sst-B5) were ordered through Molecular Instruments Inc. Briefly, the mouse was sacrificed at 20 min post injection by transcardinally injection of ice-cold PBS following 4% PFA during deep anesthesia. The brain was then dissected out and post-fixed in 4% PFA overnight, following incubation in 30% sucrose until sunken. The brain tissue was then sectioned on a cryostat to 40 um thickness and then transferred to 70% EtOH and incubated overnight. Slices containing desired amygdala regions were picked and transferred to another container and incubated with 8% SDS for 30 min at room temperature. The slices were then washed twice with fresh 2x SSC for 10 min. Using pre-warmed hybridization buffer, pre-hybridize the slices in 37 C for 10 min, then replace the buffer with pre-warmed probe solution (2 uL Ntsrl, luL Crh and 0.2 uL Sst per 100 uL buffer) in hybridization buffer. Incubate overnight in 37 CAttorney Docket No. 5470.984.WO chamber. Remove the buffer, wash the slices 3 times in fresh probe wash buffer for 10 min at 37 C, then wash twice with fresh 2x SSC at room temperature. Use the amplification buffer, preamplify the slices for 10 min at room temperature, then add the snap-cooled hairpins (B4-546, B2- 594 and B5-647). Incubate in a dark chamber at room temperature overnight. Remove amplification solution, wash twice in 2xSSC, add DAPI and mounting media, place a coverslip for microscopy. The slices were then imaged using a Leica SP8 confocal microscope in the core facility.

[0163] 3D light sheet microscopy analysis of formalin pain trapped neurons

[0164] TRAP2;Ai 14 mice were handled and habituated in clear cylinders for two days leading up to experimentation. For the formalin pain assay, 4% PFA was injected into the left hind paw of these mice. Subsequently, 20mg / kg 4-OHT was injected two hours following the hind paw injection to allow tdTomato expression. After an additional two hours, mice were returned to their home cage for one week, where they were then sacrificed, perfused, and sent to LifeCanvas for tissue clearing and initial analysis. The density of tdTomato+ neurons was calculated, normalized, and a heatmap representing all brain regions was generated. Finally, background- subtracted density for all amygdalar nuclei were plotted.

[0165] Behavioral models and tests

[0166] Hot plate. Mice were tail marked and habituated to the behavior room 30 minutes prior to experimentation. Upon habituation, mice were administered either vehicle or drug via intraperitoneal (i.p.) injection 20 minutes before hot plate. To measure affective-motivational pain, mice were individually transferred to the 52°C hotplate for one minute or until jumping behavior (to adhere to IACUC guidelines). Affective motivational behaviors, including attending the hindpaw, guarding the hindpaw, rearing, and jumping were quantified in Ethovision using a 4 camera setup.

[0167] Tail-withdrawal assay. After the hot plate assay, mice were returned to their home cage and allowed to recover for 4 minutes. At 25 minutes post-drug administration, mice were swaddled in a cloth with their tails exposed. Tails were immersed in a 52°C water bath, where the time until tail flick was recorded. This was repeated 3 times to obtain an average tail flick. A 10 second cutoff was used to prevent tissue damage.

[0168] Von Frey filaments. For the orofacial pain model, to assess mechanical sensitivity, animals received repeated application (10-20 seconds apart) of 0.16-g (light touch) von Frey filaments toAttorney Docket No. 5470.984.WO the whisker pads. Pain score was assessed considering intensity of face attending behavior response. All behavior videos were manually processed. We tracked stimulus onset moments, whisking periods, and wiping moments throughout the whole video. Specifically, stimulus onset and whisking on / off moments were tracked precisely to a single frame, and for the wiping, each time the paw touching the whiskers / face was considered as a wiping moment.

[0169] For the behavioral analysis, wiping numbers (how many times a mouse wiped the face after von Frey stimuli) was used to characterize the level of the nocifensive behavior. The mean and SE were calculated by the wiping number of all the trials.

[0170] Open field. Mice were tail marked and habituated to the behavior room 30 minutes prior to experimentation. Baseline core body temperature was taken via rectal probe, then mice were injected intraperitoneally with either vehicle or drug. Post-drug core body temperature was taken at 15 minutes post-injection, then mice were placed on a rotarod to measure motor impairment. Time on the rotarod was capped at 5 minutes.

[0171] Rotarod. At 20 minutes post-injection, mice were placed in the open field, where locomotor activity was measured by Ethovision software for 20 total minutes.

[0172] Conditioned place preference. To evaluate the rewarding effects of individual or combined drugs, a 5 days CPP protocol was set. The apparatus consists of 2 main chambers (8 inches x 8 inches x 12 inches tall) distinguished by the texture of the floor and by the wall patterns, connected to each other by a central chamber (2 inches x 8 inches x 12 inches tall). During the first day (pretest), all the mice were able to move freely during 20 minutes in the whole apparatus. Their movements were recorded with a camera connected to Ethovision software. Mice with a spontaneous preference above 75% were removed from the experiment. The next 3 days are the conditioning days. In the morning, mice were restricted for 20 minutes to one chamber after intraperitoneally injecting morphine (5 mg / kg), morphine (5 mg / kg) and SBI-553 (30 mg / kg), or the ATTA mix. In the afternoon, mice were again restricted for 20 minutes to the opposite chamber after vehicle intraperitoneal injection. Injections were switched off the next day in order to avoid time dependent conditioning. On the final day, mice were placed in the center chamber with free access to all chambers and the time spent in each chamber was recorded for 20 minutes. Ethovision software was used to track the time mice spent in each chamber, where CPP score was calculated by subtracting the time spent in the drug-paired chamber by the time spent in the vehicle chamber, divided by the overall time spent in both chambers.Attorney Docket No. 5470.984.WO

[0173] NTSR1 in vitro signaling assays

[0174] Arrestin and GqBioluminescence Resonance Energy Transfer (BRET) assays were previously described in Krumm et al. 20231---. In brief, HEK293T cells were maintained in DMEM containing 10% (v / v) FBS, 1 IU mL1Penicillin G, and 100 pg mL1Streptomycin were passed to 10 cm dishes and co-transfected withNTSRl containing C-terminal Renilla luciferase (RLuc) and Venus-tagged -arrestin2 (BRET1) for at least 24 hours. Transfected cells were then plated in poly-lysine-coated 96-well clear bottom cell culture plates in plating media (DMEM containing 1% (v / v)-dialyzed FBS, 1 IU mL Penicillin G, and 100 pg mL1Streptomycin) at a density of 40,000 cells in 200 pL per well and incubated overnight. The next day, media was aspirated, cells were washed once with 60 ul of assay buffer (20mM Hepes, lx HBSS, pH 7.4), aspirated then 60 ul of assay buffer added to each well. Drug stimulation was performed with the addition of 15 pL of 6X drug dilution of SBI-553 in drug dilution buffer (20mM Hepes, lx HBSS, 0.3% (w / v) BSA, pH 7.4) per well, and incubated at room temperature for 80 minutes. 15 pL of 6X drug dilution of NTSS-13 was then added to each well and incubated for an additional 5 minutes. The RLuc substrate, coelenterazine h (Promega), was added to a final concentration of 5 pM per well and incubated for an additional 5 minutes, plates were read at 90 minutes post-start of incubation for luminescence at 475 nm and fluorescent eYFP emission at 530 nm for 1 second per well using a PHERAstar FSX (BMG Labtech). Plates were read for multiple time points up to 30 minutes, where the BRET ratio of eYFP / RLuc was calculated per well, and the net BRET ratio was calculated by subtracting the eYFP / RLuc ratio of wells without Venus-P-Arrestin present. The net BRET ratio was normalized to wells without the addition of SBI-553 (NTS only).

[0175] To measure NTSRl-mediated G protein dissociation (BRET2), procedures were similar to NTSR1 -mediated P-Arrestin2 recruitment, except HEK293T cells were co-transfected in a 1 : 1 : 1 : 1 ratio of Gaq-RLuc, G 3, GFP2-Gy9, and NTSR1, respectively, for at least 24 hours. Transfected cells were then plated in poly-lysine-coated 96-well clear bottom cell culture plates in plating media (DMEM containing 1% (vZv)-dialyzed FBS, 1 IU mL1Penicillin G, and 100 pg mL1Streptomycin) at a density of 40,000 cells in 200 pL per well and incubated overnight. The next day, media was aspirated, cells were washed once with 70 ul of assay buffer (20mM Hepes, lx HBSS, pH 7.4), aspirated, then 70 ul of assay buffer containing BRET2 RLuc substrate Coelenterazine 400a (Nanolight) at 5 pM final concentration was added to each well and incubated for 5 minutes. Drug stimulation was performed with the addition of 30 pL of pre-mixed drugAttorney Docket No. 5470.984.WO dilutions of NTSS-B and SBT-553 in drug dilution buffer (20mM Hepes, lx HBSS, 0.3% (w / v) BSA, pH 7.4) per well, and incubated at room temperature for an additional 5 minutes. Plates were read for luminescence at 410 nm and fluorescent GFP2 emission at 515 nm for 1 second per well using a PHERAstar FSX (BMG Labtech). The ratio of GFP2 / RLuc was calculated per well and plotted as a function of drug concentration using Graphpad Prism 8 (Graphpad Software Inc., San Diego, CA).

[0176] Surgeries

[0177] All surgeries were conducted under aseptic conditions using a digital small animal stereotaxic instrument (David Kopf Instruments). Mice were anesthetized with isoflurane (3-5% induction, 1-2% maintenance). Body temperature was maintained using a heating pad.

[0178] Paw incision. C57B16 / I mice were tested on two alternating days before surgery to establish a baseline tactile withdrawal threshold using von Frey fdaments and the up-down method, as described in Bonin et al., 2014. Following baseline testing, mice were anesthetized under isoflurane, and a 0.7 cm single incision was made using a 11 scalpel blade on the left hindpaw surface, as described in Brennan et al., 1996. The subcutaneous plantaris muscle was longitudinally incised after being isolated, and the wound was sutured with two 5.0 absorbable sutures upon hemostasis. Mice were placed in recovery cages, and tactile withdrawal threshold was retested three days post-surgery. Here, mice were tested before, 20 minutes post-i.p. injection, and 60 minutes post-i.p. injection of a vehicle or 3-drug cocktail mix.

[0179] Headbar plus amygdala cannula surgery. All mice that underwent intracranial amygdalar injections were awake and headfixed during infusion, requiring an implant of bilateral cannulas and a headbar. After being placed in the stereotax and the skin cut over the skull, the remaining skin was pushed to the edges and secured with UV curable glue (Loctite 4305). A marker was used to deliniate the skull sutures adjacent to the motor cortex for later identification. We used a scalpel blade to score the skull before layering a thin layer of UV curable glue over the open skull. A 1.5mm drill bit was used to create a burr hole over each amygdala (AP: -1.22, ML: ±1.22). A 1.0 mm stainless steel cannula was gently placed in the burr hole, just above the surface of the brain. The cannula was secured with UV curable glue. Next, a small metal headbar (~0.8 cm x 0.5 cm x 0.1 cm) was secured to the glue over the posterior skull with an additional layer of UV curable glue. Finally, dental cement was layered over the UV glue and allowed to dry.Attorney Docket No. 5470.984.WO

[0180] Trigeminal neuropathic pain surgery. To induce a chronic constriction injury (CCI) of the left infraorbital branch of the trigeminal nerve, mice were anesthetized and placed in the stereotax with the right earbar stabilizing the head. We made a small cut (~3 mm) between the vibrissa pad and the eye. Nondissolvable nylon sutures (5-0, Oasis) were tied around the nerve to constrict the nerve to -80% of its original size. We ensured blood flow continued normally. We then sutured the skin closed.

[0181] Cannula injections

[0182] For local amygdala injections, we used a 1 : 10:50 ratio of ug / kg dose administered for PD149163, BAY55-9837, and LY344864 compounds, respectively, which is similar to what we used for systemic injections.Table 1. Marker catalog from integration of single-cell and spatial transcriptomics for comprehensive molecular cell atlas of the amygdala.Attorney Docket No. 5470.984.WO

[0183] References1. Treede, R.-D., Rief, W., Barke, A., Aziz, Q., Bennett, M.I., Benoliel, R., Cohen, M., Evers, S., Finnerup, N.B., First, M.B., et al. (2019). Chronic pain as a symptom or a disease: the IASP Classification of Chronic Pain for the International Classification of Diseases (ICD- 11). Pain 160, 19.2. Nahin, R.L., Feinberg, T., Kapos, F.P., and Terman, G.W. (2023). Estimated Rates of Incident and Persistent Chronic Pain Among US Adults, 2019-2020. JAMA Netw Open 6, e2313563.3. Busse, J.W., Wang, L., Kamaleldin, M., Craigie, S., Riva, J. J., Montoya, L., Mulla, S.M., Lopes, L.C., Vogel, N., Chen, E., et al. (2018). Opioids for Chronic Noncancer Pain: A Systematic Review and Meta-analysis. JAMA 320, 2448-2460.4. Els, C., Jackson, T.D., Hagtvedt, R., Kunyk, D., Sonnenberg, B., Lappi, V.G., and Straube,S. (2023). High-dose opioids for chronic non-cancer pain: an overview of Cochrane Reviews. Cochrane Database Syst. Rev. https: / / doi.org / 10.1002 / 14651858.CD012299.pub3.5. Volkow, N.D., and Blanco, C. (2021). The changing opioid crisis: development, challenges and opportunities. Mol. Psychiatry 26, 218-233.6. The Lancet Regional Health-Americas (2023). Opioid crisis: addiction, overprescription, and insufficient primary prevention. Lancet Reg Health Am 23, 100557.7. Jayakar, S., Shim, J., Jo, S., Bean, B.P., Singe?, I., and Woolf, C.J. (2021). Developing nociceptor-selective treatments for acute and chronic pain. Sci. Transl. Med. 13, eabj9837.8. Rosenfeld, M.G., Mermod, J. J., Amara, S.G., Swanson, L.W., Sawchenko, P.E., Rivier, J., Vale, W.W., and Evans, R.M. (1983). Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 304, 129-135.9. Akopian, A.N., Sivilotti, L., and Wood, J.N. (1996). A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons. Nature 379, 257-262.10. Russo, A.F., and Hay, D.L. (2023). CGRP physiology, pharmacology, and therapeutic targets: migraine and beyond. Physiol. Rev. 103, 1565-1644.11. Ferrari, M.D., Goadsby, P.J., Burstein, R., Kurth, T., Ayata, C., Charles, A., Ashina, M., van den Maagdenberg, A.M.J.M., and Dodick, D.W. (2022). Migraine. Nat Rev Dis Primers 8, 2.12. Jones, J., Correll, D.J., Lechner, S.M., Jazic, I., Miao, X., Shaw, D., Simard, C., Osteen, J.D., Hare, B., Beaton, A., et al. (2023). Selective inhibition of NaV1.8 with VX-548 for acute pain. N. Engl. J. Med. 389, 393-405.13. Melzack, R., and Casey, K.L. (1968). Sensory, motivational, and central control determinants of pain: a new conceptual model. The skin senses 1, 423-443.E htxtpogs:e / / ndoouis.o Orgp / i1o0id. !s 1 i4n6 P / aaninnu. r Perve-pnreinutr,o h-t0t8p0s3: / / 1d7o-i0.o6r 1g5 / 2120..1146 / annurev-neuro-080317-061522Attorney Docket No. 5470.984.WO14. Lumley, M.A., Cohen, J.L., Borszcz, G.S., Cano, A., Radcliffe, A M., Porter, L.S., Schubiner, H., and Keefe, F.J. (2011). Pain and emotion: a biopsychosocial review of recent research. J. Clin. Psychol. 67, 942-968.15. Gilam, G , Gross, J. J., Wager, T.D., Keefe, F.J., and Mackey, S.C. (2020). What Is the Relationship between Pain and Emotion? Bridging Constructs and Communities. Neuron 107, 17-21.16. Woo, A.K. (2010). Depression and Anxiety in Pain. Rev Pain 4, 8-12.17. De La Rosa, J.S., Brady, B.R., Ibrahim, M.M., Herder, K.E., Wallace, J.S., Padilla, A.R., and Vanderah, T.W. (2024). Co-occurrence of chronic pain and anxiety / depression symptoms in U.S. adults: prevalence, functional impacts, and opportunities. Pain 165, 666.18. Mercer Lindsay, N., Chen, C., Gilam, G., Mackey, S., and Scherrer, G. (2021). Brain circuits for pain and its treatment. Sci. Transl. Med. 13, eabj7360.19. Valentino, R.J., and Volkow, N.D. (2018). Untangling the complexity of opioid receptor function. Neuropsychopharmacology 43, 2514-2520.20. Wang, D., Tawfik, V.L., Corder, G., Low, S.A., Francois, A., Basbaum, A.I., and Scherrer, G. (2018). Functional Divergence of Delta and Mu Opioid Receptor Organization in CNS Pain Circuits. Neuron 98, 90-108. e5.21. Corder, G., Castro, D.C., Bruchas, M.R., and Scherrer, G. (2018). Endogenous and22. Cui, Y., Ostlund, S.B., James, A.S., Park, C.S., Ge, W., Roberts, K.W., Mittal, N., Murphy, N.P., Cepeda, C., Kieffer, B.L., et al. (2014). Targeted expression of p-opioid receptors in a subset of striatal direct-pathway neurons restores opiate reward. Nat. Neurosci. 17, 254-261.23. Zhu, Y., Wienecke, C.F.R., Nachtrab, G., and Chen, X. (2016). A thalamic input to the nucleus accumbens mediates opiate dependence. Nature 530, 219-222.24. Koob, G.F., and Volkow, N.D. (2016). Neurobiology of addiction: a neurocircuitry analysis. Lancet Psychiatry 3, 760-773.25. Castro, D.C., and Bruchas, M.R. (2019). A Motivational and Neuropeptidergic Hub: Anatomical and Functional Diversity within the Nucleus Accumbens Shell. Neuron 102, 529-552.26. Fields, H.L., and Margolis, E.B. (2015). Understanding opioid reward. Trends Neurosci. 38, 217-225.27. Evans, C.J., and Cahill, C.M. (2016). Neurobiology of opioid dependence in creating addiction vulnerability. FlOOORes. 5. https: / / doi.Org / 10.12688 / fl000research.8369. l .28. Ramirez, J.-M., Burgraff, N.J., Wei, A.D., Baertsch, N.A., Varga, A.G., Baghdoyan, H.A.,Attorney Docket No. 5470.984.WOLydic, R., Morris, K.F., Bolser, D C., and Levitt, E.S. (2021). Neuronal mechanisms underlying opioid-induced respiratory depression: our current understanding. J. Neurophysiol. 125, 1899-1919.29. Bachmutsky, I., Wei, X.P., Kish, E., and Yackle, K. (2020). Opioids depress breathing through two small brainstem sites. Elife 9. https: / / doi.org / 10.7554 / eLife.52694.30. Liu, S., Kim, D.-L, Oh, T.G., Pao, G.M., Kim, J.-H., Palmiter, R D., Banghart, M R., Lee, K.-E, Evans, R.M., and Han, S. (2021). Neural basis of opioid-induced respiratory depression and its rescue. Proceedings of the National Academy of Sciences 118, e2022134118.31 . Bateman, J.T., and Levitt, E.S. (2023). Opioid suppression of an excitatory pontomedullary respiratory circuit by convergent mechanisms. Elife 12. https: / / doi.org / 10.7554 / eLife.81119.32. Janak, P.H., and Tye, K.M. (2015). From circuits to behaviour in the amygdala. Nature 517, 284-292.33. Neugebauer, V., Mazzitelli, M., Cragg, B., Ji, G., Navratilova, E., and Porreca, F. (2020). Amygdala, neuropeptides, and chronic pain-related affective behaviors. Neuropharmacology 170, 108052.34. Palmiter, R.D. (2018). The Parabrachial Nucleus: CGRP Neurons Function as a General Alarm. Trends Neurosci. 41, 280-293.35. Veinante, P., Yalcin, I., and Barrot, M. (2013). The amygdala between sensation and affect: a role in pain. J Mol Psychiatry 1, 9.36. Ji, R.-R., Gereau, R.W., 4th, Malcangio, M., and Strichartz, G.R. (2009). MAP kinase and pain. Brain Res. Rev. 60, 135-148.37. Kato, F., Sugimura, Y.K., and Takahashi, Y. (2018). Pain-Associated Neural Plasticity in the Parabrachial to Central Amygdala Circuit : Pain Changes the Brain, and the Brain Changes the Pain. Adv. Exp. Med. Biol. 1099, 157-166.38. Annese, J., Schenker-Ahmed, N.M., Bartsch, H., Maechler, P., Sheh, C., Thomas, N., Kayano, J., Ghatan, A., Bresler, N., Frosch, M.P., et al. (2014). Postmortem examination of patient HM’s brain based on histological sectioning and digital 3D reconstruction. Nat. Commun. 5, 1-9.39. Hebben, N., Corkin, S., Eichenbaum, H., and Shedlack, K. (1985). Diminished ability to interpret and report internal states after bilateral medial temporal resection: Case H.M. Behav. Neurosci. 99, 1031-1039.40. Neimann, N., Beurey, J., Pierson, M., Tridon, P., Sapelier, J., and Melin, P. (1964). Familial Urbach-wiethe disease with indifference to pain. Bull. Soc. Fr. Dermatol. Syphiligr. 71, 292-294.41. Manning, B .H., and Mayer, D.J. (1995). The central nucleus of the amygdala contributes toAttorney Docket No. 5470.984.WO the production of morphine anti nociception in the formalin test. Pain 63, 141-152.42. Corder, G., Ahanonu, B., Grewe, B.F., Wang, D., Schnitzer, M.J., and Scherrer, G. (2019). An amygdalar neural ensemble that encodes the unpleasantness of pain. Science 363, 276- 281.43. Neugebauer, V. (2020). Amygdala physiology in pain. Handb. Behav. Neurosci. 26, 101— 113.44. Hua, T., Chen, B., Lu, D., Sakurai, K., Zhao, S., Han, B.-X., Kim, J., Yin, L., Chen, Y., Lu, J., et al. (2020). General anesthetics activate a potent central pain-suppression circuit in the amygdala. Nat. Neurosci. 23, 854-868.45. Han, S., Soleiman, M.T., Soden, M.E., Zweifel, L.S., and Palmiter, R.D. (2015). Elucidating an Affective Pain Circuit that Creates a Threat Memory. Cell 162, 363-374.46. Sarhan, M., Freund-Mercier, M.-J., and Veinante, P. (2005). Branching patterns of parabrachial neurons projecting to the central extended amgydala: single axonal reconstructions. J. Comp. Neurol. 491, 418-442.47. Deng, J., Zhou, H., Lin, J.-K., Shen, Z.-X., Chen, W.-Z., Wang, L.-H., Li, Q., Mu, D., Wei, Y.-C., Xu, X.-H., et al. (2020). The Parabrachial Nucleus Directly Channels Spinal Nociceptive Signals to the Intralaminar Thalamic Nuclei, but Not the Amygdala. Neuron 107, 909-923. e6.48. Gauriau, C., and Bernard, J.-F. (2002). Pain pathways and parabrachial circuits in the rat. Exp. Physiol. 87, 251-258.49. Chiang, M.C., Nguyen, E.K., Canto-Bustos, M., Papale, A.E., Oswald, A.-M.M., and Ross, S.E. (2020). Divergent Neural Pathways Emanating from the Lateral Parabrachial Nucleus Mediate Distinct Components of the Pain Response. Neuron 106, 927-939. e5.50. Li, J.-N., and Sheets, P.L. (2020). Spared nerve injury differentially alters parabrachial monosynaptic excitatory inputs to molecularly specific neurons in distinct subregions of the central amygdala. Pain 161, 166-176.51. McDonald, A. J. (1982). Neurons of the lateral and basolateral amygdaloid nuclei: a Golgi study in the rat. J. Comp. Neurol. 212, 293-312.52. Swanson, L.W., and Petrovich, G.D. (1998). What is the amygdala? Trends Neurosci. 21, 323—331.53. Raver, C., Uddin, O., Ji, Y., Li, Y., Cramer, N., Jenne, C., Morales, M., Masri, R., and Keller, A. (2020). An Amygdalo-Parabrachial Pathway Regulates Pain Perception and Chronic Pain. J. Neurosci. 40, 3424-3442.54. Sun, Y., Bianco-Centurion, C., Zou, B., Bendell, E., Shiromani, P.J., and Liu, M. (2019). Amygdala GABA Neurons Project To vlPAG And mPFC. IBRO Rep 6, 132-136.Attorney Docket No. 5470.984.WO55. Oka, T., Tsumori, T., Yokota, S., and Yasui, Y. (2008). Neuroanatomical and neurochemical organization of projections from the central amygdaloid nucleus to the nucleus retroambiguus via the periaqueductal gray in the rat. Neurosci. Res. 62, 286-298.56. Wilson, T.D., Valdivia, S., Khan, A., Ahn, H.-S., Adke, A.P., Martinez Gonzalez, S., Sugimura, Y.K., and Carrasquillo, Y. (2019). Dual and Opposing Functions of the Central Amygdala in the Modulation of Pain. Cell Rep. 29, 332-346. e5.57. Oliveira, M.A., and Prado, W.A. (2001). Role of PAG in the antinociception evoked from the medial or central amygdala in rats. Brain Res. Bull. 54, 55-63.58. Finnegan, T.F., Chen, S.-R., and Pan, H.-L. (2005). Effect of the {mu} opioid on excitatory and inhibitory synaptic inputs to periaqueductal gray-projecting neurons in the amygdala. J. Pharmacol. Exp. Ther. 312, 441-448.59. Hunskaar, S., and Hole, K. (1987). The formalin test in mice: dissociation between inflammatory and non-infl ammatory pain. Pain 30, 103-114.60. DeNardo, L.A., Liu, C.D., Allen, W.E., Adams, E.L., Friedmann, D., Fu, L., Guenthner, C.J., Tessier-Lavigne, M., and Luo, L. (2019). Temporal evolution of cortical ensembles promoting remote memory retrieval. Nat. Neurosci. 22, 460-469.61. Kim, J., Pignatelli, M., Xu, S., Itohara, S., and Tonegawa, S. (2016). Antagonistic negative and positive neurons of the basolateral amygdala. Nat. Neurosci. 19, 1636-1646.62. Zhang, X., Kim, J., and Tonegawa, S. (2020). Amygdala Reward Neurons Form and Store Fear Extinction Memory. Neuron 105, 1077-1093. e7.63. O’Leary, T.P., Sullivan, K.E., Wang, L., Clements, J., Lemire, A.L., and Cembrowski, M.S. (2020). Extensive and spatially variable within-cell-type heterogeneity across the basolateral amygdala. Elife 9. https: / / doi.org / 10.7554 / eLife.59003.64. Rhomberg, T., Rovira-Esteban, L., Vikor, A., Paradiso, E., Kremser, C., Nagy-Pai, P., Papp, O.I., Tasan, R., Erdelyi, F., Szabo, G., et al. (2018). Vasoactive Intestinal Polypeptide- Immunoreactive Interneurons within Circuits of the Mouse Basolateral Amygdala. J. Neurosci. 38, 6983-7003.65. Kemppainen, S., and Pitkanen, A. (2000). Distribution of parvalbumin, calretinin, and calbindin-D28k immunoreactivity in the rat amygdaloid complex and colocalization with y- aminobutyric acid. J. Comp. Neurol.66. McDonald, A.J., and Betette, R.L. (2001). Parvalbumin-containing neurons in the rat basolateral amygdala: morphology and co-localization of Calbindin-D28k. Neuroscience 102, 413-425.67. McDonald, A. J., and Mascagni, F. (2001). Colocalization of calcium-binding proteins and GABA in neurons of the rat basolateral amygdala. Neuroscience 105, 681-693.68. McDonald, A.J., and Mascagni, F. (2001). Localization of the CB1 type cannabinoidAttorney Docket No. 5470.984.WO receptor in the rat basolateral amygdala: high concentrations in a subpopulation of cholecystokinin-containing interneurons. Neuroscience 107, 641-652.69. Vereczki, V.K., Muller, K., Krizsan, E., Mate, Z., Fekete, Z., Rovira-Esteban, L., Veres, J.M., Erdelyi, F., and Hajos, N. (2021). Total Number and Ratio of GABAergic Neuron Types in the Mouse Lateral and Basal Amygdala. J. Neurosci. 41, 4575-4595.70. Muller, J.F., Mascagni, F., and McDonald, A. J. (2007). Postsynaptic targets of somatostatincontaining interneurons in the rat basolateral amygdala. J. Comp. Neurol. 500, 513-529.71. Muller, J.F., Mascagni, F., and McDonald, A. J. (2003). Synaptic connections of distinct intemeuronal subpopulations in the rat basolateral amygdalar nucleus. J. Comp. Neurol. 456, 217-236.72. Bocchio, M., Fisher, S.P., Unal, G., Ellender, T.J., Vyazovskiy, V.V., and Capogna, M. (2016). Sleep and Serotonin Modulate Paracapsular Nitric Oxide Synthase Expressing Neurons of the Amygdala. eNeuro 3. https: / / doi.org / 10.1523 / ENEUR0 0177-16.2016.73. Ciocchi, S., Herry, C., Grenier, F., Wolff, S.B.E., Letzkus, J.J., Vlachos, I., Ehrlich, I., Sprengel, R., Deisseroth, K., Stadler, M.B., et al. (2010). Encoding of conditioned fear in central amygdala inhibitory circuits. Nature 468, 277-282.74. Haubensak, W., Kunwar, P.S., Cai, H., Ciocchi, S., Wall, N.R., Ponnusamy, R., Biag, J., Dong, H.-W., Deisseroth, K., Callaway, E.M., et al. (2010). Genetic dissection of an amygdala microcircuit that gates conditioned fear. Nature 468, 270-276.75. Giovanniello, J., Yu, K., Furlan, A., Nachtrab, G.T., Sharma, R., Chen, X., and Li, B. (2020). A Central Amygdala-Globus Pallidus Circuit Conveys Unconditioned Stimulus- Related Information and Controls Fear Learning. J. Neurosci. 40, 9043-9054.76. Li, H., Penzo, M.A., Taniguchi, H., Kopec, C.D., Huang, Z.J., and Li, B. (2013). Experience-dependent modification of a central amygdala fear circuit. Nat. Neurosci. 16, 332-339.77. Hunt, S., Sun, Y., Kucukdereli, H., Klein, R., and Sah, P. (2017). Intrinsic Circuits in the Lateral Central Amygdala. eNeuro 4. https: / / doi.org / 10.1523 / ENEURO.0367-16.2017.78. Kim, J., Zhang, X., Muralidhar, S., LeBlanc, S.A., and Tonegawa, S. (2017). Basolateral to Central Amygdala Neural Circuits for Appetitive Behaviors. Neuron 93, 1464-1479. e5.79. Pomrenze, M.B., Giovanetti, S.M., Maiya, R., Gordon, A.G., Kreeger, L.J., and Messing, R.O. (2019). Dissecting the Roles of GABA and Neuropeptides from Rat Central Amygdala CRF Neurons in Anxiety and Fear Learning. Cell Rep. 29, 13-21. e4.80. McCullough, K.M., Morrison, F.G., Hartmann, J., Carlezon, W.A., Jr, and Ressler, K.J. (2018). Quantified Coexpression Analysis of Central Amygdala Subpopulations. eNeuro 5. https: / / doi.org / 10.1523 / ENEURO.0010-18.2018.81. Torruella-Suarez, M.L., Vandenberg, J.R., Cogan, E.S., Tipton, G.J., Teklezghi, A., Dange,Attorney Docket No. 5470.984.WOK., Patel, G.K., McHenry, J. A., Hardaway, J. A., Kantak, P.A., et al. (2020). Manipulations of Central Amygdala Neurotensin Neurons Alter the Consumption of Ethanol and Sweet Fluids in Mice. J. Neurosci. 40, 632-647.82. Perez de la Mora, M., Gallegos-Cari, A., Crespo-Ramirez, M., Marcellino, D., Hansson, A.C., and Fuxe, K. (2012). Distribution of dopamine D(2)-like receptors in the rat amygdala and their role in the modulation of unconditioned fear and anxiety. Neuroscience 20 J, 252- 266.83. De Bundel, D., Zussy, C., Espallergues, J., Gerfen, C.R., Girault, J -A., and Valjent, E. (2016). Dopamine D2 receptors gate generalization of conditioned threat responses through mTORCl signaling in the extended amygdala. Mol. Psychiatry 21, 1545-1553.84. Hein, M., Ji, G., Tidwell, D., D’ Souza, P., Kiritoshi, T., Yakhnitsa, V., Navratilova, E., Porreca, F., and Neugebauer, V. (2021). Kappa opioid receptor activation in the amygdala disinhibits CRF neurons to generate pain-like behaviors. Neuropharmacology 185, 108456.85. Gilpin, N.W., Roberto, M., Koob, G.F., and Schweitzer, P. (2014). Kappa opioid receptor activation decreases inhibitory transmission and antagonizes alcohol effects in rat central amygdala. Neuropharmacology 77, 294-302.86. Ji, G , and Neugebauer, V. (2020). Kappa opioid receptors in the central amygdala modulate spinal nociceptive processing through an action on amygdala CRF neurons. Mol. Brain 13, 128.87. Phelps, C.E., Navratilova, E., Dickenson, A.H., Porreca, F., and Bannister, K. (2019). Kappa opioid signaling in the right central amygdala causes hind paw specific loss of diffuse noxious inhibitory controls in experimental neuropathic pain. Pain 160, 1614-1621.88. Mahko, M., Geracitano, R., and Capogna, M. (2011). Functional connectivity of the main intercalated nucleus of the mouse amygdala. J. Physiol. 589, 1911-1925.89. Winters, B.L., Gregoriou, G.C., Kissiwaa, S.A., Wells, O.A., Medagoda, D.I., Hermes, S.M., Burford, N.T., Alt, A., Aicher, S.A., and Bagley, E.E. (2017). Endogenous opioids regulate moment-to-moment neuronal communication and excitability. Nat. Commun. 8, 14611.90. Marowsky, A., Yanagawa, Y., Obata, K., and Vogt, K.E. (2005). A specialized subclass of interneurons mediates dopaminergic facilitation of amygdala function. Neuron 48, 1025— 1037.91. Wood, J., Verma, D., Lach, G., Bonaventure, P., Herzog, H., Sperk, G , and Tasan, R.O. (2016). Structure and function of the amygdaloid NPY system: NPY Y2 receptors regulate excitatory and inhibitory synaptic transmission in the centromedial amygdala. Brain Struct. Funct. 221, 3373-3391.92. Nakao, A., Takahashi, Y., Nagase, M., Ikeda, R., and Kato, F. (2012). Role of capsaicinsensitive C-fiber afferents in neuropathic pain-induced synaptic potentiation in the nociceptive amygdala. Mol. Pain 8, 51.Attorney Docket No. 5470.984.WO93. Rovira-Esteban, L ., Gunduz-Cinar, O., Bukalo, O., Limoges, A., Brockway, E., Muller, K., Fenno, L., Kim, Y.S., Ramakrishnan, C., Andrasi, T., et al. (2019). Excitation of Diverse Classes of Cholecystokinin Interneurons in the Basal Amygdala Facilitates Fear Extinction. eNeuro 6. https: / / doi.org / 10.1523 / ENEURO.0220-19.2019.94. Nakashima, M., Ikegaya, Y., and Morikawa, S. (2022). Genetic labeling of axo-axonic cells in the basolateral amygdala. Neurosci. Res. 178, 33-40.95. Veres, J.M., Nagy, G.A., Vereczki, V.K., Andrasi, T., and Hajos, N. (2014). Strategically positioned inhibitory synapses of axo-axonic cells potently control principal neuron spiking in the basolateral amygdala. J. Neurosci. 34, 16194-16206.96. Muller, J.F., Mascagni, F., and McDonald, A. J. (2006). Pyramidal cells of the rat basolateral amygdala: synaptology and innervation by parvalbumin-immunoreactive interneurons. J. Comp. Neurol. 494, 635-650.97. Bienvenu, T.C.M., Busti, D., Magill, P.J., Ferraguti, F., and Capogna, M. (2012). Cell-type- specific recruitment of amygdala interneurons to hippocampal theta rhythm and noxious stimuli in vivo. Neuron 74, 1059-1074.98. Rainnie, D.G., Mania, I., Mascagni, F., and McDonald, A.J. (2006). Physiological and morphological characterization of parvalbumin-containing interneurons of the rat basolateral amygdala. J. Comp. Neurol. 498, 142-161.99. Kissiwaa, S.A., Patel, S.D., Winters, B.L., and Bagley, E.E. (2020). Opioids differentially modulate two synapses important for pain processing in the amygdala. Br. J. Pharmacol. 777, 420-431.100. Welsch, L., Bailly, J., Darcq, E., and Kieffer, B.L. (2020). The Negative Affect of Protracted Opioid Abstinence: Progress and Perspectives From Rodent Models. Biol. Psychiatry 87, 54-63.101. Navratilova, E., Ji, G., Phelps, C., Qu, C., Hein, M., Yakhnitsa, V., Neugebauer, V., and Porreca, F. (2019). Kappa opioid signaling in the central nucleus of the amygdala promotes disinhibition and aversiveness of chronic neuropathic pain. Pain 160, 824-832.102. Tejeda, H.A., Wu, J., Kornspun, A.R., Pignatelli, M., Kashtelyan, V., Krashes, M.J., Lowell, B.B., Carlezon, W.A., Jr, and Bonci, A. (2017). Pathway- and Cell-Specific Kappa-Opioid Receptor Modulation of Excitation-Inhibition Balance Differentially Gates DI and D2 Accumbens Neuron Activity. Neuron 93, 147-163.103. Smith, J.S., Schindler, A G., Martinelli, E., Gustin, R.M., Bruchas, M.R., and Chavkin, C. (2012). Stress-induced activation of the dynorphin / K-opioid receptor system in the amygdala potentiates nicotine conditioned place preference. J. Neurosci. 32, 1488-1495.104. Navratilova, E., Nation, K., Remeniuk, B., Neugebauer, V., Bannister, K., Dickenson, A.H., and Porreca, F. (2020). Selective modulation of tonic aversive qualities of neuropathic pain by morphine in the central nucleus of the amygdala requires endogenous opioid signaling in the anterior cingulate cortex. Pain 161, 609-618.Attorney Docket No. 5470.984.WO105. Wassum, K.M., Cely, I.C., Balleine, B.W., and Maidment, N.T. (2011). Micro-opioid receptor activation in the basolateral amygdala mediates the learning of increases but not decreases in the incentive value of a food reward. J. Neurosci. 31, 1591-1599.106. Lichtenberg, N.T., and Wassum, K.M. (2017). Amygdala mu-opioid receptors mediate the motivating influence of cue-triggered reward expectations. Eur. J. Neurosci. 45, 381-387.107. Randall -Thompson, J.F., Pescatore, K.A., and Unterwald, E.M. (2010). A role for delta opioid receptors in the central nucleus of the amygdala in anxiety-like behaviors. Psychopharmacology 212, 585-595.108. Rodgers, R.J. (1978). Influence of intra-amygdaloid opiate injections on shock thresholds, tail-flick latencies and open field behaviour in rats. Brain Res. 153, 21 1-216.109.Pavlovic, Z.W., Cooper, M.L., and Bodnar, R.J. (1996). Opioid antagonists in the periaqueductal gray inhibit morphine and beta-endorphin analgesia elicited from the amygdala of rats. Brain Res. 741, 13-26.110. Nation, K.M., De Felice, M., Hernandez, P.I., Dodick, D.W., Neugebauer, V., Navratilova, E., and Porreca, F. (2018). Lateralized kappa opioid receptor signaling from the amygdala central nucleus promotes stress-induced functional pain. Pain 159, 919-928.11 l. Chaudun, F., Python, L., Liu, Y., Hiver, A., Cand, J., Kieffer, B.L., Valjent, E., and Luscher, C. (2024). Distinct p-opioid ensembles trigger positive and negative fentanyl reinforcement. Nature 630, 141-148.112. Zhou, W. , Li, Y., Meng, X., Liu, A., Mao, Y„ Zhu, X., Meng, Q„ Jin, Y„ Zhang, Z„ and Tao, W. (2021). Switching of delta opioid receptor subtypes in central amygdala microcircuits is associated with anxiety states in pain. J. Biol. Chem. 296, 100277.113. Liu, S.S., Pickens, S., Burma, N.E., Ibarra-Lecue, I., Yang, H., Xue, L., Cook, C., Hakimian, J.K., Severino, A.L., Lueptow, L., et al. (2019). Kappa Opioid Receptors Drive a Tonic Aversive Component of Chronic Pain. J. Neurosci. 39, 4162-4178.114. Tejeda, H.A., Hanks, A.N., Scott, L., Mejias-Aponte, C., Hughes, Z.A., and O’Donnell, P. (2015). Prefrontal Cortical Kappa Opioid Receptors Attenuate Responses to Amygdala Inputs. Neuropsychopharmacology 40, 2856-2864.115. Crowley, N.A., Bloodgood, D.W., Hardaway, J. A., Kendra, A.M., McCall, J.G., Al-Hasani, R., McCall, N.M., Yu, W., Schools, Z.L., Krashes, M.J., et al. (2016). Dynorphin Controls the Gain of an Amygdalar Anxiety Circuit. Cell Rep. 14, 2774-2783.116. Knoll, A.T., Muschamp, J.W., Sillivan, S.E., Ferguson, D., Dietz, D.M., Meloni, E.G., Carroll, F.I., Nestler, E.J., Konradi, C., and Carlezon, W.A., Jr (2011). Kappa opioid receptor signaling in the basolateral amygdala regulates conditioned fear and anxiety in rats. Biol. Psychiatry 70, 425-433.117.Bruchas, M.R., Land, B.B., Lemos, J.C., and Chavkin, C. (2009). CRF1-R activation of the dynorphin / kappa opioid system in the mouse basolateral amygdala mediates anxiety-likeAttorney Docket No. 5470.984.WO behavior. PLoS One V, e8528.118. Liu, N.-J., Chakrabarti, S., Schnell, S., Wessendorf, M., and Gintzler, A.R. (2011). Spinal Synthesis of Estrogen and Concomitant Signaling by Membrane Estrogen Receptors Regulate Spinal K- and p-Opioid Receptor Eleterodimerization and Female-Specific Spinal Morphine Antinociception. J. Neurosci. 31, 11836-11845.119. Bloodgood, D.W., Hardaway, J.A., Stanhope, C.M., Pati, D., Pina, M.M., Neira, S., Desai, S., Boyt, K M., Palmiter, R.D., and Kash, T.L. (2021). Kappa opioid receptor and dynorphin signaling in the central amygdala regulates alcohol intake. Mol. Psychiatry 26, 2187-2199.120.Normandeau, C.P., Torruella Suarez, M.L., Sarret, P., McElligott, Z.A., and Dumont, E.C. (2018). Neurotensin and dynorphin Bi-Directionally modulate CeA inhibition of oval BNST neurons in male mice. Neuropharmacology 143, 113-121.121. Hardaway, J. A., Halladay, L.R., Mazzone, C M., Pati, D., Bloodgood, D.W., Kim, M., Jensen, J., DiBerto, J.F., Boyt, K.M., Shiddapur, A., et al. (2019). Central amygdala prepronociceptin-expressing neurons mediate palatable food consumption and reward. Neuron 102, 1037-1052.e7.122. Scherrer, G., Imamachi, N., Cao, Y.-Q., Contet, C., Mennicken, F., O’Donnell, D., Kieffer, B.L., and Basbaum, A.I. (2009). Dissociation of the opioid receptor mechanisms that control mechanical and heat pain. Cell 137, 1148-1159.123. Ong, E.W., and Cahill, C.M. (2014). Molecular Perspectives for mu / delta Opioid Receptor Heteromers as Distinct, Functional Receptors. Cells 3, 152-179.124. Sriram, K., and Insel, P.A. (2018). G Protein-Coupled Receptors as Targets for Approved Drugs: How Many Targets and How Many Drugs? Mol. Pharmacol. 93, 251-258.125. Alhosaini, K., Azhar, A., Alonazi, A., and Al-Zoghaibi, F. (2021). GPCRs: The most promiscuous druggable receptor of the mankind. Saudi Pharm. J. 29, 539-551.126.Betke, K.M., Wells, C.A., and Hamm, H.E. (2012). GPCR mediated regulation of synaptic transmission. Prog. Neurobiol. 96, 304-321.127. Huang, Y., and Thathiah, A. (2015). Regulation of neuronal communication by G protein- coupled receptors. FEBS Lett. 589, 1607-1619.128. Gerber, K.J., Squires, K.E., and Hepler, J.R. (2016). Roles for regulator of G protein signaling proteins in synaptic signaling and plasticity. Mol. Pharmacol. 89, 273-286.129. Chen, W.-H., Lien, C.-C., and Chen, C.-C. (2022). Neuronal basis for pain-like and anxietylike behaviors in the central nucleus of the amygdala. Pain 163, e463-e475.130. Ahn, D.K., Kim, K.H., Ju, J.S., Kwon, S., and Park, J.S. (2001). Microinjection of arginine vasopressin into the central nucleus of amygdala suppressed nociceptive jaw opening reflex in freely moving rats. Brain Res. Bull. 55, 117-121.Attorney Docket No. 5470.984.WO131 Cragg, B., Ji, G., and Neugebauer, V. (2016). Differential contributions of vasopressin VIA and oxytocin receptors in the amygdala to pain-related behaviors in rats. Mol. Pain 12. https: / / doi.org / 10.1177 / 1744806916676491.132. Sharfman, N.M., Kelley, L.K., Secci, M E., and Gilpin, N.W. (2022). Melanocortin-4 receptor signaling in the central amygdala mediates chronic inflammatory pain effects on nociception. Neuropharmacology 210, 109032.133. Li, J., Zhang, J.J., Xu, S.-L., and Yu, L.-C. (2012). Antinociceptive effects induced by injection of the galanin receptor 1 agonist M617 into central nucleus of amygdala in rats. Neurosci. Lett. 526, 45-48.134.Manglik, A., Lin, H., Aryal, D.K., McCorvy, J.D., Dengler, D , Corder, G., Levit, A., Kling, R.C., Bemat, V., Hubner, H., et al. (2016). Structure-based discovery of opioid analgesics with reduced side effects. Nature 537, 185-190.135. Corder, G., Tawfik, V.L., Wang, D., Sypek, E.I., Low, S.A., Dickinson, J.R., Sotoudeh, C., Clark, J.D , Barres, B.A., Bohlen, C.J., et al. (2017). Loss of p opioid receptor signaling in nociceptors, but not microglia, abrogates morphine tolerance without disrupting analgesia. Nat. Med. 23, 164-173.136. Huang, T., Lin, S.-H., Malewicz, N.M., Zhang, Y., Zhang, Y., Goulding, M., LaMotte, R.H., and Ma, Q. (2019). Identifying the pathways required for coping behaviours associated with sustained pain. Nature 565, 86-90.137. Petrie, K.A., Bubser, M., Casey, C.D., Davis, M.D., Roth, B.L., and Deutch, A.Y. (2004). The neurotensin agonist PD149163 increases Fos expression in the prefrontal cortex of the rat. Neuropsychopharmacology 29, 1878-1888.138. Tsutsumi, M., Claus, T.H., Liang, Y., Li, Y., Yang, L., Zhu, J., Dela Cruz, F., Peng, X., Chen, H., Yung, S.L., et al. (2002). A potent and highly selective VPAC2 agonist enhances glucose-induced insulin release and glucose disposal: a potential therapy for type 2 diabetes. Diabetes 51, 1453-1460.139.Phebus, L.A., Johnson, K.W., Zgombick, J.M., Gilbert, P.J., Van Belle, K., Mancuso, V., Nelson, D.L., Calligaro, D.O., Kiefer, A.D., Jr, Branchek, T.A., et al. (1997).Characterization of LY344864 as a pharmacological tool to study 5-HT1F receptors: binding affinities, brain penetration and activity in the neurogenic dural inflammation model of migraine. Life Sci. 61, 2117-2126.140. Ankersen, M., Crider, M., Liu, S., Ho, B., Andersen, H.S., and Stidsen, C. (1998). Discovery of a Novel Non-Peptide Somatostatin Agonist with SST4 Selectivity. J. Am. Chem. Soc. 120, 1368-1373.14LMonn, J. A., Valli, M.J., Massey, S.M., Hansen, M.M., Kress, T.J., Wepsiec, J.P., Harkness, A.R., Grutsch, J.L., Wright, R.A., Johnson, B.G., et al. (1999). Synthesis, Pharmacological Characterization, and Molecular Modeling of Heterobicyclic Amino Acids Related to (+)-2- Aminobicyclo[3.1 ,0]hexane- 2,6-dicarboxylic Acid (LY354740): Identification of Two NewAttorney Docket No. 5470.984.WOPotent, Selective, and Systemically Active Agonists for Group IT Metabotropic Glutamate Receptors. J. Med. Chem. 42, 1027-1040.142. Slosky, L.M., Bai, Y., Toth, K., Ray, C., Rochelle, L.K., Badea, A., Chandrasekhar, R., Pogorelov, V.M., Abraham, D.M., Atluri, N., et al. (2020). P-Arrestin-Biased Allosteric Modulator of NT SRI Selectively Attenuates Addictive Behaviors. Cell 181, 1364— 1379. el4.143. Sharpe, A.L., Varela, E., and Beckstead, M.J. (2017). Systemic PD149163, a neurotensin receptor 1 agonist, decreases methamphetamine self-administration in DBA / 2J mice without causing excessive sedation. PLoS One 12, e0180710.144. Ehrlich, A.T., Couvineau, P., Schamiloglu, S., Wojcik, S., Da Fonte, D., Mezni, A., von Zastrow, M., Bender, K.J., Bouvier, M., and Kieffer, B.L. (2022). Visualization of real-time receptor endocytosis in dopamine neurons enabled by NTSR1 -Venus knock-in mice. Front. Cell. Neurosci. 16, 1076599.145. Caceda, R., Kinkead, B., and Nemeroff, C.B. (2006). Neurotensin: role in psychiatric and neurological diseases. Peptides 27, 2385-2404.146. Ferraro, L., Tiozzo Fasiolo, L., Beggiato, S., Borelli, A C., Pomierny-Chamiolo, L., Frankowska, M., Antonelli, T., Tomasini, M.C., Fuxe, K., and Filip, M. (2016). Neurotensin: A role in substance use disorder? J. Psychopharmacol. 30, 112-127.147. Gereau, G.B., Zhou, D., Van Voorhies, K., Tyler, R.E., Campbell, J., Murray, J.G., Alvarez- Pamir, A., Wykoff, L.A., Companion, M.A., Jackson, M.R., et al. (2024). P-arrestin-biased Allosteric Modulator of Neurotensin Receptor 1 Reduces Ethanol Drinking and Responses to Ethanol Administration in Rodents. bioRxiv. https: / / doi.org / 10.! 101 / 2024.04.10.588903.148. Pinkerton, A.B., Peddibhotla, S., Yamamoto, F., Slosky, L.M., Bai, Y., Maloney, P., Hershberger, P., Hedrick, M.P., Falter, B., Ardecky, R.J., et al. (2019). Discovery of P- arrestin biased, orally bioavailable, and CNS penetrant neurotensin receptor 1 (NTR1) allosteric modulators. J. Med. Chem. 62, 8357-8363.149.Krumm, B E., DiBerto, J.F., Olsen, R.H.J., Kang, H.J., Slocum, S T., Zhang, S., Strachan, R.T., Huang, X.-P., Slosky, L.M., Pinkerton, A.B., et al. (2023). Neurotensin receptor allosterism revealed in complex with a biased allosteric modulator. Biochemistry 62, 1233— 1248.150. Menendez- Arias, L., and Delgado, R. (2022). Update and latest advances in antiretroviral therapy. Trends Pharmacol. Sci. 43, 16-29.151. Al-Lazikani, B., Baneiji, U., and Workman, P. (2012). Combinatorial drug therapy for cancer in the post-genomic era. Nat. Biotechnol. 30, 679-692.152. Capi, M., de Andres, F , Lionetto, L., Gentile, G., Cipolla, F., Negro, A., Borro, M., Martelletti, P., and Curto, M. (2017). Lasmiditan for the treatment of migraine. Expert Opin. Investig. Drugs 26, 227-234.Attorney Docket No. 5470.984.WO153. Brennan, T.I., Vandermeulen, E.P., and Gebhart, G.F. (1996). Characterization of a rat model of incisional pain. Pain 67, 493-502.154. Chou, R., Turner, J. A., Devine, E.B., Hansen, R.N., Sullivan, S.D., Blazina, I., Dana, T., Bougatsos, C., and Deyo, R.A. (2015). The Effectiveness and Risks of Long-Term Opioid Therapy for Chronic Pain: A Systematic Review for a National Institutes of Health Pathways to Prevention Workshop. Ann. Intern. Med. 162, 276-286.155.Benyamin, R., Trescot, A M., Datta, S., Buenaventura, R., Adlaka, R., Sehgal, N., Glaser, S.E., and Vallejo, R. (2008). Opioid complications and side effects. Pain Physician 11, S105-S120.156.1asmin, L., Burkey, A R., Card, J.P., and Basbaum, A. I. (1997). Transneuronal labeling of a nociceptive pathway, the spino-(trigemino-)parabrachio-amygdaloid, in the rat. J. Neurosci. 17, 3751-3765.157.Kiritoshi, T., Yakhnitsa, V., Singh, S., Wilson, T.D., Chaudhry, S., Neugebauer, B., Torres- Rodriguez, J.M., Lin, J.L., Carrasquillo, Y., and Neugebauer, V. (2024). Cells and circuits for amygdala neuroplasticity in the transition to chronic pain. Cell Rep. 43, 114669.158. Wang, Y., Krabbe, S., Eddison, M., Henry, F.E., Fleishman, G., Lemire, A.L., Wang, L., Korff, W ., Tillberg, P.W., Liithi, A., et al. (2023). Multimodal mapping of cell types and projections in the central nucleus of the amygdala. Elife 12. https: / / doi.org / 10.7554 / eLife.84262.159. Poulin, J.-F., Caronia, G., Hofer, C., Cui, Q., Helm, B , Ramakrishnan, C., Chan, C.S., Dombeck, D.A., Deisseroth, K., and Awatramani, R. (2018). Mapping projections of molecularly defined dopamine neuron subtypes using intersectional genetic approaches. Nat. Neurosci. 21, 1260-1271.160. Zhao, Q., Ito, T., Soko, C., Hori, Y., Furuyama, T., Hioki, H., Konno, K., Yamasaki, M., Watanabe, M., Ohtsuka, S., et al. (2022). Histochemical characterization of the dorsal raphe- periaqueductal grey dopamine transporter neurons projecting to the extended amygdala. eNeuro 9, ENEUR0.0121-0122.2022.161. Welch, J.D., Kozareva, V., Ferreira, A., Vanderburg, C., Martin, C., and Macosko, E.Z. (2019). Single-cell multi-omic integration compares and contrasts features of brain cell identity. Cell 177, 1873-1887.el7.162. Yu, W., Pati, D., Pina, M.M., Schmidt, K.T., Boyt, K.M., Hunker, A.C., Zweifel, L.S., McElligott, Z.A., and Kash, T.L. (2021). Periaqueductal gray / dorsal raphe dopamine neurons contribute to sex differences in pain-related behaviors. Neuron 109, 1365-1380. e5.163. Li, C., Sugam, J.A., Lowery-Gionta, E.G., McElligott, Z.A., McCall, N.M., Lopez, A.J., McKlveen, J.M., Pled, K.E., and Kash, T.L. (2016). Mu opioid receptor modulation of dopamine neurons in the periaqueductal gray / dorsal raphe: A role in regulation of pain. Neuropsychopharmacology 41, 2122-2132.164. Li, H., Namburi, P., Olson, J.M., Borio, M., Lemieux, M.E., Beyeler, A., Calhoon, G.G.,Attorney Docket No. 5470.984.WOHitora-Imamura, N., Coley, A. A., Libster, A., et al. (2022). Neurotensin orchestrates valence assignment in the amygdala. Nature 608, 586-592.165.Mazzitelli, M., and Neugebauer, V. (2019). Amygdala group II mGluRs mediate the inhibitory effects of systemic group II mGluR activation on behavior and spinal neurons in a rat model of arthritis pain. Neuropharmacology J 58, 107706.166. Woo, M.S. (2021). Neuronal Metabotropic Glutamate Receptor 8 Protects Against Neurodegeneration in CNS Inflammation (Universitat Hamburg).167. Palazzo, E., Marabese, I., Soukupova, M., Luongo, L., Boccella, S., Giordano, C., de Novellis, V., Rossi, F., and Maione, S. (2011). Metabotropic glutamate receptor subtype 8 in the amygdala modulates thermal threshold, neurotransmitter release, and rostral ventromedial medulla cell activity in inflammatory pain. J. Neurosci. 31, 4687-4697.168. Palazzo, E., Fu, Y., Ji, G., Maione, S., and Neugebauer, V. (2008). Group III mGluR7 and mGluR8 in the amygdala differentially modulate nocifensive and affective pain behaviors. Neuropharmacology 55, 537-545.169. Jung, M., Dourado, M., Maksymetz, J., Jacobson, A., Laufer, B.I., Baca, M., Foreman, O., Hackos, D.H., Riol-Blanco, L., and Kaminker, J.S. (2023). Cross-species transcriptomic atlas of dorsal root ganglia reveals species-specific programs for sensory function. Nat. Common. 14, 366.170. Mosley, R.L., Lu, Y., Olson, K.E., Machhi, J., Yan, W ., Namminga, K.L., Smith, J.R., Shandler, S.J., and Gendelman, H E. (2019). A Synthetic Agonist to Vasoactive Intestinal Peptide Receptor-2 Induces Regulatory T Cell Neuroprotective Activities in Models of Parkinson’s Disease. Front. Cell. Neurosci. 13, 421.171. Olson, K.E., Kosloski-Bilek, L.M., Anderson, K.M., Diggs, B.J., Clark, B E., Gledhill, J.M., Jr, Shandler, S.J., Mosley, R.L., and Gendelman, H.E. (2015). Selective VIP Receptor Agonists Facilitate Immune Transformation for Dopaminergic Neuroprotection in MPTP- Intoxicated Mice. J. Neurosci. 35, 16463-16478.172. Lamb, Y.N. (2019). Lasmiditan: First Approval. Drugs 79, 1989-1996.173. Allen, W.E., DeNardo, L.A., Chen, M.Z., Liu, C.D., Loh, K.M., Fenno, L.E., Ramakrishnan, C., Deisseroth, K., and Luo, L. (2017). Thirst-associated preoptic neurons encode an aversive motivational drive. Science 357, 1149-1155.174.Madisen, L., Zwingman, T.A., Sunkin, S.M., Oh, S.W., Zariwala, H.A., Gu, H., Ng, L.L., Palmiter, R D , Hawrylycz, M.J., Jones, A.R., et al. (2010). A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133-140.175. Cai, X., Huang, H., Kuzirian, M.S., Snyder, L.M., Matsushita, M., Lee, M.C., Ferguson, C., Homanics, G.E., Barth, A.L., and Ross, S.E. (2016). Generation of a KOR-Cre knockin mouse strain to study cells involved in kappa opioid signaling. Genesis 54, 29-37.Attorney Docket No. 5470.984.WO176. Chevalier, C., Nicolas, J.-F., and Petit, A.-C. (2014). Preparation and Delivery of 4- Hydroxy-Tamoxifen for Clonal and Polyclonal Labeling of Cells of the Surface Ectoderm, Skin, and Hair Follicle. In Epidermal Cells: Methods and Protocols, K. Turksen, ed. (Springer New York), pp. 239-245.177. Nichols, M.L., Allen, B.J., Rogers, S.D., Ghilardi, J.R., Honore, P., Luger, N.M., Finke, M.P., Li, J., Lappi, D.A., Simone, D.A., et al. (1999). Transmission of chronic nociception by spinal neurons expressing the substance P receptor. Science 286, 1558-1561.178.Dubuisson, D., and Dennis, S.G. (1977). The formalin test: a quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats. Pain 4, 161-174.179. Tj01sen, A., Berge, O.-G , Hunskaar, S., Rosland, J.H., and Hole, K. (1992). The formalin test: an evaluation of the method. Pain 51, 5-17.180. Ossipov, M.H., Dussor, G.O., and Porreca, F. (2010). Central modulation of pain. J. Clin. Invest. 120, 3779-3787.181. Nakamura, K., and Morrison, S.F. (2007). Central efferent pathways mediating skin cooling- evoked sympathetic thermogenesis in brown adipose tissue. Am. I. Physiol. Regul. Integr. Comp. Physiol. 292, R127-R136.182. Picelli, S., Faridani, O.R., Bjbrklund, A.K., Winberg, G., Sagasser, S., and Sandberg, R. (2014). Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171-181.183. Chen, M.B., Jiang, X., Quake, S.R., and Stidhof, T.C. (2020). Persistent transcriptional programmes are associated with remote memory. Nature 587, 437-442.184. Liao, Y., Smyth, G.K., and Shi, W. (2014). featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923-930.185. Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., and Gingeras, T.R. (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21.186. Butler, A., Hoffman, P., Smibert, P., Papalexi, E., and Satija, R. (2018). Integrating singlecell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36, 411-420.187. Stuart, T., Butler, A., Hoffman, P., Hafemeister, C., Papalexi, E., Mauck, W.M., 3rd, Hao, Y., Stoeckius, M., Smibert, P., and Satija, R. (2019). Comprehensive Integration of SingleCell Data. Cell 177, 1888-19O2.e21.188. Conway, J.R., Lex, A., and Gehlenborg, N. (2017). UpSetR: an R package for the visualization of intersecting sets and their properties. Bioinformatics 33, 2938-2940.189. Stringer, C., Wang, T., Michaelos, M., and Pachitariu, M. (2021). Cellpose: a generalist algorithm for cellular segmentation. Nat. Methods 18, 100-106.Attorney Docket No. 5470.984.WO190. Petukhov, V., Xu, R.J., Soldatov, R.A., Cadinu, P., Khodosevich, K., Moffitt, J.R., and Kharchenko, P.V. (2022). Cell segmentation in imaging-based spatial transcriptomics. Nat. Biotechnol. 40, 345-354.191 . Ahlers, J., Althviz More, D., Amsalem, O., and Anderson, A. napari: a multi-dimensional image viewer for Python (vO. 4.18). Zenodo.Example 2. NTSR1 Small Molecules to Treat Pain Unpleasantness

[0184] Synthesis of SBI-553 and Analogs. SBI-553 and analogs thereof were synthesized as shown in Scheme 1.SCHEME 1

[0185] NTSR1 in vitro signaling assays. Arrestin and Gq Bioluminescence Resonance Energy Transfer (BRET) assays were previously described in Krumm et al. (2023) Biochemistry 62: 1233- 1248. In brief, HEK293T cells were maintained in DMEM containing 10% (v / v) FBS, 1 IU m ’1Penicillin G, and 100 pg m '1Streptomycin were passed to 10 cm dishes and co-transfected with NTSR1 containing C-terminal Renilla luciferase (ALuc) and Venus-tagged [3-arrestin2 (BRET1) for at least 24 hours. Transfected cells were then plated in poly-lysine-coated 96-well clear bottom cell culture plates in plating media (DMEM containing 1% (v / v)-dialyzed FBS, 1 IU mb'1Penicillin G, and 100 pg mL'1Streptomycin) at a density of 40,000 cells in 200 pL per well and incubated overnight. The next day, media was aspirated, cells were washed once with 60 pL of assay buffer (20mM HEPES, lx HBSS, pH 7.4), aspirated then 60 pL of assay buffer added to each well. Drug stimulation was performed with the addition of 15 pL of 6X drug dilution of modulatory compound in drug dilution buffer (20mM HEPES, lx HBSS, 0.3% (w / v) BSA, pH 7.4) per well, and incubated at room temperature for 80 minutes. 15 pL of 6X drug dilution of NTSs-13 was then added to each well and incubated for an additional 5 minutes. The ALuc substrate,Attorney Docket No. 5470.984.WO coelenterazine h (Promega), was added to a final concentration of 5 pM per well and incubated for an additional 5 minutes, plates were read at 90 minutes post-start of incubation for luminescence at 475 nm and fluorescent eYFP emission at 530 nm for 1 second per well using a PHERAstar FSX (BMG Labtech). Plates were read for multiple time points up to 30 minutes, where the BRET ratio of eYFPZRLuc was calculated per well, and the net BRET ratio was calculated by subtracting the eYFP RLuc ratio of wells without Venus-P- Arrestin present. The net BRET ratio was normalized to wells without the addition of SBI-553 (NTS only).

[0186] To measure NT SRI -mediated G protein dissociation (BRET2), procedures were similar to NTSR1 -mediated P-Arrestin2 recruitment, except HEK293T cells were co-transfected in a 1 : 1 : 1 : 1 ratio of Gaq- / ?Luc, G 3, GFP2-Gy9, and NTSR1, respectively, for at least 24 hours. Transfected cells were then plated in poly-lysine-coated 96-well clear bottom cell culture plates in plating media (DMEM containing 1% (v / v)-dialyzed FBS, 1 IU mL'1Penicillin G, and 100 pg mL'1Streptomycin) at a density of 40,000 cells in 200 pL per well and incubated overnight. The next day, media was aspirated, cells were washed once with 70 pL of assay buffer (20mM HEPES, lx HBSS, pH 7.4), aspirated, then 70 pL of assay buffer containing BRET2 Luc substrate Coelenterazine 400a (Nanolight) at 5 pM final concentration was added to each well and incubated for 5 minutes. Drug stimulation was performed with the addition of 30 pL of pre-mixed drug dilutions of NTSs-13 and SBI-553 in drug dilution buffer (20mM HEPES, lx HBSS, 0.3% (w / v) BSA, pH 7.4) per well, and incubated at room temperature for an additional 5 minutes. Plates were read for luminescence at 410 nm and fluorescent GFP2 emission at 515 nm for 1 second per well using a PHERAstar FSX (BMG Labtech). The ratio of GFP2 / Luc was calculated per well and plotted as a function of drug concentration using Graphpad Prism 8 (Graphpad Software Inc., San Diego, CA). The results of this analysis are presented in FIGS. 15-17.

[0187] Animals. All procedures followed animal care guidelines approved by the University of North Carolina at Chapel Hill and Stanford University Administrative Panel on Laboratory Animal Care, in accordance with American Veterinary Medical Association guidelines and the International Association for the Study of Pain. Mice were housed 2-5 per cage and maintained on a 12 hr light / dark cycle in a temperature controlled environment with ad lib access to food and water. Male and female C57BL / 6 wild-type mice from 8 to 15 weeks old were used for behavioral pharmacology experiments. No sex differences were observed across behavioral studies.Attorney Docket No. 5470.984.WO

[0188] Hot Plate. Mice were tail marked and habituated to the behavior room 30 minutes prior to experimentation. Upon habituation, mice were administered either vehicle or drug via intraperitoneal (i.p.) injection 20 minutes before hot plate. To measure affective-motivational pain, mice were individually transferred to the 52°C hotplate for one minute or until jumping behavior (to adhere to IACUC guidelines). Affective motivational behaviors, including attending the hindpaw, guarding the hindpaw, rearing, and jumping were quantified in Ethovision using a 2 or 4 camera setup. The results of this analysis are presented in FIG. 18.

[0189] Paw incision. We conducted all surgeries under aseptic conditions using a digital small animal stereotaxic instrument (David Kopf Instruments). Mice were anesthetized with isoflurane (3-5% induction, 1-2% maintenance). Body temperature was maintained using a heating pad. C57B16 / J mice were tested on two alternating days before surgery to establish a baseline tactile withdrawal threshold using von Frey filaments and the up-down method, as described in Chaplan et al. 1994 202. Following baseline testing, mice were anesthetized under isoflurane, and a 0.7 cm single incision was made using a 11 scalpel blade on the left hindpaw surface, as described in 203. The subcutaneous plantaris muscle was longitudinally incised after being isolated, and the wound was sutured with two 5.0 absorbable sutures upon hemostasis. Mice were placed in recovery cages, and tactile withdrawal threshold was retested three days post-surgery. Here, mice were tested before, 20 minutes post-i.p. injection, and 60 minutes post-i.p. injection of a vehicle, 30 mg / kg SBI-553, or ATTA.

[0190] Von Frey filaments. To assess mechanical sensitivity of the paw, the tactile withdrawal threshold was determined in response to probing of the hindpaw with eight calibrated von Frey filaments (Stoelting, Wood Dale, IL, USA in logarithmically spaced increments ranging from 0.04 to 8 g. Filaments were applied perpendicularly to the plantar surface of the paw. The 50 % paw withdrawal threshold was determined in grams by the Dixon nonparametric test (Dixon (1980) Annu. Rev. Pharmacol. Toxicol. 20:441-462). The protocol was repeated (10-20 seconds apart) until four changes in behavior occurred. Further, a 0.6 g filament was applied 3 times (10-20 seconds apart) and averaged to evaluate pain scores where 0=no response, l=response without withdrawal, 2=slow withdrawal, 3=sharp withdrawal, and 4=strong withdrawal together with another nocifensive response (e.g., licking, guarding, jumping). The results of this analysis are presented in FIGS. 19-20.Attorney Docket No. 5470.984.WO

[0191] Conditioned place preference testing. Conditioned place preference (CPP) was used to measure the conditioned rewarding effects of morphine in accordance with methods known in the art. The results of this analysis are presented in FIG. 21. An illustrative method for carrying out CPP includes conditioning mice with a counterbalanced place conditioning paradigm (see, e.g., Varadi et al. (2016) J. Med. Chem. 59:8381-8397). Groups of C57BL / 6J mice (n = 18-24) are allowed to freely explore a three-compartment apparatus for 30 minutes. The amount of time subjects spend in each compartment is measured over a 30 minute testing period. Prior to place conditioning, the animals do not demonstrate significant differences in their time spent exploring the left vs right compartments. During each of the next 2 days, mice are administered vehicle (0.9% saline) and consistently confined in a randomly assigned outer compartment for 40 minutes, half of each group in the right chamber, half in the left chamber. Four hours later, mice are administered morphine (5 mg / kg, icv) alone or in combination with a compound that modulates NTSR1 activity (30 mg / kg, icv) or vehicle (blank, BL) and are placed in the opposite compartment for 40 minutes. Mice are allowed to move freely and CPP scores are calculated as time spent on the respective side on test day minus the average time spent on the same side during preconditioning.

[0192] The foregoing examples are illustrative of the present invention and are not to be construed as limiting thereof. Although the invention has been described in detail with reference to preferred embodiments, variations and modifications exist within the scope and spirit of the invention as described and defined in the following claims.

Claims

Attorney Docket No. 5470.984.WOWhat is Claimed:

1. A method of treating and / or preventing pain in a subject, comprising administering to the subject a therapeutically effective amount of a G protein-coupled receptor (GPCR) ligand or pharmaceutically acceptable salt thereof, wherein the GPCR ligand targets a non-opioid GPCR in the amygdalar region of the brain, and wherein pain is alleviated and / or minimized in the subject.

2. A method of treating unpleasantness related to pain in a subject, comprising administering to the subject a therapeutically effective amount of G protein-coupled receptor (GPCR) ligand or pharmaceutically acceptable salt thereof, wherein the GPCR ligand targets a non-opioid GPCR in the amygdalar region of the brain, and wherein unpleasantness related to pain is alleviated and / or minimized in the subject.

3. The method of claims 1 or 2, wherein the therapeutically effective amount of the GPCR ligand is a dose of about 0.1 mg / kg / day to about 100 mg / kg / day.

4. The method of any one of claims 1-3, wherein the GPCR ligand specifically binds to a non-opioid GPCR.

5. The method of any one of claims 1-4, wherein the non-opioid GPCR is NTSR1, VIPR2, SSTR4, mGluR2, mGluR3, or 5HT1F.

6. The method of any one of claims 1-5, wherein the GPCR ligand is PD149163, SBI-553, BAY 55-9837, LY379268, NNC-269100, or LY344864.

7. The method of any one of claims 1-6, further comprising repeating the administering of the ligand to the subject.

8. The method of any one of claims 1-7, wherein two or more GPCR ligands are administered to the subject.Attorney Docket No. 5470.984.WO9. The method of claim 8, wherein one or more of the ligands is administered at subthreshold doses.

10. The method of any one of claims 1-9, wherein administering the ligand comprises oral administration, intravenous administration, intrathecal administration, intraperitoneal administration, intra-articular administration, epidural administration, or any combination thereof.

11. The method of any one of claims 1-10, wherein the pain is peripheral or central neuropathic pain, inflammatory pain, migraine-related pain, headache-related pain, irritable bowel syndrome-related pain, fibromyalgia-related pain, arthritic pain, skeletal pain, joint pain, gastrointestinal pain, muscle pain, angina pain, facial pain, pelvic pain, claudication, postoperative pain, post traumatic pain, tension-type headache, obstetric pain, gynecological pain, or chemotherapy-induced pain.

12. The method of any one of claims 1-11, wherein the subject has acute pain, chronic pain, or both.

13. The method of any one of claims 1-12, wherein the subject has sustained one or more injuries and / or one or more physical injuries.

14. The method of any one of claims 1-13, wherein the subject has opioid dependence and / or opioid use disorder (OUD).

15. The method of any one of claims 1-14, further comprising administering to the subject a therapeutically effective amount of one or more therapeutic agents.

16. The method of claim 15, wherein the one or more therapeutic agents are opioids, narcotic analgesics, Mu receptor agonists, Kappa receptor antagonists, non-narcotic analgesics, monoamine uptake inhibitors, adenosine regulating agents, cannabinoid derivatives, Substance PAttorney Docket No. 5470.984.WO antagonists, neurokinin-1 receptor antagonists, calcitonin gene-related peptide (CGRP) and its receptor inhibitors, sodium channel blockers, non-steroid anti-inflammatory drugs (NSAIDs), and any combination thereof.

17. The method of any one of claims 1-16, further comprising monitoring the subject for adverse effects.

Images